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General Tests, Processes and Apparatus PDF

This document describes general testing methods used for drugs and related articles. It includes common analytical techniques such as liquid chromatography, gas chromatography, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The tests are categorized by number and include methods for determining alcohol content, residue on ignition, loss on drying, pH, particle size, sterility, and more. The document provides detailed procedures for various tests like the alcohol number determination test.

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General Tests, Processes and Apparatus PDF

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GENERAL TESTS, PROCESSES

AND APPARATUS
General Tests, Processes and Apparatus includes common netic resonance (NMR) spectroscopy are performed as di-
methods for tests, useful test methods for quality recognition rected in the corresponding items under the Crude Drugs
of drugs and other articles related to them. Unless otherwise Test.
specified, acid-neutralizing capacity determination of gastro- The number of each test method is a category number
intestinal medicines, alcohol number determination, amino given individually. The number in blackets (< >) appeared
acid analysis of proteins, ammonium determination, arsenic in monograph indicates the number corresponding to the
determination, atomic absorption spectrophotometry, boil- general test method.
ing point determination, chloride determination, conduc-
tivity measurement, congealing point determination, deter-
mination of bulk and tapped densities, digestion test,
disintegration test, dissolution test, distilling range determi- 1. Chemical Methods
nation, endpoint determination in titrimetry, flame colora-
tion, fluorometry, foreign insoluble matter test for injec-
tions, foreign insoluble matter test for ophthalmic liquids 1.01 Alcohol Number
and solutions, gas chromatography, glycosylation analysis of Determination
glycoprotein, heavy metal determination, inductively cou-
pled plasma-atomic emission spectrometry and inductively Alcohol Number Determination represents the number of
coupled plasma-mass spectrometry, infrared spectropho- milliliters of ethanol at 159C obtained from 10 mL of tin-
tometry, insoluble particulate matter test for injections, in- cture or other preparations containing ethanol by the follow-
soluble particulate matter test for ophthalmic liquids and so- ing procedures.
lutions, iron determination, liquid chromatography, loss on
1. Method 1 Distilling method
drying determination, loss on ignition determination, mass
This is a method to determine the Alcohol Number by
spectrometry, melting point determination, methanol deter-
reading the number of milliliters of ethanol distillate at 159C
mination, methods for color matching, methods of adhesion
obtained from 10 mL of a sample measured at 159C by the
testing, microbial assay for antibiotics, mineral oil determi-
following procedures.
nation, nitrogen determination, nuclear magnetic resonance
1.1. Apparatus
spectroscopy, optical rotation determination, osmolarity de-
Use hard glass apparatus as illustrated in Fig. 1.01-1.
termination, oxygen flask combustion method, particle size
Ground glass may be used for the joints.
determination, particle size distribution test for prepara-
1.2. Reagent
tions, pH determination, powder particle density determina-
Alkaline phenolphthalein solution: To 1 g of phenol-
tion, qualitative test, refractive index determination, release
phthalein add 7 mL of sodium hydroxide TS and water to
test for preparations for cutaneous application, residual sol-
make 100 mL.
vents test, residue on ignition determination, specific gravity
1.3. Procedure
and density determination, specific surface area determina-
Transfer 10 mL of the sample preparation, accurately
tion, sulfate determination, test for bacterial endotoxins, test
measured at 15 ± 29C, to the distilling flask A, add 5 mL of
for glass containers for injections, test for metal particles in
water and boiling chips. Distil ethanol carefully into the
ophthalmic ointments, test for microbial limit, test for
glass-stoppered, volumetric cylinder D.
microbial limit for crude drugs, test for plastic containers,
By reference to Table 1.01-1, a suitable volume of distil-
test for pyrogen, test for readily carbonizable substances,
late (mL) should be collected, according to the content of
test for rubber closure for aqueous infusions, test for sterili-
ethanol in the sample preparation.
ty, test for total organic carbon, test of extractable volume
Prevent bumping during distillation by rendering the sam-
for injection, thermal analysis, thin-layer chromatography,
ple strongly acidic with phosphoric acid or sulfuric acid, or
turbidity measurement, ultravioletvisible spectrophotome-
by adding a small amount of paraffin, beeswax or silicone
try, uniformity of dosage units (test for content uniformity,
resin before starting the distillation.
mass variation test), viscosity determination, vitamin A
assay, water determination, and X-ray powder diffraction
are performed as directed in the corresponding articles under
the General Tests, Processes and Apparatus. The tests for Table 1.01-1
melting point of fats, congealing point of fatty acids, spe- Ethanol content in Distillate
cific gravity, acid value, saponification value, ester value, the sample (volz) to be collected
hydroxyl value, unsaponifiable matter and iodine value of (mL)
fats and fatty oils are performed as directed in the corre- more than 80 13
sponding items under Fats and Fatty Oils Test, and sam- 80 – 70 12
pling, preparation of sample for analysis, microscopic ex- 70 – 60 11
amination, purity test, loss on drying, total ash, acid-insolu- 60 – 50 10
ble ash, extract content, essential oil content of crude drugs 50 – 40 9
and assay of marker compounds for the assay of crude drugs 40 – 30 8
and extracts of Kampo Formulations utilizing nuclear mag- less than 30 7

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs, 25
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
26 1.01 Alcohol Number Determination / General Tests JP XVII
(iii).
To the distillate add 4 to 6 g of potassium carbonate and 1
to 2 drops of alkaline phenolphthalein solution, and shake
vigorously. If the aqueous layer shows no white turbidity,
agitate the distillate with additional potassium carbonate.
After allowing to stand in water at 15 ± 29C for 30 minutes,
read the volume of the upper reddish ethanol layer in mL,
and regard it as the Alcohol Number. If there is no clear
boundary surface between these two layers, shake vigorously
after addition of a few drops of water, then observe in the
same manner.
2. Method 2 Gas chromatography
This is a method to determine the alcohol number by de-
termining ethanol (C2H5OH) content (volz) from a sample
measured at 159C by the following procedures.
2.1. Reagent
Ethanol for alcohol number: Ethanol (99.5) with deter-
mined ethanol (C2H5OH) content. The relation between
specific gravity d 15
15 of ethanol and content of ethanol
(C2H5OH) is 0.797:99.46 volz, 0.796:99.66 volz, and
0.795:99.86 volz.
2.2. Preparation of sample solution and standard solution
Sample solution: Measure accurately a volume of sample
at 15 ± 29C equivalent to about 5 mL of ethanol (C2H5OH),
and add water to make exactly 50 mL. Measure accurately
25 mL of this solution, add exactly 10 mL of the internal
standard solution, and add water to make 100 mL.
Standard solution: Measure accurately 5 mL of ethanol
for alcohol number at the same temperature as the sample,
and add water to make exactly 50 mL. Measure accurately
25 mL of this solution, add exactly 10 mL of the internal
standard solution, and add water to make 100 mL.
2.3. Procedure
Place 25 mL each of the sample solution and the standard
solution in a 100-mL, narrow-mouthed, cylindrical glass bot-
tle sealed tightly with a rubber closure and aluminum band,
immerse the bottle up to the neck in water, allowed to stand
at room temperature for more than 1 hour in a room with lit-
tle change in temperature, shake gently so as not to splash
Fig. 1.01-1 the solution on the closure, and allow to stand for 30
minutes. Perform the test with 1 mL each of the gas in the
bottle with a syringe according to the Gas Chromatography
<2.02> under the following conditions, and calculate the
When the samples contain the following substances, carry
ratios, QT and QS, of the peak height of ethanol to that of
out pretreatment as follows before distillation.
the internal standard.
(i) Glycerin: Add sufficient water to the sample so that
the residue in the distilling flask, after distillation, contains QT 5 (mL)
Alcohol number ＝ ×
at least 50z of water. QS a volume (mL) of sample
(ii) Iodine: Decolorize the sample with zinc powder.
(iii) Volatile substances: Preparations containing appre- ethanol (C2H5OH) content (volz) of
ethanol for alcohol number
ciable proportions of essential oil, chloroform, diethyl ether ×
9.406
or camphor require treatment as follows. Mix 10 mL of the
sample, accurately measured, with 10 mL of saturated so- Internal standard solution—A solution of acetonitrile (3 in
dium chloride solution in a separator, add 10 mL of petro- 50).
leum benzin, and shake. Collect the separated aqueous layer. Operating conditions—
The petroleum benzin layer was extracted with two 5 mL Detector: A hydrogen flame-ionization detector.
portions of saturated sodium chloride solution. Combine the Column: A glass tube about 3 mm in inside diameter and
aqueous layers, and distill. According to the ethanol content about 1.5 m in length, packed with 150- to 180-mm porous
in the sample, collect a volume of distillate 2 to 3 mL more ethylvinylbenzene-divinylbenzene copolymer (mean pore
than that shown in the above Table. size: 0.0075 mm, 500 – 600 m2/g) for gas chromatography.
(iv) Other substances: Render preparations containing Column temperature: A constant temperature between
free ammonia slightly acidic with dilute sulfuric acid. If 1059C and 1159C.
volatile acids are present, render the preparation slightly Carrier gas: Nitrogen.
alkaline with sodium hydroxide TS, and if the preparations Flow rate: Adjust so that the retention time of ethanol is 5
contain soap along with volatile substances, decompose the to 10 minutes.
soap with an excess of dilute sulfuric acid before the ex- Selection of column: Proceed with 1 mL of the gas ob-
traction with petroleum benzin in the treatment described in tained from the standard solution in the bottle under the

above operating conditions, and calculate the resolution. For the distillation under reduced pressure, take the
Use a column giving elution of ethanol and the internal amount of sample specified in the monograph to the vacuum
standard in this order with the resolution between these distillation flask L, add 70 mL of water and 1 g of magne-
peaks being not less than 2.0. sium oxide, and connect to the apparatus (Fig. 1.02-2). To
the receiver M add 20 mL of a solution of boric acid (1 in
200) as absorbing liquid, put the end of the branch tube of
the distillation flask L in the absorbing liquid, and keep at
1.02 Ammonium Limit Test 609C using a water bath or alternative equipment. Adjust
the reduced pressure to get the distillate at a rate of 1 to 2
Ammonium Limit Test is a limit test for ammonium salt
mL per minute, and continue the distillation until to get 30
contained in drugs.
mL of the distillate. Cool the receiver M with running water
In each monograph, the permissible limit for ammonium
during the distillation. Get off the end of the branch tube
(as NH＋ 4 ) is described in terms of percentage (z) in paren-
from surface of the absorbing liquid, rinse in the end with a
theses.
small amount of water, then add water to the liquid to make
1. Apparatus 100 mL, and perform the test using this solution as the test
Use a distilling apparatus for ammonium limit test as illus- solution.
trated in Fig. 1.02-1. For the distillation under reduced pres- Place a volume of Standard Ammonium Solution, di-
sure, use the apparatus shown in Fig. 1.02-2. Either appa- rected in the monograph, in the distilling flask A or the
ratus are composed of hard glass, and ground-glass joints vacuum distillation flask L, proceed as for the preparation
may be used. All rubber parts used in the apparatus should of the test solution, and designate it as the control solution.
be boiled for 10 to 30 minutes in sodium hydroxide TS and 2.2. Test of the test solution and the control solution
for 30 to 60 minutes in water, and finally washed thoroughly Unless otherwise specified, proceed as directed in the fol-
with water before use. lowing.
Place 30 mL each of the test solution and the control solu-
2. Procedure
tion in Nessler tubes, add 6.0 mL of phenol-sodium penta-
2.1. Preparation of test solution and control solution
cyanonitrosylferrate (III) TS to each solution, and mix. Then
Unless otherwise specified, test solutions and control solu-
add 4 mL of sodium hypochlorite-sodium hydroxide TS and
tion are prepared as directed in the following.
water to make 50 mL, mix, and allow to stand for 60
Place an amount of the sample, directed in the mono-
minutes. Compare the color of both solutions against a
graph, in the distilling flask A. Add 140 mL of water and 2 g
white background by viewing downward or transversely: the
of magnesium oxide, and connect the distillation apparatus.
color developed in the test solution is not more intense than
To the receiver (measuring cylinder) F add 20 mL of boric
that of the control solution.
acid solution (1 in 200) as an absorbing solution, and im-
merse the lower end of the condenser. Adjust the heating to
give a rate of 5 to 7 mL per minute of distillate, and distill
until the distillate measures 60 mL. Remove the receiver
from the lower end of the condenser, rinsing the end part
with a small quantity of water, add sufficient water to make
100 mL and designate it as the test solution.

Fig. 1.02-2 Vacuum distilling apparatus for ammonium

Fig. 1.02-1 Distilling apparatus for ammonium limit test limit test

1.05 Mineral Oil Test

Mineral Oil Test is a method to test mineral oil in nona-
queous solvents for injections and for eye drops.
1. Procedure
Pour 10 mL of the sample into a 100-mL flask, and add 15
mL of sodium hydroxide solution (1 in 6) and 30 mL of
ethanol (95). Put a short-stemmed, small funnel on the neck Fig. 1.06-1

bent cotton with filter paper, 50 mm in length and 5 mm in prepared with 5 mL of water in the same manner. Determine
width, so that the end part of the paper is left to a length of the absorbances, AT, AC and AS, of the subsequent solutions
about 20 mm as a fuse-strip, and place the parcel in a plati- of the test solution, the correction solution and the standard
num basket or cylinder B. Place the sample in a suitable solution at 600 nm.
glass tube, weigh accurately, and moisten the cotton with the
Amount (mg) of fluorine (F) in the test solution
quantity of the sample specified in the monograph, bringing
＝ amount (mg) of fluorine in 5 mL of
the edge of the sample in contact with the cotton.
2.2. Method of combustion AT － AC 50
the standard solution × ×
Place the absorbing liquid specified in the monograph in AS V
flask A, fill it with oxygen, moisten the ground part of the
Standard Fluorine Solution: Dry sodium fluoride (stand-
stopper C with water, then ignite the fuse-strip, immediatey
ard reagent) in a platinum crucible between 5009C and
transfer it to the flask, and keep the flask airtight until the
5509C for 1 hour, cool it in a desiccator (silica gel), weigh ac-
combustion is completed. Shake the flask occasionally until
curaly about 66.3 mg of it, and dissolve in water to make ex-
the white smoke in A vanishes completely, allow to stand for
actly 500 mL. Pipet 10 mL of this solution, and dilute with
15 to 30 minutes, and designate the resulting solution as the
sufficient water to make exactly 100 mL.
test solution. Prepare the blank solution in the same manner,
3.4. Sulfur
without sample.
Apply a small amount of water to the upper part of A,
3. Procedure of determination pull out C carefully, and wash C, B and the inner side of A
Unless otherwise specified in the monograph, perform the with 15 mL of methanol. To this solution add 40 mL of
test as follows. methanol, then add exactly 25 mL of 0.005 mol/L barium
3.1. Chlorine and bromine perchlorate VS, allow to stand for 10 minutes, add 0.15 mL
Apply a small amount of water to the upper part of A, of arsenazo III TS with a measuring pipet, and titrate <2.50>
pull out C carefully, and transfer the test solution to a with 0.005 mol/L sulfuric acid VS. Perfrom the test with the
beaker. Wash C, B and the inner side of A with 15 mL of 2- blank solution in the same manner.
propanol, and combine the washings with the test solution.
Each mL of 0.005 mol/L barium perchlorate VS
To this solution add 1 drop of bromophenol blue TS, add
＝ 0.1604 mg of S
dilute nitric acid dropwise until a yellow color develops, then
add 25 mL of 2-propanol, and titrate <2.50> with 0.005
mol/L silver nitrate VS according to the potentiometric titra-
tion. Perform the test with the blank solution in the same 1.07 Heavy Metals Limit Test
manner, and make any necessary correction.
Heavy Metals Limit Test is a limit test of the quantity of
Each mL of 0.005 mol/L silver nitrate VS
heavy metals contained as impurities in drugs. The heavy
＝ 0.1773 mg of Cl
metals are the metallic inclusions that are darkened with so-
Each mL of 0.005 mol/L silver nitrate VS dium sulfide TS in acidic solution, as their quantity is ex-
＝ 0.3995 mg of Br pressed in terms of the quantity of lead (Pb).
In each monograph, the permissible limit for heavy metals
3.2. Iodine
(as Pb) is described in terms of ppm in parentheses.
Apply a small amount of water to the upper part of A,
pull out C carefully, add 2 drops of hydrazine monohydrate 1. Preparation of test solutions and control solutions
to the test solution, put C on A, and decolorize the solution Unless otherwise specified, test solutions and control solu-
by vigorous shaking. Transfer the content of A to a beaker, tions are prepared as directed in the following:
wash C, B and the inner side of A with 25 mL of 2-propanol, 1.1. Method 1
and transfer the washings to the above beaker. To this solu- Place an amount of the sample, directed in the mono-
tion add 1 drop of bromophenol blue TS, then add dilute graph, in a Nessler tube. Dissolve in water to make 40 mL.
nitric acid dropwise until a yellow color develops, and titrate Add 2 mL of dilute acetic acid and water to make 50 mL,
<2.50> with 0.005 mol/L silver nitrate VS according to the and designate it as the test solution.
potentiometric tiration. Perform the test with the blank solu- The control solution is prepared by placing the volume of
tion in the same manner, and make any necessary correction. Standard Lead Solution directed in the monograph in a
Nessler tube, and adding 2 mL of dilute acetic acid and
Each mL of 0.005 mol/L silver nitrate VS
water to make 50 mL.
＝ 0.6345 mg of I
1.2. Method 2
3.3. Fluorine Place an amount of the sample, directed in the mono-
Apply a small amount of water to the upper part of A, graph, in a quartz or porcelain crucible, cover loosely with a
pull out C carefully, transfer the test solution and the blank lid, and carbonize by gentle ignition. After cooling, add 2
solution to 50 mL volumetric flasks separately, wash C, B mL of nitric acid and 5 drops of sulfuric acid, heat cau-
and the inner side of A with water, add the washings and tiously until white fumes are no longer evolved, and inciner-
water to make 50 mL, and use these solutions as the test so- ate by ignition between 5009 C and 6009C. Cool, add 2 mL
lution and the correction solution. Pipet the test solution of hydrochloric acid, evaporate to dryness on a water bath,
(V mL) equivalent to about 30 mg of fluorine, V mL of the moisten the residue with 3 drops of hydrochloric acid, add
correction solution and 5 mL of standard fluorine solution, 10 mL of hot water, and warm for 2 minutes. Then add 1
transfer to 50-mL volumetric flasks separately, add 30 mL of drop of phenolphthalein TS, add ammonia TS dropwise
a mixture of alizarin complexone TS, acetic acid-potassium until the solution develops a pale red color, add 2 mL of
acetate buffer solution (pH 4.3) and cerium (III) nitrate TS dilute acetic acid, filter if necessary, and wash with 10 mL of
(1:1:1), add water to make 50 mL, and allow to stand for 1 water. Transfer the filtrate and washings to a Nessler tube,
hour. Perform the test with these solutions as directed under and add water to make 50 mL. Designate it as the test solu-
Ultraviolet-visible Spectrophotometry <2.24>, using a blank tion.

Fig. 1.08-1

the following method: (2) When an acetate is warmed with sulfuric acid and a
Weigh accurately about 1.7 g of amidosulfuric acid (stand- small quantity of ethanol (95), the odor of ethyl acetate is
ard reagent), previously dried in a desiccator (in vacuum, evolved.
silica gel) for about 48 hours, dissolve in water to make ex- (3) Neutral solutions of acetates produce a red-brown
actly 200 mL. Pipet 2 mL of this solution, and transfer to a color with iron (III) chloride TS, and a red-brown precipitate
digestion flask. When the test is performed as directed in the when boiled. The precipitate dissolves and the color of the
instrumental manual the nitrogen content (z) in amidosul- solution changes to yellow upon addition of hydrochloric
furic acid should be determined between 14.2z and 14.6z. acid.
3. Reagents, Test Solutions Aluminum salt
Decomposition accelerator: Unless otherwise specified, (1) Solutions of aluminum salts, when treated with am-
use 1 g of a powdered mixture of 10 g of potassium sulfate monium chloride TS and ammonia TS, yield a gelatinous,
and 1 g of cupper (II) sulfate pentahydrate. The composition white precipitate which does not dissolve in an excess of am-
and amount of the digestion accelerator may be modified if monia TS.
it is confirmed that the modified one give almost the same (2) Solutions of aluminum salts, when treated with so-
results using the sample as those obtained from the conven- dium hydroxide TS, yield a gelatinous, white precipitate
tional catalyst. which dissolves in an excess of the reagent.
(3) Solutions of aluminum salts, when treated with so-
4. Procedure
dium sulfide TS, yield a gelatinous, white precipitate which
Unless otherwise specified, proceed by the following
dissolves in an excess of the reagent.
method. Weigh accurately or pipet a quantity of the sample
(4) Add ammonia TS to solutions of aluminum salts
corresponding to 2 to 3 mg of nitrogen (N:14.01), and place
until a gelatinous, white precipitate is produced. The color
in the Kjeldahl flask A. Add the decomposition accelerator
of the precipitate changes to red upon addition of 5 drops of
and wash down any adhering sample from the neck of the
alizarin red S TS.
flask with a small quantity of water. Add 7 mL of sulfuric
acid, allowing it to flow down the inside wall of the flask. Ammonium salt
Then, while shaking the flask, add cautiously 1 mL of When heated with an excess of sodium hydroxide TS, am-
hydrogen peroxide (30) drop by drop along the inside wall of monium salts evolve the odor of ammonia. This gas changes
the flask. Heat the flask gradually, then heat so strong that moistened red litmus paper to blue.
the vapor of sulfuric acid is condensed at the neck of the
Antimony salt, primary
flask, until the solution changes through a blue and clear to
(1) When primary antimony salts are dissolved in a slight
a vivid green and clear, and the inside wall of the flask is free
excess of hydrochloric acid for the test and then diluted with
from a carbonaceous material. If necessary, add a small
water, a white turbidity is produced. The mixture produces
quantity of hydrogen peroxide (30) after cooling, and heat
an orange precipitate upon addition of 1 to 2 drops of so-
again. After cooling, add cautiously 20 mL of water, cool
dium sulfide TS. When the precipitate is separated, and so-
the solution, and connect the flask to the distillation appa-
dium sulfide TS is added to one portion of the precipitate
ratus (Fig. 1.08-1) washed beforehand by passing steam
and sodium hydroxide TS is added to another portion, it dis-
through it. To the absorption flask K add 15 mL of boric
solves in either of these reagents.
acid solution (1 in 25), 3 drops of bromocresol green-methyl
(2) Add water to acidic solutions of primary antimony
red TS and sufficient water to immerse the lower end of the
salts in hydrochloric acid until a small quantity of precipitate
condenser tube J. Add 30 mL of sodium hydroxide solution
is produced, and then add sodium thiosulfate TS: the pre-
(2 in 5) through the funnel F, rinse cautiously the funnel with
cipitate dissolves. A red precipitate is reproduced when the
10 mL of water, close the clamp attached to the rubber tub-
solution is heated.
ing G, then begin the distillation with stream, and continue
until the distillate measures 80 to 100 mL. Remove the ab- Aromatic amines, primary
sorption flask from the lower end of the condenser tube J, Acidic solutions of primary aromatic amines, when cooled
rinsing the end part with a small quantity of water, and in ice, mixed with 3 drops of sodium nitrite TS under agita-
titrate <2.50> the distillate with 0.005 mol/L sulfuric acid VS tion, allowed to stand for 2 minutes, mixed well with 1 mL
until the color of the solution changes from green through of ammonium amidosulfate TS, allowed to stand for 1
pale grayish blue to pale grayish red-purple. Perform a blank minute, and then mixed with 1 mL of N, N-diethyl-N?-1-
determination in the same manner, and make any necessary naphtylethylenediamine oxalate TS, exhibit a red-purple
correction. color.
Each mL of 0.005 mol/L sulfuric acid VS Arsenate
＝ 0.1401 mg of N (1) Neutral solutions of arsenates produce no precipitate
with 1 to 2 drops of sodium sulfide TS, but produce a yellow
If an automated apparatus is used, proceed as directed in
precipitate with hydrochloric acid subsequently added. The
the instrumental procedure.
separated precipitate dissolves in ammonium carbonate TS.
(2) Neutral solutions of arsenates produce a dark red-
brown precipitate with silver nitrate TS. When dilute nitric
1.09 Qualitative Tests acid is added to one portion of the suspension, and ammonia
TS is add to another portion, the precipitate dissolves in
Qualitative Tests are applied to the identification of drugs either of these reagents.
and are done generally with quantities of 2 to 5 mL of the (3) Neutral or ammonia alkaline solutions of arsenates
test solution. produce with magnesia TS a white, crystalline precipitate,
which dissolves by addition of dilute hydrochloric acid.
Acetate
(1) When warmed with diluted sulfric acid (1 in 2), ace- Arsenite
tates evolve the odor of acetic acid. (1) Acidic solutions of arsenites in hydrochloric acid

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
32 1.09 Qualitative Tests / General Tests JP XVII
produce a yellow precipitate with 1 to 2 drops of sodium sul- tate, which dissolves upon heating. When 1 drop of sodium
fide TS. When hydrochloric acid is added to one portion of nitrite TS is added to this solution, a pale yellow precipitate
the separated precipitate, it does not dissolve. When ammo- is produced.
nium carbonate TS is added to another portion, the precipi- (2) Acidic solutions of bromates in nitric acid exhibit a
tate dissolves. yellow to red-brown color upon addition of 5 to 6 drops of
(2) Slightly alkaline solutions of arsenites produce a yel- sodium nitrite TS. When 1 mL of chloroform is added to the
lowish white precipitate with silver nitrate TS. When ammo- mixture and shaken, the chloroform layer exhibits a yellow
nia TS is added to one portion of the suspension, and dilute to red-brown color.
nitric acid is added to another portion, the precipitate dis-
Bromide
solves in either of these reagents.
(1) Solutions of bromides yield a pale yellow precipitate
(3) Slightly alkaline solutions of arsenites produce a
with silver nitrate TS. Upon addition of dilute nitric acid to a
green precipitate with copper (II) sulfate TS. When the sepa-
portion of the separated precipitate, it dose not dissolve.
rated precipitate is boiled with sodium hydroxide TS, it
When ammonia solution (28) is added to another portion
changes to red-brown.
and shaken, the separated solution yields a white turbidity
Barium salt upon acidifying with dilute nitric acid.
(1) When the Flame Coloration Test (1) <1.04> is applied (2) Solutions of bromides exhibit a yellow-brown color
to barium salts, a persistent yellow-green color develops. with chlorine TS. The mixture is separated into 2 portions.
(2) Solutions of barium salts produce with dilute sulfuric When one portion is shaken with chloroform, the chlo-
acid a white precipitate, which does not dissolve upon addi- roform layer exhibits a yellow-brown to red-brown color.
tion of dilute nitric acid. When phenol is added to the other portion, a white precipi-
(3) Acidic solutions of barium salts in acetic acid tate is produced.
produce a yellow precipitate with potassium chromate TS.
Calcium salt
The precipitate dissolves by addition of dilute nitric acid.
(1) When the Flame Coloration Test (1) <1.04> is applied
Benzoate to calcium salts, a yellow-red color develops.
(1) Concentrated solutions of benzoates produce a white, (2) Solutions of calcium salts yield a white precipitate
crysralline precipitate with dilute hydrochloric acid. The with ammonium carbonate TS.
separated precipitate, washed with cold water and dried, (3) Solutions of calcium salts yield a white precipitate
melts between 1209C and 1249C <2.60>. with ammonium oxalate TS. The separated precipitate does
(2) Neutral solutions of benzoates produce a pale yellow- not dissolve in dilute acetic acid, but dissolves in dilute hy-
red precipitate upon dropwise addition of iron (III) chloride drochloric acid.
TS. The precipitate changes to white on subsequent addition (4) Neutral solutions of calcium salts produce no precipi-
of dilute hydrochloric acid. tate, when mixed with 10 drops of potassium chromate TS
and heated (discrimination from strontium salts).
Bicarbonate
(1) Bicarbonates effervesce upon addition of dilute hy- Carbonate
drochloric acid, generating a gas, which produces a white (1) Carbonates effervesce upon addition of dilute hydro-
precipitate immediately, when passed into calcium hydroxide chloric acid, generating a gas, which produces a white pre-
TS (common with carbonates). cipitate immediately, when passed into calcium hydroxide
(2) Solutions of bicarbonates produce no precipitate with TS (common with bicarbonates).
magnesium sulfate TS, but produce a white precipitate when (2) Solutions of carbonates yield with magnesium sulfate
boiled subsequently. TS a white precipitate, which dissolves by addition of dilute
(3) Cold solutions of bicarbonates remain unchanged or acetic acid.
exhibits only a slightly red color upon addition of 1 drop of (3) Cold solutions of carbonates exhibit a red color with
phenolphthalein TS (discrimination from carbonates). 1 drop of phenolphthalein TS (discrimination from bicar-
bonates).
Bismuth salt
(1) Bismuth salts, dissolved in a slight excess of hydro- Ceric salt
chloric acid, yield a white turbidity upon dilution with water. (1) When a cerous salt is mixed with 2.5 times its mass of
A dark brown precipitate is produced with 1 to 2 drops of lead (IV) oxide, nitric acid is added and the solution is
sodium sulfide TS subsequently added. boiled, it exhibits a yellow color.
(2) Acidic solutions of bismuth salts in hydrochloric acid (2) Solutions of cerous salts yield a yellow to red-brown
exhibit a yellow color upon addition of thiourea TS. precipitate upon addition of hydrogen peroxide TS and am-
(3) Solution of bismuth salts in dilute nitric acid or in monia TS.
dilute sulfuric acid yield with potassium iodide TS a black
Chlorate
precipitate, which dissolves in an excess of the reagent to
(1) Solutions of chlorates yield no precipitate with silver
give an orange-colored solution.
nitrate TS. When 2 to 3 drops of sodium nitrite TS and
Borate dilute nitric acid are added to the mixture, a white precipitate
(1) When ignite a mixture of a borate with sulfuric acid is produced gradually, which dissolves by addition of ammo-
and methanol, it burns with a green flame. nia TS.
(2) Turmeric paper, when moistened with acidic solu- (2) When indigocarmine TS is added dropwise to neutral
tions of borates in hydrochloric acid and dried by warming, solutions of chlorates until a pale blue color appears, and the
exhibits a red color, which changes to blue with ammonia TS mixture is acidified with dilute sulfuric acid, the blue color
added dropwise. vanishes promptly upon subsequent dropwise addition of so-
dium hydrogensulfite TS.
Bromate
(1) Acidic solutions of bromates in nitric acid yield with Chloride
2 to 3 drops of silver nitrate TS a white, crystalline precipi- (1) Solutions of chlorides evolve an odor of chlorine,

when mixed with sulfuric acid and potassium permanganate, dium hydroxide TS, and then acidified with dilute sulfuric
and heated. The gas evolved turns moistened potassium acid.
iodide starch paper blue.
Dichromate
(2) Solutions of chlorides yield a white precipitate with
(1) Solutions of dichromates exhibit a yellow-red color.
silver nitrate TS. When dilute nitric acid is added to a por-
(2) Solutions of dichromates produce a yellow precipi-
tion of the separated precipitate, it does not dissolve. When
tate with lead (II) acetate TS. When acetic acid (31) is added
an excess of ammonia TS is added to another portion, the
to one portion of the suspension, the precipitate dose not
precipitate dissolves.
dissolve. When dilute nitric acid is added to another portion,
Chromate the precipitate dissolves.
(1) Solutions of chromates exhibit a yellow color. (3) When acidic solutions of dichromates in sulfuric acid
(2) Solutions of chromates produce a yellow precipitate are mixed with an equal volume of ethyl acetate and with 1
with lead (II) acetate TS. When acetic acid (31) is added to a to 2 drops of hydrogen peroxide TS, shaken immediately
portion of the suspension, the precipitate does not dissolve. and allowed to stand, the ethyl acetate layer exhibits a blue
When dilute nitric acid is added to another portion, the pre- color.
cipitate dissolves.
Ferric salt
(3) When acidic solutions of chromates in sulfuric acid
(1) Slightly acidic solutions of ferric salts yield with po-
are mixed with an equal volume of ethyl acetate and 1 to 2
tassium hexacyanoferrate (II) TS a blue precipitate, which
drops of hydrogen peroxide TS, shaken immediately and
does not dissolve in dilute hydrochloric acid subsequently
allowed to stand, the ethyl acetate layer exhibits a blue color.
added.
Citrate (2) Solutions of ferric salts yield with sodium hydroxide
(1) When 20 mL of a mixture of pyridine and acetic an- TS a gelatinous, red-brown precipitate, which changes to
hydride (3:1) is added to 1 or 2 drops of a solution of citrate, black upon addition of sodium sulfide TS. The separated
and the solution is allowed to stand for 2 to 3 minutes, a red- precipitate dissolves in dilute hydrochloric acid, yielding a
brown color develops. white turbidity.
(2) Neutral solutions of citrates, when mixed with an (3) Slightly acidic solutions of ferric salts exhibit a purple
equal volume of dilute sulfuric acid and two-thirds volume color with 5-sulfosalicylic acid TS.
of potassium permanganate TS, heated until the color of
Ferricyanide
permanganate is discharged, and then treated dropwise with
(1) Solutions of ferricyanides exhibit a yellow color.
bromine TS to one-tenth of total volume, yield a white pre-
(2) Solutions of ferricyanides yield with iron (II) sulfate
cipitate.
TS a blue precipitate, which does not dissolve in dilute hy-
(3) Neutral solutions of citrates, when boiled with an
drochloric acid subsequently added.
excess of calcium chloride TS, yield a white crystalline pre-
cipitate. When sodium hydroxide TS is added to a portion of Ferrocyanide
the separated precipitate, it does not dissolve. When dilute (1) Solutions of ferrocyanides yield with iron (III) chlo-
hydrochloric acid is added to another portion, the precipitate ride TS a blue precipitate, which does not dissolve in dilute
dissolves. hydrochloric acid subsequently added.
(2) Solutions of ferrocyanides yield with copper (II) sul-
Cupric salt
fate TS a red-brown precipitate, which does not dissolve in
(1) When a well polished iron plate is immersed in acidic
dilute hydrochloric acid subsequently added.
solutions of cupric salts in hydrochloric acid, a red metallic
film appears on its surface. Ferrous salt
(2) Solutions of cupric salts produce a pale blue precipi- (1) Slightly acidic solutions of ferrous salts yield with po-
tate with a small quantity of ammonia TS. The precipitate tassium hexacyanoferrate (III) TS a blue precipitate, which
dissolves in an excess of the reagent, yielding a deep blue- does not dissolve in dilute hydrochloric acid subsequently
colored solution. added.
(3) Solutions of cupric salts yield a red-brown precipitate (2) Solutions of ferrous salts yield with sodium hydrox-
with potassium hexacyanoferrate (II) TS. When dilute nitric ide TS a greenish gray, gelatinous precipitate, which changes
acid is added to a portion of the suspension, the precipitate to black with sodium sulfide TS. The separated precipitate
does not dissolve. When ammonia TS is added to another dissolves in dilute hydrochloric acid.
portion, the precipitate dissolves, yielding a deep blue- (3) Neutral or slightly acidic solutions of ferrous salts ex-
colored solution. hibit an intense red color upon dropwise addition of a solu-
(4) Solutions of cupric salts produce a black precipitate tion of 1,10-phenanthroline monohydrate in ethanol (95) (1
with sodium sulfide TS. When dilute hydrochloric acid, in 50).
dilute sulfuric acid or sodium hydroxide TS is added to a
Fluoride
portion of the separated precipitate, it does not dissolve.
(1) When solutions of fluorides are heated with chromic
When hot dilute nitric acid is added to another portion, the
acid-sulfuric acid TS, the inside of the test tube is not
precipitate dissolves.
moistened uniformly.
Cyanide (2) Neutral or slightly acidic solutions of fluorides ex-
(1) Solutions of cyanides yield a white precipitate with an hibit a blue-purple color after standing with 1.5 mL of a
excess of silver nitrate TS. When dilute nitric acid is added to mixture of alizarin complexone TS, acetic acid-potassium
a portion of the separated precipitate, it does not dissolve. acetate buffer solution (pH 4.3) and cerium (III) nitrate TS
When ammonia TS is added to another portion, the precipi- (1:1:1).
tate dissolves.
Glycerophosphate
(2) Solutions of cyanides yield a blue precipitate, when
(1) Solutions of glycerophosphates remain unaffected by
mixed by shaking with 2 to 3 drops of iron (II) sulfate TS, 2
addition of calcium chloride TS, but yield a precipitate when
to 3 drops of dilute iron (III) chloride TS and 1 mL of so-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
34 1.09 Qualitative Tests / General Tests JP XVII
boiled. acid exhibit a purple-red color with a small quantity of pow-
(2) Solutions of glycerophosphates yield no precipitate dered bismuth sodium trioxide.
with hexaammonium heptamolybdate TS in the cold, but
Mercuric salt
yield a yellow precipitate when boiled for a long time.
(1) A copper plate is immersed in solutions of mercuric
(3) When glycerophosphates are mixed with an equal
salts, allowed to stand, taken out, and then washed with
mass of powdered potassium hydrogen sulfate and heated
water. The plate becomes bright and silvery white in appear-
gently over a free flame, the pungent odor of acrolein is
ance, when rubbed with paper or cloth (common with mer-
evolved.
curous salts).
Iodide (2) Solutions of mercuric salts yield with a small quantity
(1) Solutions of iodides yield a yellow precipitate with of sodium sulfide TS a black precipitate, which dissolves in
silver nitrate TS. When dilute nitric acid is added to one por- an excess of the reagent. The black precipitate is reproduced
tion of the suspension, and ammonia solution (28) to by subsequent addition of ammonium chloride TS.
another portion, the precipitates do not dissolve in either of (3) When potassium iodide TS is added dropwise to neu-
these reagents. tral solutions of mercuric salts, a red precipitate is produced.
(2) Acidic solutions of iodides exhibit a yellow-brown The precipitate dissolves in an excess of the reagent.
color with 1 to 2 drops of sodium nitrite TS and then yield a (4) Acidic solutions of mercuric salts in hydrochloric
black-purple precipitate. The solutions exhibit a deep blue acid yield with a small quantity of tin (II) chloride TS a white
color with starch TS subsequently added. precipitate, which changes to grayish black upon addition of
an excess of the reagent.
Lactate
Acidic solutions of lactates in sulfuric acid, when heated Mercurous salt
with potassium permanganate TS, evolve the odor of acetal- (1) A copper plate is immersed in solutions of mercurous
dehyde. salts, allowed to stand, taken out, and then washed with
water. The plate becomes bright and silvery white in appear-
Lead salt
ance, when rubbed with paper or cloth (common with mer-
(1) Solutions of lead salts yield a white precipitate with
curic salts).
dilute sulfuric acid. When dilute nitric acid is added to a por-
(2) Mercurous salts or their solutions exhibit a black
tion of the separated precipitate, it does not dissolve. When
color with sodium hydroxide TS.
sodium hydroxide TS is added to another portion and
(3) Solutions of mercurous salts yield a white precipitate
warmed, or when ammonium acetate TS is added to another
with dilute hydrochloric acid. The separated precipitate
portion, the precipitate dissolves.
changes to black upon addition of ammonia TS.
(2) Solutions of lead salts yield with sodium hydroxide
(4) Solutions of mercurous salts yield with potassium
TS a white precipitate, which dissolves in an excess of so-
iodide TS a yellow precipitate, which changes to green, when
dium hydroxide TS, and yields a black precipitate upon sub-
allowed to stand, and changes again to black upon subse-
sequent addition of sodium sulfide TS.
quent addition of an excess of the reagent.
(3) Acidic solutions of lead salts in dilute acetic acid
yield with potassium chromate TS a yellow precipitate, Mesilate
which does not dissolve in ammonia TS but dissolves in so- (1) To mesilates add twice its mass of sodium hydroxide,
dium hydroxide TS subsequently added. heat gently to melt, and continue heating for 20 to 30
seconds. After cooling, add a little amount of water, then
Lithium salt
add dilute hydrochloric acid, and warm: the gas evolved
(1) When the Flame Coloratuion Test (1) <1.04> is ap-
changes moistened potassium iodate-starch paper to blue.
plied to lithium salts, a persistent red color develops.
(2) To mesilates add threefold its mass of sodium nitrate
(2) Solutions of lithium salts yield with disodium
and anhydrous sodium carbonate, mix, and heat gradually.
hydrogenphosphate TS a white precipitate, which dissolves
After cooling, dissolve the residue in diluted hydrochloric
upon subsequent addition of dilute hydrochloric acid.
acid (1 in 5), and filter if necessary. The filtrate yields a
(3) Solutions of lithium salts yield no precipitate with
white precipitate upon addition of barium chloride TS.
dilute sulfuric acid (discrimination from strontium salts).
Nitrate
Magnesium salt
(1) Solutions of nitrates, when mixed with an equal
(1) Solutions of magnesium salts yield upon warming
volume of sulfuric acid, the mixture is cooled, and iron (II)
with ammonium carbonate TS a white precipitate, which dis-
sulfate TS is superimposed, a dark brown ring is produced at
solves in ammonium chloride TS. A white, crystalline pre-
the junction of the two liquids.
cipitate is reproduced by subsequent addition of disodium
(2) Solutions of nitrates exhibit a blue color with
hydrogenphosphate TS.
diphenylamine TS.
(2) Solutions of magnesium salts yield with sodium hy-
(3) When potassium permanganate TS is added to acidic
droxide TS a white, gelatinous precipitate. When iodine TS
solutions of nitrates in sulfuric acid, the red-purple color of
is added to one portion of the suspension, the precipitate de-
the reagent does not fade (discrimination from nitrites).
velops a dark-brown color. When excess sodium hydroxide
TS is added to another portion, the precipitate does not dis- Nitrite
solve. (1) Solutions of nitrites, when acidified with dilute sulfu-
ric acid, evolve a yellow-brown gas with a characteristic
Manganese salt
odor. The solutions exhibit a dark brown color upon addi-
(1) Solutions of manganese salts yield a white precipitate
tion of a small quantity of iron (II) sulfate crystals.
with ammonia TS. When silver nitrate TS is added to a por-
(2) Solutions of nitrites, when 2 to 3 drops of potassium
tion of the suspension, the precipitate changes to black.
iodide TS and dilute sulfuric acid are added dropwise,
When another portion is allowed to stand, the upper part of
exhibit a yellow-brown color, and then yield a black-purple
the precipitate exhibits a brownish color.
precipitate. When the mixture is shaken with 2 mL of chlo-
(2) Acidic solutions of manganese salts in dilute nitric

roform, the chloroform layer exhibits a purple color. (2) Concentrated solutions of salicylates yield a white,
(3) Solutions of nitrites, when mixed with thiourea TS crystalline precipitate with dilute hydrochloric acid. The
and acidified with dilute sulfuric acid, and iron (III) chloride separated precipitate, washed well with cold water and dried,
TS is added dropwise, exhibit a dark red color. When the melts <2.60> at about 1599C.
mixture is shaken with 2 mL of diethyl ether, the diethyl (3) Neutral solutions of salicylates exhibit with 5 to 6
ether layer exhibits a red color. drops of dilute iron (III) chloride TS a red color, which
changes to purple and then fades when dilute hydrochloric
Oxalate
acid is added dropwise.
(1) When potassium permanganate TS is added dropwise
to warm acidic solutions of oxalates in sulfuric acid, the rea- Silver salt
gent is decolorized. (1) Solutions of silver salts yield a white precipitate with
(2) Solutions of oxalates yield a white precipitate with dilute hydrochloric acid. When dilute nitric acid is added
calcium chloride TS. The separated precipitate does not dis- subsequently to a portion of the suspension, the precipitate
solve in dilute acetic acid but dissolves upon subsequent ad- does not dissolve. When an excess of ammonia TS is added
dition of dilute hydrochloric acid. to another portion, the precipitate dissolves.
(2) Solutions of silver salts yield with potassium chro-
Permanganate
mate TS a red precipitate, which dissolves upon addition of
(1) Solutions of permanganates exhibit a red-purple
dilute nitric acid.
color.
(3) Solutions of silver salts yield a brownish gray precipi-
(2) When an excess of hydrogen peroxide TS is added to
tate with ammonia TS added dropwise. When ammonia TS
acidic solutions of permanganates in sulfuric acid, the solu-
is added dropwise until the precipitate dissolves, then 1 to 2
tions effervesce and decolorize permanganates.
drops of formaldehyde solution are added and warmed, a
(3) Acidic solutions of permanganates in sulfuric acid
mirror of metallic silver is deposited on the inside wall of the
are decolorized, when an excess of oxalic acid TS is added
container.
and heated.
Sodium salt
Peroxide
(1) When the Flame Coloration Test (1) <1.04> is applied
(1) Solutions of peroxides are mixed with an equal
to sodium salts, a yellow color develops.
volume of ethyl acetate and 1 to 2 drops of potassium
(2) Concentrated, neutral or slightly alkaline solutions of
dichromate TS, and then acidified with dilute sulfuric acid.
sodium salts yield a white, crystalline precipitate with potas-
When the mixture is shaken immediately and allowed to
sium hexahydroxoantimonate (V) TS. The formation of the
stand, the ethyl acetate layer exhibits a blue color.
precipitate is accelerated by rubbing the inside wall of the
(2) Acidic solutions of peroxides in sulfuric acid decolo-
test tube with a glass rod.
rize dropwise added potassium permanganate TS, and effer-
vesce to evolve a gas. Stannic salt
(1) When the outside bottom of a test tube containing
Phosphate (Orthophosphate)
water is moistened with acidic solutions of stannic salts in
(1) Neutral solutions of phosphates yield with silver ni-
hydrochloric acid and is placed in a nonluminous flame of a
trate TS a yellow precipitate, which dissolves upon addition
Bunsen burner, a blue flame mantle is seen around the bot-
of dilute nitric acid or ammonia TS.
tom of the test tube (common with stannous salts).
(2) Acidic solutions in dilute nitric acid of phosphates
(2) When granular zinc is immersed in acidic solutions of
yield a yellow precipitate with hexaammonium heptamolyb-
stannic salts in hydrochloric acid, a spongy, gray substance
date TS on warming. The precipitate dissolves upon subse-
is deposited on the surface of the granules (common with
quent addition of sodium hydroxide TS or ammonia TS.
stannous salts).
(3) Neutral or ammonia-alkaline solutions of phosphates
(3) Add iron powder to acidic solutions of stannic salts
yield with magnesia TS a white, crystalline precipitate, which
in hydrochloric acid, allow to stand, and then filter. When
dissolves upon subsequent addition of dilute hydrochloric
iodine-starch TS is added dropwise to the filtrate, the color
acid.
of the test solution disappears.
Potassium salt (4) Acidic solutions of stannic salts in hydrochloric acid,
(1) When the Flame Coloration Test (1) <1.04> is applied to which ammonia TS is added dropwise until a small quan-
to potassium salts, a pale purple color develops. When it tity of precipitate is produced, yield a pale yellow precipitate
gives a yellow color, a red-purple color can be seen through with 2 to 3 drops of sodium sulfide TS. The separated pre-
cobalt glass. cipitate dissolves upon addition of sodium sulfide TS and
(2) Neutral solutions of potassium salts yield a white, pale yellow precipitate is reproduced by subsequent addition
crystalline precipitate with sodium hydrogen tartrate TS. of hydrochloric acid.
The formation of the precipitate is accelerated by rubbing
Stannous salt
the inside wall of the test tube with a glass rod. The sepa-
(1) When the outside bottom of a test tube containing
rated precipitate dissolves upon addition of any of ammonia
water is moistened with acidic solutions of stannous salts in
TS, sodium hydroxide TS or sodium carbonate TS.
hydrochloric acid and is placed in a nonluminous flame of a
(3) Acidic solutions of potassium salts in acetic acid (31)
Bunsen burner, a blue flame mantle is seen around the bot-
yield a yellow precipitate with sodium hexanitrocobaltate
tom of the test tube (common with stannic salts).
(III) TS.
(2) When granular zinc is immersed in acidic solutions of
(4) Potassium salts do not evolve the odor of ammonia,
stannous salts in hydrochloric acid, a spongy, gray substance
when an excess of sodium hydroxide TS is added and
is deposited on the surface of the granules (common with
warmed (discrimination from ammonium salts).
stannic salts).
Salicylate (3) When iodine-starch TS is added dropwise to solu-
(1) Salicylates evolve the odor of phenol, when an excess tions of stannous salts, the color of the test solution disap-
of soda-lime is added and heated. pears.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
36 1.10 Iron Limit Test / General Tests JP XVII
(4) Acidic solutions of stannous salts in hydrochloric added, solutions of thiosulfates evolve the odor of sulfur di-
acid, to which ammonia TS is added dropwise until a small oxide, and yield gradually a white turbidity, which changes
quantity of precipitate is produced, yield a dark brown pre- to yellow on standing.
cipitate with 2 to 3 drops of sodium sulfide TS. When so- (3) Solutions of thiosulfates yield with an excess of silver
dium sulfide TS is added to a portion of the separated pre- nitrate TS a white precipitate, which changes to black on
cipitate, it does not dissolve. When ammonium polysulfide standing.
TS is added to another portion, the precipitate dissolves.
Zinc salt
Sulfate (1) Neutral to alkaline solutions of zinc salts yield a whit-
(1) Solutions of sulfates yield with barium chloride TS a ish precipitate with ammonium sulfide TS or sodium sulfide
white precipitate, which does not dissolve upon addition of TS. The separated precipitate does not dissolve in dilute
dilute nitric acid. acetic acid but dissolves upon subsequent addition of dilute
(2) Neutral solutions of sulfates yield with lead (II) ace- hydrochloric acid.
tate TS a white precipitate, which dissolves upon subsequent (2) Solutions of zinc salts yield a white precipitate with
addition of ammonium acetate TS. potassium hexacyanoferrate (II) TS. When dilute hydrochlo-
(3) When an equal volume of dilute hydrochloric acid is ric acid is added to a portion of the suspension, the precipi-
added, solutions of sulfates yield no white turbidity (dis- tate does not dissolve. When sodium hydroxide TS is added
crimination from thiosulfates), and do not evolve the odor to another portion, the precipitate dissolves.
of sulfur dioxide (discrimination from sulfites). (3) Neutral to weakly acidic solutions of zinc salts yield a
white precipitate, when 1 or 2 drops of pyridine and 1 mL of
Sulfide
potassium thiocyanate TS are added.
(1) Most kinds of sulfides evolve the odor of hydrogen
sulfide with dilute hydrochloric acid. This gas blackens lead
(II) acetate paper moistened with water.
Sulfite and Bisulfite 1.10 Iron Limit Test
(1) When iodine TS is added dropwise to acidic solutions
Iron Limit Test is a limit test for iron contained in drugs.
of sulfites or bisulfites in acetic acid (31), the color of the
The limit is expressed in term of iron (Fe).
reagent fades.
In each monograph, the permissible limit for iron (as Fe)
(2) When an equal volume of dilute hydrochloric acid is
is described in terms of ppm in parentheses.
added, solutions of sulfites or bisulfites evolve the odor of
sulfur dioxide but yield no turbidity (discrimination from 1. Preparation of test solutions and control solutions
thiosulfates). The solutions yield immediately with 1 drop of Unless otherwise specified, test solutions and control solu-
sodium sulfide TS a white turbidity, which changes gradually tions are prepared as follows:
to a pale yellow precipitate. 1.1. Method 1
Weigh the amount of sample specified in indivisual mono-
Tartrate
graph, add 30 mL of acetic acid-sodium acetate buffer solu-
(1) Neutral tartrate solutions yield a white precipitate
tion for iron limit test (pH 4.5), dissolve by warming if nec-
with silver nitrate TS. When nitric acid is added to a portion
essary, and designate this solution as the test solution.
of the separated precipitate, it dissolves. When ammonia TS
Prepare the control solution as follows: To the amount of
is added to another portion and warmed, the precipitate dis-
Standard Iron Solution specified in individual monograph
solves and metallic silver is deposited gradually on the inside
add 30 mL of acetic acid-sodium acetate buffer solution for
wall of the test tube, forming a mirror.
iron limit test (pH 4.5).
(2) Solutions of tartrates exhibit a red-purple to purple
1.2. Method 2
color, when 2 drops of acetic acid (31), 1 drop of iron (II)
Weigh the amount of sample specified in individual mono-
sulfate TS, 2 to 3 drops of hydrogen peroxide TS and an
graph, add 10 mL of dilute hydrochloric acid, and dissolve
excess of sodium hydroxide TS are added.
by warming if necessary. Dissolve 0.5 g of L-tartaric acid,
(3) When a solution, prepared by mixing 2 to 3 drops of
and add one drop of phenolphthalein TS. Add ammonia TS
a solution of resorcinol (1 in 50) and 2 to 3 drops of a solu-
dropwise untill the solution develops a pale red color. Add
tion of potassium bromide (1 in 10) with 5 mL of sulfuric
20 mL of acetic acid-sodium acetate buffer solution for iron
acid, is added to 2 to 3 drops of solutions of tartrates, and
limit test (pH 4.5) and designate this solution as the test solu-
then heated for 5 to 10 minutes on a water bath, a deep blue
tion.
color is produced. The solution exhibits a red to red-orange
Prepare the control solution as follows: To the amount of
color when poured to 3 mL of water after cooling.
Standard Iron Solution specified in individual monograph
Thiocyanate add 10 mL of dilute hydrochloric acid, and proceed as di-
(1) Solutions of thiocyanates yield a white precipitate rected for the test solution.
with an excess of silver nitrate TS. When dilute nitric acid is 1.3. Method 3
added to a portion of the suspension, the precipitate does Place the amount of sample specified in individual mono-
not dissolve. When ammonia solution (28) is added to graph in a crucible, moisten with a small amount of sulfuric
another portion, the precipitate dissolves. acid, heat cautiously and gently at first, and then incinerate
(2) Solutions of thiocyanates produce with iron (III) by ignition. After cooling, add 1 mL of diluted hydrochloric
chloride TS a red color, which is not decolored by addition acid (2 in 3) and 0.5 mL of diluted nitric acid (1 in 3), evapo-
of hydrochloric acid. rate on a water bath to dryness, and to the residue add 0.5
mL of diluted hydrochloric acid (2 in 3) and 10 mL of water.
Thiosulfate
After dissolving by warming, add 30 mL of acetic acid-
(1) When iodine TS is added dropwise to acidic solutions
sodium acetate buffer solution for iron limit test (pH 4.5),
of thiosulfates in acetic acid (31), the color of the reagent
and designate this solution as the test solution.
fades.
Prepare the control solution as follows: Transfer the
(2) When an equal volume of dilute hydrochloric acid is

amount of Standard Iron Solution specified in indivisual

monograph to a crucible, and add 1 mL of diluted hydro-
chloric acid (2 in 3) and 0.5 mL of diluted nitric acid (1 in 3),
evaporate on a water bath to dryness, and proceed
as directed for the test solution.
In this procedure, use a quartz or porcelain crucible,
which is immersed in boiling dilute hydrochloric acid for 1
hour and washed throughly with water and dried.
2. Procedure
Unless otherwise specified, proceed as follows:
2.1. Method A
Transfer the test solution and the control solution to sepa-
rate Nessler tubes, to each add 2 mL of a solution of L-ascor-
bic acid (1 in 100), mix well, and allow to stand for 30
minutes. Add 1 mL of a solution of a, a?-dipyridyl in
ethanol (95) (1 in 200), add water to make 50 mL, and allow
to stand for 30 minutes. Then compare the colors developed
in both solutions against a white background. The test solu-
tion has no more color than the control solution.
2.2. Method B
Dissolve 0.2 g of L-ascorbic acid in the test solution and
the control solution, and allow to stand for 30 minutes. Add
1 mL of a solution of a, a?-dipyridyl in ethanol (95) (1 in
200), and allow to stand for 30 minutes. Then add 2 mL of a
solution of 2,4,6-trinitrophenol (3 in 1000) and 20 mL of
1,2-dichloroethane, shake vigorously, collect the 1,2-
dichloroethane layer, and filter through a pledget of absor-
bent cotton in a funnel on which 5 g of anhydrous sodium
sulfate is placed if necessary. Then compare the colors deve-
loped in both solutions against a white background. The test
solution has no more color than the control solution.

1.11 Arsenic Limit Test

Arsenic Limit Test is a limit test for arsenic contained in
drugs. The limit is expressed in terms of arsenic trioxide
(As2O3).
In each monograph, the permissible limit for arsenic (as
As2O3) is described in terms of ppm in parentheses.
1. Apparatus
Use the apparatus illustrated in Fig. 1.11-1.
Place glass wool F in the exit tube B up to about 30 mm in
height, moisten the glass wool uniformly with a mixture of
an equal volume of lead (II) acetate TS and water, and apply
gentle suction to the lower end to remove the excess of the
mixture. Insert the tube vertically into the center of the rub-
ber stopper H, and attach the tube to the generator bottle A
so that the small perforation E in the lower end of B extends
slightly below. At the upper end of B, attach the rubber
stopper J to hold the tube C vertically. Make the lower end
to the exit tube of C level with that of the rubber stopper J.
2. Preparation of the test solution
Unless otherwise specified, proceed as directed in the fol-
lowing.
2.1. Method 1
Fig. 1.11-1 Arsenic limit test apparatus
Weigh the amount of the sample directed in the mono-
graph, add 5 mL of water, dissolve by heating if necessary,
and designate the solution as the test solution.
2.2. Method 2 from sulfurous acid and is reduced to about 2 mL in volume.
Weigh the amount of the sample directed in the mono- Dilute with water to make 5 mL, and designate it as the test
graph, add 5 mL of water, and add 1 mL of sulfuric acid ex- solution.
cept in the cases that the samples are inorganic acids. Add 10 2.3. Method 3
mL of sulfurous acid solution, transfer to a small beaker, Weigh the amount of the sample directed in the mono-
and evaporate the mixture on a water bath until it is free graph, and place it in a crucible of platinum, quartz or por-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
38 1.12 Methanol Test / General Tests JP XVII
celain. Add 10 mL of a solution of magnesium nitrate hexa- ride TS, and allow to stand for 10 minutes. Then add water
hydrate in ethanol (95) (1 in 50), ignite the ethanol, and heat to make 40 mL, add 2 g of zinc for arsenic analysis, and im-
gradually to incinerate. If carbonized material still remains mediately connect the rubber stopper H fitted with B and C
by this procedure, moisten with a small quantity of nitric with the generator bottle A. Transfer 5 mL of the absorbing
acid, and ignite again to incinerate. After cooling, add 3 mL solution for hydrogen arsenide to the absorber tube D, insert
of hydrochloric acid, heat on a water bath to dissolve the the tip of C to the bottom of the absorber tube D, then im-
residue, and designate it as the test solution. merse the generator bottle A up to the shoulder in water
2.4. Method 4 maintained at 259C, and allow to stand for 1 hour. Discon-
Weigh the amount of the sample directed in the mono- nect the absorber tube, add pyridine to make 5 mL, if neces-
graph, and place it in a crucible of platinum, quartz or por- sary, and observe the color of the absorbing solution: the
celain. Add 10 mL of a solution of magnesium nitrate hexa- color produced is not more intense than the standard color.
hydrate in ethanol (95) (1 in 10), burn the ethanol, heat Preparation of standard color: Measure accurately 2 mL
gradually, and ignite to incinerate. If carbonized material of Standard Arsenic Solution in the generator bottle A. Add
still remains by this procedure, moisten with a small quantity 5 mL of diluted hydrochloric acid (1 in 2) and 5 mL of potas-
of nitric acid, and ignite again to incinerate in the same man- sium iodide TS, and allow to stand for 2 to 3 minutes. Add
ner. After cooling, add 3 mL of hydrochloric acid, heat on a 5 mL of acidic tin (II) chloride TS, allow to stand at room
water bath to dissolve the residue, and designate it as the test temperature for 10 minutes, and then proceed as directed
solution. above. The color produced corresponds to 2 mg of arsenic
2.5. Method 5 trioxide (As2O3) and is used as the standard.
Weigh the amount of the sample directed in the mono-
5. Note
graph, add 10 mL of N, N-dimethylformamide, dissolve by
Apparatus, reagents and test solutions used in the test
heating if necessary, and designate the solution as the test so-
should contain little or no arsenic. If necessary, perform a
lution.
blank determination.
3. Test solutions
(i) Absorbing solution for hydrogen arsenide: Dissolve
0.50 g of silver N,N-diethyldithiocarbamate in pyridine to
make 100 mL. Preserve this solution in a glass-stoppered 1.12 Methanol Test
bottle protected from light, in a cold place.
Methanol Test is a method to determine methanol adher-
(ii) Standard Arsenic Stock Solution: Weigh exactly
ing in ethanol.
0.100 g of finely powdered arsenic trioxide dried at 1059C
for 4 hours, and add 5 mL of sodium hydroxide solution 1. Reagents
(1 in 5) to dissolve. Add dilute sulfuric acid to neutralize, (i) Standard Methanol Solution—To 1.0 g of methanol,
add further 10 mL of dilute sulfuric acid, add freshly boiled accurately measured, add water to make exactly 1000 mL.
and cooled water to make exactly 1000 mL, and preserve in a To 5 mL of this solution, exactly measured, add 2.5 mL of
glass-stoppered bottle. methanol-free ethanol and water to make exactly 50 mL.
(iii) Standard Arsenic Solution: Pipet 10 mL of Standard (ii) Solution A—To 75 mL of phosphoric acid add water
Arsenic Stock Solution, add 10 mL of dilute sulfuric acid, to make 500 mL, then dissove 15 g of potassium permanga-
and add freshly boiled and cooled water to make exactly nate in this solution.
1000 mL. Each mL of the solution contains 1 mg of arsenic (iii) Solution B—Add sulfuric acid carefully to an equal
trioxide (As2O3). Prepare Standard Arsenic Solution just volume of water, cool, and dissolve 25 g of oxalic acid dihy-
before use. drate in 500 mL of this dilute sulfuric acid.
In the case where the preparation of Standard Arsenic
2. Procedure
Stock Solution is difficult, Certified Standard Arsenic Solu-
Pipet 1 mL of the sample, and add water to make exactly
tion may be used to prepare Standard Arsenic Solution as
20 mL. Use this solution as the sample solution. Transfer 5
follows: Pipet 15 mL of Certified Standard Arsenic Solu-
mL each of the sample solution and the Standard Methanol
tion, add 1 mL of dilute sulfuric acid, and add freshly boiled
Solution, accurately measured, to test tubes, add 2 mL of
and cooled water to make exactly 100 mL. Pipet 5 mL of this
Solution A to each solution, and allow to stand for 15
solution, add 1 mL of dilute sulfuric acid, and add freshly
minutes. Decolorize these solutions by adding 2 mL of Solu-
boiled and cooled water to make exactly 100 mL. Prepare
tion B, and mix with 5 mL of fuchsin-sulfurous acid TS.
just before use.
Allow to stand for 30 minutes at ordinary temperature. The
(iv) Certified Standard Arsenic Solution: JCSS Arsenic
sample solution has no more color than the Standard
Standard Solution (100 mg/L) Each mL of this solution
Methanol Solution.
contains 0.1 mg of arsenic (AS).
JCSS (Japan Calibration Service System) is a registration
system of calibration service.
4. Procedure 1.13 Fats and Fatty Oils Test
Unless otherwise specified, proceed using apparatus
Fats and Fatty Oils Test is a method applied to fats, fatty
shown in Fig. 1.11-1. Carry out the preparation of the stand-
oils, waxes, fatty acids, higher alcohols, and related sub-
ard color at the same time.
stances.
Place the test solution in the generator bottle A and, if
necessary, wash down the solution in the bottle with a small 1. Preparation of test sample
quantity of water. Add 1 drop of methyl orange TS, and For a solid sample, melt with care, and, if necessary, filter
after neutralizing with ammonia TS, ammonia solution (28) the melted sample with a dry filter paper by warming. For a
or dilute hydrochloric acid, add 5 mL of diluted hydrochlo- turbid liquid sample, heat at about 509C. If it is still turbid,
ric acid (1 in 2) and 5 mL of potassium iodide TS, and allow filter it with a dry filter paper while warm. In either case,
to stand for 2 to 3 minutes. Add 5 mL of acidic tin (II) chlo- mix the sample to make it homogeneous.

2. Melting point Table 1.13-1

Proceed by the method described in Method 2 of Melting
Acid value Amount (g) of sample
Point Determination <2.60>.
Less than 5 20
3. Congealing point of fatty acids 5 to 15 10
3.1. Preparation of fatty acids 15 to 30 5
Dissolve 25 g of potassium hydroxide in 100 g of glycerin. 30 to 100 2.5
Transfer 75 g of this solution into a 1-L beaker, and heat at More than 100 1.0
1509C. Add 50 g of the sample to this solution, and heat at a
temperature not higher than 1509 C for 15 minutes under fre-
quent stirring to saponify completely. Cool the solution to
until the solvent remains light red for 30 seconds.
1009C, dissolve by addition of 500 mL of hot water, and add
slowly 50 mL of diluted sulfuric acid (1 in 4). Heat the solu- consumed volume (mL) of 0.1 mol/L ×
potassium hydroxide-ethanol VS 5.611
tion under frequent stirring until the clear layer of fatty acid
Acid value ＝
is separated distinctly. Separate the fatty acid layer, and amount (g) of sample
wash the fatty acid with hot water until the washing shows
6. Saponification value
no acidity to methyl orange TS. Transfer the fatty acid layer
The saponification value is the number of milligrams of
to a small beaker, and heat on a water bath until the fatty
potassium hydroxide (KOH) required to saponify the esters
acid becomes clear owing to the separation of water. Filter
and to neutralize the free acids in 1 g of the sample.
the warm solution, and complete the evaporation of water
6.1. Procedure
by carefully heating the filtered solution to 1309 C.
Unless otherwise specified, weigh accurately 1 to 2 g of the
3.2. Measurement of congealing point
sample, transfer to a 200-mL flask, and add exactly 25 mL
Proceed by the method described in Congealing Point De-
of 0.5 mol/L potassium hydroxide-ethanol VS. Attach a
termination <2.42>.
short reflux condenser or an air condenser 750 mm in length
4. Specific gravity and 6 mm in diameter to the neck of the flask, and heat
4.1. Liquid sample at ordinary temperature gently in a water bath for 1 hour with frequent shaking.
Proceed by the method described in Determination of Cool the solution, add 1 mL of phenolphthalein TS, and
Specific Gravity and Density <2.56>. titrate <2.50> immediately the excess potassium hydroxide
4.2. Solid sample at ordinary temperature with 0.5 mol/L hydrochloric aicd VS. If the sample solution
Unless otherwise specified, fill a pycnometer with water at is turbid at lower temperature, titration should be done while
209 C. Weigh accurately the pycnometer, and, after dis- warm. Perform a blank determination.
carding the water and drying, weigh accurately the empty
(a － b) × 28.05
pycnometer. Then, fill the pycnometer with the melted sam- Saponification value ＝
amount (g) of sample
ple to about three-fourths of the depth, and allow to stand at
a temperature a little higher than the melting temperature of a: Volume (mL) of 0.5 mol/L hydrochloric acid VS con-
the sample for 1 hour to drive off the air in the sample. After sumed in the blank determination
keeping at the specified temperature, weigh accurately the b: Volume (mL) of 0.5 mol/L hydrochloric acid VS con-
pycnometer. Fill up the pycnometer with water over the sam- sumed for titration of the sample
ple at 209C, and weigh accurately again.
7. Ester value
The other procedure is the same as described in Method 1
The ester value is the number of milligrams of potassium
of Determination of Specific Gravity and Density <2.56>.
hydroxide (KOH) required to saponify the esters in 1 g of
M1 － M sample.
d＝
(M2 － M ) － (M3 － M1) 7.1. Procedure
Unless otherwise specified, designate the difference be-
M: Mass (g) of the empty pycnometer
tween the saponification value and the acid value determined
M1: Mass (g) of the pycnometer filled with the sample
as the ester value.
M2: Mass (g) of the pycnometer filled with water
M3: Mass (g) of the pycnometer filled with the sample and 8. Hydroxyl value
water The hydroxyl value is the number of milligrams of potas-
sium hydroxide (KOH) required to neutralize acetic acid
5. Acid value
combined with hydroxyl groups, when 1 g of the sample is
The acid value is the number of milligrams of potassium
acetylated by the following procedure.
hydroxide (KOH) required to neutralize the free acids in 1 g
8.1. Procedure
of sample.
Place about 1 g of the sample, weighed accurately, in a
5.1. Procedure
200-mL round-bottom flask (shown in Fig. 1.13-1), and add
Unless otherwise specified, weigh accurately the amount
exactly 5 mL of pyridine-acetic anhydride TS. Place a small
of sample shown in Table 1.13-1, according to the expected
funnel on the neck of the flask, and heat by immersing the
acid value of the sample, in a glass-stoppered, 250-mL flask,
flask up to 1 cm from the bottom in an oil bath between
add 100 mL of a mixture of diethyl ether and ethanol (95)
959C and 1009C. Put a thick, round paper with a round hole
(1:1 or 2:1) as the solvent, and dissolve the sample by warm-
on the joint of the neck of the flask to protect the neck from
ing, if necessary. Then, add a few drops of phenolphthalein
the heat of the oil bath. After heating for 1 hour, take the
TS, and titrate <2.50> with 0.1 mol/L potassium hydroxide-
flask from the oil bath, and cool by standing. Add 1 mL of
ethanol VS until the solution develops a light red color which
water to the flask, and shake to decompose acetic anhydride.
persists for 30 seconds. If the sample solutions is turbid at
Heat the flask in the oil bath for 10 minutes again. After
lower temperature, titration should be done while warm. To
cooling, wash the funnel and neck with 5 mL of neutralized
the solvent used add phenolphthalein TS as an indicator, and
ethanol down into the flask, and titrate <2.50> with 0.5
add 0.1 mol/L potassium hydroxide-ethanol VS before use,
mol/L potassium hydroxide-ethanol VS (indicator: 1 mL of

and 809C under reduced pressure (about 2.67 kPa) for 30

minutes, allow to stand for cooling in a desiccator (reduced
pressure, silica gel) for 30 minutes, and then weigh. After
weighing, add 2 mL of diethyl ether and 10 mL of neutral-
ized ethanol, and dissolve the residue by shaking well. Add a
few drops of phenolphthalein TS, and titrate <2.50> the
remaining fatty acids in the residue with 0.1 mol/L potas-
sium hydroxide-ethanol VS until the solution develops a light
Fig. 1.13-1 Hydroxyl value determination flask
red color which persists for 30 seconds.
a － (b × 0.0282)
Unsaponifiable matter (z) ＝ × 100
amount (g) of sample
phenolphthalein TS). Perform a blank determination.
a: Amount (g) of the extracts
(a － b) × 28.05
Hydroxyl value ＝＋ acid value b: Volume (mL) of 0.1 mol/L potassium hydroxide-
amount (g) of sample
ethanol VS consumed for titration
a: Volume (mL) of 0.5 mol/L potassium hydroxide-
10. Iodine value
ethanol VS consumed in the blank determination
The iodine value, when measured under the following con-
b: Volume (mL) of 0.5 mol/L potassium hydroxide-
ditions, is the number of grams of iodine (I), representing
ethanol VS consumed for titration of the sample
the corresponding amount of halogen, which combines with
9. Unsaponifiable matter 100 g of sample.
Unsaponifiable matter is calculated as the difference be- 10.1. Procedure
tween the amount of materials, which are unsaponifiable by Unless otherwise specified, weigh accurately the amount
the procedure described below, soluble in diethyl ether and of sample shown in Table 1.13-2, according to the expected
insoluble in water, and the amount of fatty acids expressed iodine value of the sample, in a small glass container. In a
in terms of the amount of oleic acid. Its limit is expressed as 500-mL glass-stoppered flask place the container containing
a percentage in the monograph. the sample, add 20 mL of cyclohexane to dissolve the sam-
9.1. Procedure ple, then add exactly 25 mL of Wijs' TS, and mix well. Stop-
Transfer about 5 g of the sample, accurately weighed, to a per the flask, and allow to stand, protecting against light, be-
250-mL flask. Add 50 mL of potassium hydroxide-ethanol tween 209 C and 309 C for 30 minutes (when the expected
TS, attach a reflux condenser to the flask, boil gently on a iodine value is more than 100, for 1 hour) with occasional
water bath for 1 hour with frequent shaking, and then trans- shaking. Add 20 mL of potassium iodide solution (1 in 10)
fer to the first separator. Wash the flask with 100 mL of and 100 mL of water, and shake. Then, titrate <2.50> the lib-
warm water, and transfer the washing to the separator. Fur- erated iodine with 0.1 mol/L sodium thiosulfate VS (indica-
ther, add 50 mL of water to the separator, and cool to room tor: 1 mL of starch TS). Perform a blank determination.
temperature. Wash the flask with 100 mL of diethyl ether,
(a － b) × 1.269
add the washing to the separator, extract by vigorous shak- Iodine value ＝
amount (g) of sample
ing for 1 minute, and allow to stand until both layers are
separated clearly. Transfer the water layer to the second a: Volume (mL) of 0.1 mol/L sodium thiosulfate VS con-
separator, add 50 mL of diethyl ether, shake, and allow to sumed in the blank determination
stand in the same manner. Transfer the water layer in the b: Volume (mL) of 0.1 mol/L sodium thiosulfate VS con-
second separator to the third separator, add 50 mL of diethyl sumed for titration of the sample
ether, and extract by shaking again in the same manner.
Combine the diethyl ether extracts in the second and third
separators into the first separator, wash each separator with
a small amount of diethyl ether, and combine the washings 1.14 Sulfate Limit Test
into the first separator. Wash the combined extracts in the
Sulfate Limit Test is a limit test for sulfate contained in
first separator with 30 mL portions of water successively,
drugs.
until the washing does not develop a light red color with 2
In each monograph, the permissible limit for sulfate (as
drops of phenolphthalein TS. Add a small amount of anhy-
SO4) is described in terms of percentage (z) in parentheses.
drous sodium sulfate to the diethyl ether extracts, and allow
to stand for 1 hour. Filter the diethyl ether extracts with dry 1. Procedure
filter paper, and collect the filtrates into a tared flask. Wash Unless otherwise specified, transfer the quantity of the
well the first separator with diethyl ether, and add the wash- sample, directed in the monograph, to a Nessler tube, dis-
ing to the flask through the above filter paper. After evapo- solve it in sufficient water, and add water to make 40 mL.
ration of the filtrate and washing almost to dryness on a Add 1 mL of dilute hydrochloric acid and water to make 50
water bath, add 3 mL of acetone, and evaporate again to mL, and use this solution as the test solution. Transfer the
dryness on a water bath. Complete the drying between 709C volume of 0.005 mol/L sulfuric acid VS, directed in the

monograph, to another Nessler tube, add 1 mL of dilute hy- the time for which the mobile phase is passed through the
drochloric acid and water to make 50 mL, and use this solu- column (t0: time measured from the time of injection of a
tion as the control solution. When the test solution is not compound with k ＝ 0 to the time of elution at the peak
clear, filter both solutions according to the same procedure. maximum), and the retention time (tR: time measured from
Add 2 mL of barium chloride TS to the test solution and the time of injection of a compound to be determined to the
to the control solution, mix well, and allow to stand for 10 time of elution at the peak maximum), the retention time for
minutes. Compare the white turbidity produced in both solu- a compound on a column has a characteristic value under
tions against a black background by viewing downward or fixed chromatographic conditions.
transversely.
tR ＝ (1 ＋ k) t0
The turbidity produced in the test solution is not thicker
than that of the control solution. 1. Apparatus
Basically, the apparatus required for the liquid chromato-
graphic procedure consists of a pumping system for the mo-
bile phase, a sample injection port, a column, a detector and
1.15 Readily Carbonizable a recorder. A mobile phase component regulator, a ther-
Substances Test mostat for the column, a pumping system for reaction rea-
gents and a chemical reaction chamber are also used, if nec-
Readily Carbonizable Substances Test is a method to exa- essary. The pumping system serves to deliver the mobile
mine the minute impurities contained in drugs, which are phase and the reagents into the column and connecting tube
readily colored by addition of sulfuric acid. at a constant flow rate. The sample injection port is used to
deliver a quantity of the sample to the apparatus with high
1. Procedure
reproducibility. The column is a tube with a smooth interior,
Before use, wash the Nessler tubes thoroughly with sulfu-
made of inert metal, etc., in which a packing material for
ric acid for readily carbonizable substances. Unless other-
liquid chromatography is uniformly packed. A column with
wise specified, proceed as follows. When the sample is solid,
a stationary phase chemically bound on the inside wall in-
place 5 mL of sulfuric acid for readily carbonizable sub-
stead of the column packed with the packing material may
stances in a Nessler tube, to which add a quantity of the
be used. The detector is used to detect a property of the sam-
finely powdered sample, little by little, as directed in the
ples which is different from that of the mobile phase, and
monograph, and dissolve it completely by stirring with a
may be an ultraviolet or visible spectrophotometer, fluoro-
glass rod. When the sample is liquid, transfer a volume of
metric detector, differential refractometer, electrochemical
the sample, as directed in the monograph, to a Nessler tube,
detector, chemiluminescence detector, electric conductivity
add 5 mL of sulfuric acid for readily carbonizable sub-
detector, mass spectrophotometer, etc. The output signal is
stances, and mix by shaking. If the temperature of the con-
usually proportional to the concentration of samples at
tent of the tube rises, cool the content; maintain it at the
amounts of less than a few mg. The recorder is used to record
standard temperature, if the reaction may be affected by the
the output signals of the detector. As required, a data
temperature. Allow to stand for 15 minutes, and compare
processor may be used as the recorder to record or output
the color of the liquid with that of the matching fluid in the
the chromatogram, retention times or amounts of the com-
Nessler tube specified in the monograph, by viewing trans-
ponents. The mobile phase component regulator is used to
versely against a white background.
vary the ratio of the mobile phase components in a stepwise
or gradient fashion.
2. Procedure
2. Physical Methods Fix the detector, column and mobile phase to the appa-
ratus, and adjust the flow rate and the column temperature
to the values described in the operating conditions specified
Chromatography in the individual monograph. Inject a volume of the sample
solution or the standard solution specified in the individual
monograph with the sample injector into the column
2.01 Liquid Chromatography through the sample injection port. The separated compo-
nents are detected by the detector, and recorded by the
Liquid Chromatography is a method to develop a mixture
recorder as a chromatogram. If the components to be ana-
injected into a column prepared with a suitable stationary
lyzed have no readily detectable physical properties such as
phase by passing a liquid as a mobile phase through the
absorbance or fluorescence, the detection is achieved by
column, in order to separate the mixture into its components
changing the components to suitable derivatives. Usually,
by making use of the difference of retention capacity against
the derivatization is performed as a pre- or post-column
the stationary phase, and to determine the components. This
labeling.
method can be applied to a liquid or soluble sample, and is
used for identification, purity test, and quantitative determi- 3. Identification and purity test
nation. When Liquid Chromatography is used for identification
A mixture injected into the column is distributed between of a component of a sample, it is performed by confirming
the mobile phase and the stationary phase with a characteris- identity of the retention time of the component and that of
tic ratio (k) for each component. an authentic specimen, or by confirming that the peak shape
of the component is unchanged after mixing the sample with
amount of compound in the stationary phase
k＝ an authentic specimen. If a detector which is able to obtain
amount of compound in the mobile phase
chemical structural information of the component at the
The ratio k represents the mass distribution ratio in liquid same time is used, highly specific identification can be
chromatography. achieved by confirming identity of the chemical structure of
Since the relation given below exists among the ratio (k), the component and that of an authentic specimen, in addi-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
42 2.01 Liquid Chromatography / General Tests JP XVII
tion to the identity of their retention times. amount of the compound from the calibration curve.
When Liquid Chromatography is used for purity test, it is In an individual monograph, generally one of the standard
generally performed by comparing the peak area of target solutions with a concentration within the linear range of the
impurity from the sample solution with that of the main calibration curve and a sample solution with a concentration
component from a standard solution, which is prepared by close to that of the standard solution are prepared, and the
diluting the sample solution to a concentration correspond- chromatography is performed with these solutions under a
ing to the specified limit of the impurity, or by calculating fixed condition to obtain the amount of the component. In
target impurity content using the peak area percentage this method, all procedures, such as the injection procedure,
method. Unless otherwise specified, if a sample is separated must be carried out under a strictly constant condition.
into isomers in the chromatogram, the isomer ratio is calcu-
5. Method for peak measuring
lated by using the peak area percentage method.
Generally, the following methods are used.
The peak area percentage method is a method to calculate
5.1. Peak height measuring method
the proportion of the components from the ratio of the peak
(i) Peak height method: Measure the distance between
area of each component to the sum of the peak areas of
the maximum of the peak and the intersecting point of a per-
every peak recorded in the chromatogram. In order to obtain
pendicular line from the maximum of the peak to the
accurate results in evaluating the proportion of the compo-
horizontal axis of recording paper with a tangent linking the
nents, it is necessary to correct the area of each component
baselines on both sides of the peak.
based on its relative response factor to the principal compo-
(ii) Automatic peak height method: Measure the signals
nent.
from the detector as the peak height using a data processing
4. Assay system.
4.1. Internal standard method 5.2. Peak area measuring method
In the internal standard method, choose a stable com- (i) Width at half-height method: Multiply the peak width
pound as an internal standard which shows a retention time at the half-height by the peak height.
close to that of the compound to be assayed, and whose peak (ii) Automatic integration method: Measure the signals
is well separated from all other peaks in the chromatogram. from the detector as the peak area using a data processing
Prepare several kinds of standard solutions containing a system.
fixed amount of the internal standard and several graded
6. System suitability
amounts of the authentic specimen specified in the individual
System suitability testing is an integral part of test
monograph. Based on the chromatogram obtained by injec-
methods using chromatography, and is used to ensure that
tion of a fixed volume of individual standard solutions, cal-
the performance of the chromatographic systems used is as
culate the ratio of peak area or peak height of the authentic
suitable for the analysis of the drug as was at the time when
specimen to that of the internal standard, and prepare a
the verification of the test method was performed using the
calibration curve by plotting these ratios on the ordinate
system. System suitability testing should be carried out at
against the amount of the authentic specimen or the ratio of
every series of drug analysis. The test procedures and accep-
the amount of the authentic specimen to that of the internal
tance criteria of system suitability testing must be prescribed
standard on the abscissa. The calibration curve is usually
in the test method of drugs. The results of drug analyses are
obtained as a straight line passing through the origin. Then,
not acceptable unless the requirements of system suitability
prepare a sample solution containing the internal standard in
have been met.
the same amount as in the standard solutions used for the
In system suitability testing of the chromatographic sys-
preparation of the calibration curve according to the method
tems, the evaluation of ``System performance'' and ``System
specified in the individual monograph, perform the liquid
repeatability'' is usually required. For quantitative purity
chromatography under the same operating conditions as for
tests, the evaluation of ``Test for required detectability'' may
the preparation of the calibration curve, calculate the ratio
also be required.
of the peak area or peak height of the objective compound
6.1. Test for required detectability
to that of the internal standard, and read the amount of the
For purity tests, when it is confirmed that the target impu-
compound from the calibration curve.
rity is distinctly detected at the concentration of its spe-
In an individual monograph, generally one of the standard
cification limit, it is considered verified that the system used
solutions with a concentration within the linear range of the
has adequate performance to achieve its intended use.
calibration curve and a sample solution with a concentration
For quantitative purity tests, ``Test for required detecta-
close to that of the standard solution are prepared, and the
bility'' is usually required, and in order to confirm, in some
chromatography is performed with these solutions under
degree, the linearity of response near its specification limit,
fixed conditions to determine the amount of the objective
the range of expected response to the injection of a certain
compound.
volume of target impurity solution at the concentration of its
4.2. Absolute calibration curve method
specification limit should be prescribed. For limit test, ``Test
Prepare standard solutions with several graded amounts
for required detectability'' is not required, if the test is per-
of the authentic specimen, and inject accurately a fixed
formed by comparing the response from sample solution
volume of these standard solutions. With the chromatogram
with that from standard solution at the concentration of its
obtained, prepare a calibration curve by plotting the peak
specification limit. ``Test for required detectability'' is also
areas or peak heights on the ordinate against the amount of
not required, if it is confirmed that the impurity can be de-
the authentic specimen on the abscissa. The calibration curve
tected at its specification limit by the evaluation of ``System
is generally obtained as a straight line passing through the
repeatability'' or some other procedure.
origin. Then, prepare a sample solution according to the
6.2. System performance
method specified in the individual monograph, perform the
When it is confirmed that the specificity for determining
liquid chromatography under the same conditions as for the
the test ingredient is ensured, it is considered verified that the
preparation of the calibration curve, measure the peak area
system used has adequate performance to achieve its in-
or peak height of the objective compound, and read the
tended use.

In assay, ``System performance'' should be defined by the

resolution between the test ingredient and a target substance
to be separated (a closely eluting compound is preferable),
and when appropriate, by their order of elution. In purity
tests, both the resolution and the order of elution between
the test ingredient and a target substance to be separated
(a closely eluting compound is preferable) should be
prescribed. In addition, if necessary, the symmetry factor of
the test ingredient should be prescribed together with them.
However, if there is no suitable target substance to be sepa-
rated, it is acceptable to define ``System performance'' using
the number of theoretical plates and the symmetry factor of
the test ingredient.
6.3. System repeatability
When it is confirmed that the degree of variation (preci-
sion) of the response of the test ingredient is at a level that
meets the requirement of ``System repeatability'', it is consi- The baseline and background noise are measured over a
dered verified that the system used has adequate per- range 20 times of peak width at the center point of peak
formance to achieve its intended use. height of the target ingredient. When a solvent blank is used,
The allowable limit of ``System repeatability'' is normally measure over almost the same range as mentioned above
defined as the relative standard deviation (RSD) of the around the point where the target ingredient elutes.
response of the test ingredient in replicate injections of
(ii) Symmetry factor: It shows the degree of symmetry of
standard solution. It is acceptable to confirm the repeata-
a peak in the chromatogram, and is defined as S in the fol-
bility of the system not only by replicate injections of stand-
lowing equation.
ard solution before sample injections, but also by divided
injections of standard solution before and after sample injec- W0.05 h
S＝
tions, or by interspersed injections of standard solution 2f
among sample injections.
W0.05 h: Width of the peak at one-twentieth of the peak
In principle, total number of replicate injections should be
height
6. However, in the case that a long time is necessary for one
f: Distance between the perpendicular from the peak max-
analysis, such as the analysis using the gradient method, or
imum and the leading edge of the peak at one-twentieth
the analysis of samples containing late eluting components,
of the peak height
it may be acceptable to decrease the number of replicate
injections by adopting new allowable limit of ``System Where W0.05 h and f have the same unit.
repeatability'' which can guarantee a level of ``System
(iii) Relative standard deviation: Generally, it is defined
repeatability'' equivalent to that at 6 replicate injections.
as RSD (z) in the following equation.
The allowable limit of ``System repeatability'' should be
set at an appropriate level based on the data when suitability n
of the method for the evaluation of quality of the drug was
100
S(x － X̃ )
i＝1
i
2

verified, and the precision necessary for the quality test. RSD (z) ＝ ×
X̃ n－1
7. Point to consider on changing the operating conditions
xi: Observed value
Among the operating conditions specified in the individual
X̃: Mean of observed values
monograph, inside diameter and length of the column,
n: Number of replicate measurements
particle size of the packing material, column temperature,
composition ratio of the mobile phase, composition of (iv) Complete separation of peak: It means that the reso-
buffer solutions in the mobile phase, pH of the mobile lution between two peaks is not less than 1.5. It is also called
phase, concentration of ion-pair forming agents in the as ``baseline separation''.
mobile phase, ionic strength of the mobile phase, flow rate
(v) Peak-valley ratio: It indicates the degree of separa-
of the mobile phase, number and timing of mobile phase
tion between 2 peaks on a chromatogram when baseline
composition changes in gradient program, flow rate of
separation cannot be attained, and is defined as p/v by the
mobile phase in gradient program, composition and flow
following formula.
rate of derivatizing reagents, and reaction time and chamber
temperature in chemical reaction may be modified within the Hp
p/v ＝
ranges in which the liquid chromatographic system used Hv
conforms to the requirements of system suitability.
Hp: peak height from the baseline of the minor peak
8. Terminology Hv: height from the baseline of the lowest point (peak
(i) SN ratio: It is defined by the following formula. valley) of the curve between major and minor peaks
2H (vi) Separation factor: It shows the relation between the
S/N ＝
h retention times of peaks in the chromatogram, and is defined
as a in the following equation.
H: Peak height of the target ingredient peak from the
baseline tR2 － t0
a＝
h: Width of background noise of the chromatogram of tR1 － t0
sample solution or solvent blank around the peak of the
tR1, tR2: Retention times of two compounds used for the
target ingredient
resolution measurement (tR1 ＜ tR2)

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
44 2.02 Gas Chromatography / General Tests JP XVII
tic ratio (k) for each component.
amount of compound in the stationary phase
k＝
amount of compound in the mobile phase
Since the relation given below exists among the ratio (k),
the time for which the mobile phase is passed through the
column (t0: time measured from the time of injection of a
compound with k ＝ 0 to the time of elution at the peak
maximum), and the retention time (tR: time measured from
the time of injection of a compound to be determined to the
time of elution at the peak maximum), the retention time for
a compound on a column has a characteristic value under
fixed chromatographic conditions.
tR ＝ (1 ＋ k) t0
1. Apparatus
t0: Time of passage of the mobile phase through the Basically, the apparatus required for the gas chromato-
column (time measured from the time of injection of a graphic procedure consists of a carrier gas-introducing port
compound with k ＝ 0 to the time of elution at the peak and flow regulator, a sample injection port, a column, a
maximum) column oven, a detector and a recorder. Gas introducing
port and flow regulator for a combustion gas, a burning sup-
The separation factor (a) indicates thermodynamic differ-
porting gas and an accessory gas and sample injection port
ence in partition of two compounds. It is basically the ratio
for headspace are also used, if necessary. The carrier gas-
of their partition equilibrium coefficients or of their mass-
introducing port and flow regulator serves to deliver the car-
distribution ratios, and is obtained from the chromatogram
rier gas into the column at a constant flow rate, and usually
as the ratio of the retention times of the two compounds.
consist of a pressure regulation valve, a flow rate regulation
(vii) Resolution: It shows the relation between the reten- valve and a pressure gauge. The sample injection port is used
tion time and the peak width of peaks in the chromatogram, to deliver a quantity of the sample to the flow line of carrier
and is defined as RS in the following equation. gas with high reproducibility. There are sample injection
ports for packed column and for capillary column. There are
tR2 － tR1
RS ＝ 1.18 × both divided injection mode and non-divided injection mode
W0.5 h1 ＋ W0.5 h2
to sample injection port for capillary column. The columns
tR1, tR2: Retention times of two compounds used for the are usually classified as packed column or capillary column.
measurement of resolution (tR1 ＜ tR2) The packed column is a tube made of inert metal, glass or
W0.5 h1, W0.5 h2: Peak widths at half peak height synthetic resin, in which a packing material for gas chroma-
tography is uniformly packed. The packed column with not
Where tR1, tR2, W0.5 h1 and W0.5 h2 have the same unit.
more than 1 mm in inside diameter is also called a packed
(viii) Number of theoretical plates: It indicates the extent capillary column (micro packed column). A capillary column
of band broadening of a compound in the column, and is is a tube made of inert metal, glass, quartz or synthetic resin,
generally defined as N in the following equation. whose inside wall is bound chemically with stationary phase
for gas chromatography. The column oven has the setting
tR 2
N ＝ 5.54 × capacity for a column with required length and the tem-
W0.5 h2
perature regulation system for keeping the constant column
tR: Retention time of compound temperature. The detector is used to detect a component
W0.5 h: Width of the peak at half peak height separated on the column, and may be an alkaline thermal
ionization detector, a flame photometry detector, mass
Where tR and W0.5 h have the same unit
spectrophotometer, hydrogen flame-ionization detector, an
9. Note electron capture detector, a thermal conductivity detector,
Avoid the use of authentic specimens, internal standards, etc. The recorder is used to record the output signals of the
reagents or solvents containing substances that may interfere detector.
with the determination.
2. Procedure
Unless otherwise specified, proceed by the following
method. Fix the detector, column and carrier gas to the ap-
2.02 Gas Chromatography paratus, and adjust the flow rate and the column tempera-
ture to the values described in the operating conditions speci-
Gas Chromatography is a method to develop a mixture in- fied in the individual monograph. Inject a volume of the
jected into a column prepared with a suitable stationary sample solution or the standard solution specified in the
phase by passing a gas (carrier gas) as a mobile phase individual monograph with the sample injector into the
through the column, in order to separate the mixture into its column system through the sample injection port. The sepa-
components by making use of the difference of retention rated components are detected by the detector, and recorded
capacity against the stationary phase, and to determine the by the recorder as a chromatogram.
components. This method can be applied to a gaseous or
3. Identification and purity test
vaporizable sample, and is used for identification, purity
Identification of a component of a sample is performed by
test, and quantitative determination.
confirming identity of the retention time of the component
A mixture injected into the column is distributed between
and that of an authentic specimen, or by confirming that the
the mobile phase and the stationary phase with a characteris-
peak shape of the component is unchanged after mixing the

sample with an authentic specimen. is generally obtained as a straight line passing through the
In general, the purity of the sample is determined by com- origin. Then, prepare a sample solution according to the
paring the peak area of target impurity from the sample method specified in the individual monograph, perform the
solution with that of the main component from a standard gas chromatography under the same conditions as for the
solution, which is prepared by diluting the sample solution preparation of the calibration curve, measure the peak area
to a concentration corresponding to the specified limit of the or peak height of the objective compound, and read the
impurity, or by calculating target impurity content using the amount of the compound from the calibration curve.
peak area percentage method. Unless otherwise specified, if In an individual monograph, generally one of the standard
a sample is separated into isomers in the chromatogram, the solutions with a concentration within the linear range of the
isomer ratio is calculated by using the peak area percentage calibration curve and a sample solution with a concentration
method. close to that of the standard solution are prepared, and the
The peak area percentage method is a method to calculate chromatography is performed with these solutions under a
the proportion of the components from the ratio of the peak fixed condition to obtain the amount of the component. In
area of each component to the sum of the peak areas of this method, all procedures, such as the injection procedure,
every peak recorded in the chromatogram. In order to obtain must be carried out under a strictly constant condition.
accurate results in evaluating the proportion of the compo- 4.3. Standard addition method
nents, it is necessary to correct the area of each component Pipet a fixed volume of more than 4 sample solutions, add
based on its response factor to the principal component. exactly the standard solution so that stepwise increasing
amounts of the object compound are contained in the solu-
4. Assay
tions except 1 sample solution, diluted exactly each solution
In general, perform the assay by using the internal stand-
with and without standard solution to a definite volume, and
ard method. The absolute calibration curve method is used
use each solution as the sample solution. Based on the chro-
when a suitable internal standard is not available. Perform
matogram obtained by exact injection of a fixed volume of
the assay by using the standard addition method when the
individual sample solutions, measure the peak area or peak
effect of the component other than the compound to be
height of individual sample solutions. Calculate the concen-
assayed on the quantitative determination is not negligible
tration of standard objective compound added into each
against a result of the determination.
sample solution, plot the amounts (concentration) of added
4.1. Internal standard method
standard object compound on the abscissa and the peak area
In the internal standard method, choose a stable com-
or peak height on the ordinate on the graph, extend the
pound as an internal standard which shows a retention time
calibration curve obtained by linking the plots, and deter-
close to that of the compound to be assayed, and whose peak
mine the amount of object compound to be assayed from the
is well separated from all other peaks in the chromatogram.
distance between the origin and the intersecting point of the
Prepare several kinds of standard solutions containing a
calibration curve with the abscissa. This method is available
fixed amount of the internal standard and several graded
only in the case that the calibration curve is a straight line,
amounts of the authentic specimen specified in the individual
and passes through the origin when the absolute calibration
monograph. Based on the chromatogram obtained by injec-
curve method is employed. In this method, all procedures
tion of a fixed volume of individual standard solutions, cal-
must be carried out under a strictly constant condition.
culate the ratio of peak area or peak height of the authentic
specimen to that of the internal standard, and prepare a 5. Method for peak measuring
calibration curve by plotting these ratios on the ordinate Generally, the following methods are used.
against the amount of the authentic specimen or the ratio of 5.1. Peak height measuring method
the amount of the authentic specimen to that of the internal (i) Peak height method: Measure the distance between
standard on the abscissa. The calibration curve is usually ob- the maximum of the peak and the intersecting point of a per-
tained as a straight line passing through the origin. Then, pendicular line from the maximum of the peak to the
prepare a sample solution containing the internal standard in horizontal axis of recording paper with a tangent linking the
the same amount as in the standard solutions used for the baselines on either side of the peak.
preparation of the calibration curve according to the method (ii) Automatic peak height method: Measure the signals
specified in the individual monograph, perform the gas chro- from the detector as the peak height using a data processing
matography under the same operating conditions as for the system.
preparation of the calibration curve, calculate the ratio of 5.2. Peak area measuring method
the peak area or peak height of the objective compound to (i) Width at half-height method: Multiply the peak width
that of the internal standard, and read the amount of the at the half-height by the peak height.
compound from the calibration curve. (ii) Automatic integration method: Measure the signals
In an individual monograph, generally one of the standard from the detector as the peak area using a data processing
solutions with a concentration within the linear range of the system.
calibration curve and a sample solution with a concentration
6. System suitability
close to that of the standard solution are prepared, and the
Refer to ``System suitability'' described under 2.01 Liquid
chromatography is performed with these solutions under
Chromatography.
fixed conditions to determine the amount of the objective
compound. 7. Point to consider in changing the operating conditions
4.2. Absolute calibration curve method Among the operating conditions specified in the individual
Prepare standard solutions with several graded amounts monograph, inside diameter and length of column, particle
of the authentic specimen, and inject accurately a fixed size of packing material, concentration or thickness of sta-
volume of these standard solutions. With the chromatogram tionary phase, column temperature, temperature-rising rate,
obtained, prepare a calibration curve by plotting the peak kind and flow rate of carrier gas, and split ratio may be mo-
areas or peak heights on the ordinate against the amount of dified within the ranges in which the gas chromatographic
the authentic specimen on the abscissa. The calibration curve system used conforms to the requirements of system suita-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
46 2.03 Thin-layer Chromatography / General Tests JP XVII
bility. Headspace sample injection device and its operating Amino acid analysis can be used to quantify protein and
conditions may be also modified, provided that they give peptides, to determine the identity of proteins or peptides
equivalent or more accuracy and precision. based on their amino acid composition, to support protein
and peptide structure analysis, to evaluate fragmentation
8. Terminology
strategies for peptide mapping, and to detect atypical amino
The definition of terms described under 2.01 Liquid Chro-
acids that might be present in a protein or peptide. It is nec-
matography shall apply in 2.02 Gas Chromatography.
essary to hydrolyze a protein/peptide to its individual amino
9. Note acid constituents before amino acid analysis. Following pro-
Avoid the use of authentic specimens, internal standards, tein/peptide hydrolysis, the amino acid analysis procedure
reagents or solvents containing substances that may interfere can be the same as that practiced for free amino acids in
with the determination. other pharmaceutical preparations. The amino acid constitu-
ents of the test sample are typically derivatized for analysis.
1. Hydrolysis of Protein and Peptide
2.03 Thin-layer Chromatography Acid hydrolysis at 1109C for 24 hours using 6 mol/L hy-
drochloric acid containing phenol (Method 1) is the most
Thin-layer Chromatography is a method to separate each common method for hydrolyzing protein and samples. The
ingredient by developing a mixture in a mobile phase, using a result should be analyzed carefully because several amino
thin-layer made of a suitable stationary phase, and is applied acids are chemically modified during the acid hydrolysis and
for identification, purity test, etc. of substances. thus not recovered quantitatively. Tryptophan is destroyed;
serine and threonine are partially destroyed; methionine
1. Preparation of thin-layer plate
might undergo oxidation; and cysteine is typically recovered
Generally, proceed by the following method.
as cystine (but cystine recovery is usually poor because of
A smooth and uniformly thick glass plate having a size of
partial destruction or reduction to cysteine). Peptide bonds
50 mm × 200 mm or 200 mm × 200 mm is used for prepar-
involving isoleucine and valine are partially cleaved; and
ing a thin-layer plate. Using a suitable apparatus, apply a
asparagine and glutamine are deamidated, resulting in aspar-
water suspension of powdered solid substance for the sta-
tic acid and glutamic acid, respectively.
tionary phase, directed in the monograph, on one side of the
The hydrolysis techniques, Methods 2 to 11, are used to
glass plate to make a uniform layer of 0.2 to 0.3 mm in
address these concerns. Some of the hydrolysis techniques,
thickness. After air-drying, dry further by heating at a fixed
Methods 4 to 11, may cause modifications of cysteine,
temperature between 1059 C and 1209C for 30 to 60 minutes.
methionine, asparagines and glutamine to other amino acids.
A suitable plastic plate may be used instead of the glass
Therefore, the benefits of using a given hydrolysis technique
plate. Preserve the dried plate with protection from mois-
are weighed against the concerns with the technique and are
ture.
tested adequately before employing a method other than acid
2. Procedure hydrolysis by Method 1.
Unless otherwise specified, proceed by the following
(i) Method 1: Hydrolysis using hydrochloric acid con-
method.
taining phenol (liquid phase hydrolysis, vapor phase hy-
Designate a line about 20 mm distant from the bottom of
drolysis)
the thin-layer plate as the starting line, spot 2 to 6 mm in di-
Prevention of tryptophan oxidation
ameter the directed volumes of the sample solution or the
(ii) Method 2: Mercaptoethanesulfonic acid hydrolysis
standard solution in the monograph using micropipets at
(vapor phase hydrolysis)
points on this line, separated by more than 10 mm, and air-
(iii) Method 3: Hydrolysis using hydrochloric acid con-
dry. Unless otherwise specified, attach the filter paper along
taining thioglycolic acid (vapor phase hydrolysis)
with the inside wall of the container, and wet the filter paper
Cysteine-cystine and methionine oxidation
with the developingt solvent. In the container, the develop-
(iv) Method 4: Hydrolysis by Method 1 or Method 2 after
ing solvent is placed up to about 10 mm in height from the
oxidation with performic acid
bottom beforehand, seal the container closely, and allow it
Cysteine-cystine oxidation
to stand for 1 hour at ordinary temperature. Place the plate
(v) Method 5: Hydrolysis using hydrochloric acid contain-
in the container, avoiding contact with the inside wall, and
ing sodium azide (liquid phase hydrolysis)
seal the container. Develop it at ordinary temperature.
(vi) Method 6: Hydrolysis using hydrochloric acid contain-
When the solvent front has ascended from the starting line
ing dimethylsulfoxide (vapor phase hydrolysis)
to the distance directed in the monograph, remove the plate
Cysteine-cystine reduction and alkylation
from the container. Immediately put a mark at the solvent
(vii) Method 7: Hydrochloric acid hydrolysis after a vapor
front. After air-drying, observe the location, color, etc., of
phase pyridylethylation reaction
each spot by the method specified in the monograph. Calcu-
(viii) Method 8: Hydrochloric acid hydrolysis after a liquid
late the R f value by using the following equation:
phase pyridylethylation reaction
distance from the starting line to the center of the spot (ix) Method 9: Hydrochloric acid hydrolysis after a liquid
Rf ＝
distance from the starting line to the solvent front phase carboxymethylation reaction
Conversion of cysteine-cystine to mixed disulfide
(x) Method 10: Hydrochloric acid hydrolysis after a reac-
tion with dithiodiglycolic acid or dithiodipropionic acid
2.04 Amino Acid Analysis of Derivatization of asparagine and glutamine
Proteins (xi) Method 11: Hydrochloric acid hydrolysis after reac-
tion with bis(1,1-trifuoroacetoxy) iodobenzene
Amino acid analysis of proteins refers to the methodology
A time-course study is often employed to analyze the start-
used to determine the amino acid composition or content of
ing concentration of amino acids that are partially destroyed
proteins, peptides, and other pharmaceutical preparations.

or slow to cleave. An acceptable alternative to the time- where H0 is the strength of the applied external magnetic
course study is to subject an amino acid calibration standard field and g is the gyromagnetic ratio, a constant characteriz-
to the same hydrolysis conditions as the test sample. This ing a particular isotope. The absorption of radiation (NMR
technique will allow the analyst to account for some residue signal) can occur only when the irradiating radio frequency
destruction. satisfies the resonance condition. Since the absorption
Microwave acid hydrolysis has been used and is rapid but coefficient (the transition probability) does not depend on
requires special equipment as well as special precautions. the environment in which the nuclei are located, the intensity
Complete proteolysis, using a mixture of proteases, has been is basically proportional to the number of nuclei. The excess
used but can be complicated, requires the proper controls, spins shifted to the higher energy levels by the transition
and is typically more applicable to peptides than proteins. process return to the thermal equilibrium state at various
rates determined by a characteristic time constant (known as
2. Methodologies of Amino Acid Analysis
the relaxation time).
The amino acid analysis techniques include the postcol-
A nucleus is shielded from the applied magnetic field by
umn derivatization for detection (Methods 1 to 2) after the
the electrons belonging to its own atom and to the molecule.
separation of the free amino acids by ion-exchange chroma-
Therefore nuclei in different environments are shielded to
tography and the precolumn derivatization of the free amino
different extents and resonate at different frequencies. The
acids (Methods 2 to 7) followed by reversed-phase HPLC.
difference in resonance frequencies is defined as chemical
(i) Method l: Ninhydrin shift (d), which is independent of the strength of the mag-
(ii) Method 2: o-Phthalaldehyde (OPA) netic field, and is given by
(iii) Method 3: Phenylisothiocyanate (PITC)
nS － nR
(iv) Method 4: 6-Aminoquinolyl-N-hydroxysuccinimidyl d＝＋ dR
nR
carbamate (AQC)
(v) Method 5: (Dimethylamino)azobenzenesulfonyl chlo- where,
ride (DABS-Cl) nS: The resonance frequency of the observed signal,
(vi) Method 6: 9-Fluorenylmethyl chloroformate (FMOC- nR: The resonance frequency of the reference signal,
Cl) dR: The chemical shift of the reference signal (in the case
(vii) Method 7: 7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazole of the value not being 0).
(NBD-F)
The chemical shifts are normally expressed in ppm, a
Among these methods, ion-exchange chromatography dimensionless unit, by assuming the chemical shift of the ref-
with postcolumn ninhydrin derivertization is one of the most erence compound as 0 ppm. When the chemical shift of the
common methods employed for quantitative amino acid reference compound is not assumed to be 0 ppm, chemical
analysis. The choice of any one technique often depends on shifts of samples are corrected accordingly.
the sensitivity required from the assay. Instruments and rea- In addition to the shielding due to electrons, the nucleus is
gents for these procedures are available commercially. Fur- subjected to effects due to the spin orientations of other
thermore, many modifications of these methodologies exist magnetic nuclei through chemical bonds, resulting in an ad-
with different reagent preparations, reaction procedures, ditional splitting of the signal. The spacing between two ad-
chromatographic systems, etc. Specific parameters may vary jacent components of the signal is known as the spin-spin
according to the exact equipment and procedure used. coupling constant (J ). Coupling constants are measured in
hertz and are independent of the strength of the external
magnetic field. The increased number of interacting nuclei
will make the multiplet pattern more complex.
Spectroscopic Methods From the NMR spectrum the following four parameters
can be obtained: chemical shift, spin-spin coupling constant,
resonance intensity (intensities of 1H are proportional to the
2.21 Nuclear Magnetic Resonance number of nuclei and those of 13C and others are susceptible
Spectroscopy to the nuclear Overhauser effect (NOE) and relaxation) and
relaxation time. These parameters are useful for structural
Nuclear Magnetic Resonance (NMR) Spectroscopy is determination, identification and quantitative analysis of
based on the phenomenon that specific radio frequency ra- molecules. Spin decoupling, NOE, and two-dimensional
diation is absorbed by magnetic nuclei in a sample placed in NMR techniques are also available for structural analysis.
a magnetic field; target nuclei are 1H, 13C, 15N, 19F, 31P, etc.
1. Spectrometer
These nuclei have intrinsic spin angular momentum, of
There are two types of spectrometers.
which the magnitude is given by I (I ＋ 1)/h/2p, where I is
1.1. Fourier transform NMR (FT-NMR) spectrometers
the spin quantum number and is integral or half-integral (I
(Fig. 2.21-1)
＝ 1/2 for 1H and 13C). When the magnetic nuclei are placed
Target nuclei are simultaneously excited in all frequency
in a magnetic field, they are oriented in 2I ＋ 1 possible
range of the nuclei by means of an intense radio frequency
orientations corresponding to 2I ＋ 1 equally spaced energy
pulse. The FID (free induction decay) after the pulse is
levels (two energy levels for 1H and 13C). The transition be-
detected, which is a time domain signal, is converted to a
tween two successive quantized energy levels corresponding
frequency domain spectrum by Fourier transformation.
to adjacent orientations can be induced by electromagnetic
Number of data points suitable for the spectral range, flip
radiation with a suitable frequency. The precise relation be-
angle, acquisition time, delay time and number of scans
tween the field strength and the resonant frequency n is given
should be set appropriately.
by
Recently FT-NMR is commonly used because of its high
H0 sensitivity and various advanced applications.
n ＝ g・
2p

Fig. 2.21-1 FT-NMR spectrometer Fig. 2.21-2 CW-NMR spectrometer

1.2. Continuous wave NMR (CW-NMR) spectrometers methane, trichlorofluoromethane and phosphoric acid are
(Fig. 2.21-2) used as reference compounds for 15N, 19F and 31P, respec-
In the case of the CW method, a spectrum is obtained by tively. Furthermore, chemical shifts of residual protons in
sweeping the radio frequency or magnetic field continuously deuterated solvents and 13C in the solvent instead of a refer-
over the frequency range of the nuclei being observed. ence compound can be used for 1H and 13C NMR.
2. Measurement 3. Record of apparatus and measurement conditions
For NMR measurements, the sensitivity and resolution of Type of instrument, frequency, solvent, temperature, sam-
the instrument must be adjusted to the optimum levels. The ple concentration, reference compound, experimental tech-
excitation and observation of magnetization are provided by nique, etc. should be recorded to allow appropriate compari-
using coils. The coils need the optimization to the Larmor son of spectra, because NMR spectra depend on the meas-
frequency of the targeted nuclear spin called as ``Tuning'' urement conditions.
and sensitivity setting called as ``Matching''. An additional
4. Identification
operation is to apply an electronic current to plural shim
The sample solution is prepared and tested by the method
coils wrapping the sample and/or a correction current for
directed in each monograph. Usually in the case of 1H NMR,
optimization of resolution. This is necessary for adjusting
the sample is identified by the following method.
unevenness of spatial strength in the static magnetic field
4.1. Identification by the use of chemical shift, signal mul-
around the sample. After optimizing the sensitivity and
tiplicity and signal relative intensity
resolution of NMR instrument using ethylbenzene, 1,2-
When chemical shifts, multiplicities and relative intensities
dichlorobenzene, etc. dissolved in an appropriate deuterated
of signals are defined, the sample can be identified as being
NMR solvent, spectrum is usually measured by the following
the same substance when all chemical shifts, multiplicities
method.
and relative intensities are the same as those prescribed in
An NMR tube should be prepared for internal reference
principle. However, when NMR spectra of the same sample
method (a solution which is the sample and a drop of the ref-
are measured in the different magnitude of magnetic field, it
erence compound dissolved in a suitable solvent is directly
should be noted that the multiplicities of signals sometimes
transferred into the NMR tube) or external reference method
are not be identical. This is due to the difference in resolu-
(a sealed capillary tube containing the reference compound is
tion ability among instruments and the relative relation be-
inserted into the NMR tube with the sample solution). The
tween the size of spin-spin coupling and the difference in
measurement should be conducted by setting the NMR tube
resonance frequency of spin-spin coupled nuclei. Therefore,
into the NMR probe. The sample solutions should be
the multiplicities of signals should be evaluated, considering
completely homogeneous. In particular, solid contaminants
the magnitude of magnetic field of NMR instrument.
should be removed in order to obtain good spectra.
4.2. Identification by the use of a Reference Standard
Various deuterated NMR solvents are commonly used for
Measurement conditions should be the same as those used
NMR measurement and the following points should be con-
in the case of the Reference Standard. When the spectra of a
sidered in selecting an appropriate solvent: (i) The solvent
sample and the Reference Standard exhibit the same mul-
signals do not overlap with the sample signals. (ii) The sam-
tiplicities and relative intensities of signal at the same chemi-
ple must be soluble in the solvent selected. (iii) The solvent
cal shifts, the sample can be identified as being the same sub-
does not react with the sample. Furthermore, it should be
stance as the Reference Standard.
noted that chemical shifts can depend upon the solvent
employed, sample concentration and deuterium ion concen- 5. Experimental techniques of 1H and 13C NMR spectros-
tration, and that viscous solutions usually give rather broad, copy
poorly resolved spectra. NMR spectroscopy includes one-, two- and multi-dimen-
For the reference standards use the reagents for nuclear sional techniques, which are used for various purposes.
magnetic resonance spectroscopy. For 1H and 13C spectra, Spin decoupling, and NOE are available in one-dimen-
tetramethylsilane (TMS) is usually used as the reference sional 1H NMR spectroscopy. Spin decoupling can assign
compound for samples dissolved in organic solvents. For coupling correlations. NOE (nuclear Overhauser effect) can
samples dissolved in deuterium oxide, sodium 2,2-dimethyl- show correlations among spatially proximal protons, and
2-silapentane-5-sulfonate (DSS) or sodium 3-(trimethyl- provide information about configuration or conformation.
silyl)propionate (TSP) is used. For other nuclei, nitro- In one-dimensional 1H NMR spectroscopy, the peak inten-

sity in a fully relaxed spectrum of a compound is directly F ＝ kI0qecl

proportional to the number of 1H nuclei, when NMR instru-
k: Constant
ment and experimental conditions are optimized for quan-
I0: Intensity of exciting light
titative analysis. Accordingly, high-reliable purity, content
q: Quantum yield of fluorescence or phosphorescence
etc. based on molar amount (mol) can be determined by
using the internal reference materials having traceability to Quantum yield of fluorescence or phosphorescence
International System of Units (SI). This measurement number of quanta as fluorescence or phosphorescence
＝
method is called quantitative 1H NMR. number of quanta absorbed
Broadband decoupling, INEPT and DEPT are usually ap-
e: Molar extinction coefficient of the substance at the exci-
plied in one-dimensional 13C spectroscopy. The broadband
tation wavelength
decoupling technique simplifies a spectrum and achieves
enhancement of sensitivity. INEPT (insensitive nuclei en- 1. Apparatus
hanced by polarization transfer) and DEPT (distortionless Spectrofluorometer is usually used. Generally, a xenon
enhancement of polarization transfer) enhance the sensitivity lamp, laser, an alkaline halide lamp, etc. which provide sta-
of 13C by means of polarization transfer from directly bond- ble exciting light are used as the light source. Usually, a non-
ed 1H with a large magnetic moment. They can be applied to fluorescent quarz cell (1 cm × 1 cm) with four transparent
identify primary, secondary, tertiary or quarternary carbon. sides is used as the container for sample solution.
Two-dimensional spectroscopy can observe all correlation
2. Procedure
peaks between nuclei through spin-spin coupling or NOE in
Excitation spectrum is obtained by measuring fluorescence
a single experiment, and there are many techniques for
intensities of sample solution with varying excitation wave-
homonuclear and heteronuclear measurements. Representa-
lengths at a fixed emission wavelength (in the vicinity of the
tive techniques are described below.
fluorescence maximum) and drawing a curve showing the
(i) COSY (2D correlation spectroscopy), TOCSY (total
relationship between the excitation wavelength and the
correlation spectroscopy), HOHAHA (homonuclear Har-
fluorescence intensity. Fluorescence spectrum is obtained by
tmann-Hahn spectroscopy): Correlation between protons
measuring fluorescence intensities of sample solution with
through scalar spin-spin coupling is obtained and in-
varying emission wavelengths at a fixed excitation wave-
tramolecular connectivities of hydrogen atoms are revealed.
length (in the vicinity of the excitation maximum) and draw-
(ii) NOESY (2D nuclear Overhauser enhancement and
ing the same curve as described for the excitation spectrum.
exchange spectroscopy): NOE is measured by a two-dimen-
If necessary, the spectra are corrected with regard to the op-
sional technique. Approximate distances between spatially
tical characteristics of the apparatus.
proximate hydrogen atoms are obtained to analyze the three-
The fluorescence intensity is usually measured at the exci-
dimensional structure.
tation and the emission wavelengths in the vicinity of excita-
(iii) INADEQUATE (incredible natural abundance dou-
tion and emission maxima of the fluorescent substance. The
ble quantum transfer experiment): Although this technique
fluorescence intensity is expressed as a value relative to that
is insensitive because it involves double quantum transfer by
13C-13C scalar coupling in a sample with natural isotopic of a standard solution, because it is readily affected even by
a slight change in the condition for the measurement.
abundance, the connectivities of all neighboring 13C nuclei
Unless otherwise specified, the instrument is operated as
can be obtained to analyze the carbon skeleton.
follows with standard, sample, and reference solutions pre-
(iv) HMQC (heteronuclear multiple quantum coher-
pared as directed in the monograph: Fix the excitation and
ence): This technique observes correlations between 1H and
13C with direct spin-spin coupling using 1H detection and re- fluorescence wavelength scales at the designated positions,
adjust the dark current to zero, put the quarz cell containing
veals intramolecular chemical bonds between hydrogen and
the standard solution in the light path, and adjust the instru-
carbon atoms.
ment so that the standard solution shows the fluorescence in-
(v) HMBC (heteronuclear multiple bond connectivity):
tensity of 60z to 80z of full scale. Then perform the meas-
This technique observes correlations between 1H and 13C
urements with the cells containing the sample solution and
with long range spin-spin coupling using 1H detection and re-
the control solution, and read the fluorescence intensity as
veals intramolecular connectivities of hydrogen and carbon
z under the same condition. Set the width of the wavelength
atoms.
properly unless otherwise specified.
There are many other techniques such as 2D J-resolved
spectroscopy, DQF-COSY (double quantum filtered COSY), 3. Note
HSQC (heteronuclear single quantum coherence) and DOSY The fluorescence intensity is readily affected by the con-
(diffusion ordered spectroscopy). Furthermore, multidimen- centration, temperature and pH of the solution, and nature
sional NMR techniques are used to analyze macromolecules. and purity of solvents or reagents used.

2.22 Fluorometry 2.23 Atomic Absorption

Fluorometry is a method to measure the intensity of Spectrophotometry
fluorescence emitted from a solution of fluorescent sub-
Atomic Absorption Spectrophotometry is a method to de-
stance irradiated with an exciting light in a certain wave-
termine the amount or the concentration of an element in a
length range. This method is also applied to the phosphores-
sample specimen being examined, by utilizing the phenome-
cent substances.
non that atoms being in the ground state absorb the light of
Fluorescence intensity F in a dilute solution is propor-
specific wavelength, characteristic of the respective atom,
tional to the concentration c in mol per liter of the solution
when the light passes through an atomic vapor layer of the
and the pathlength l of light through the solution in cen-
element to be determined.
timeter.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
50 2.23 Atomic Absorption Spectrophotometry / General Tests JP XVII
1. Apparatus atomization-vaporization method, a mercury containing
Usually, the apparatus consists of a light source, a sample compound in the sample solution, prepared by the specified
atomizer, a spectroscope, a photometer and a recording sys- procedure, is chemically reduced to metallic mercury by add-
tem. Some are equipped with a background compensation ing a proper reducing reagent to the closed vessel and the
system. As a light source, usually a hollow cathode lamp spe- generated mercury is vaporized and introduced into the
cified for each element is used and sometimes a discharge absorption cell with a flow of inert gas. In the thermal
lamp is also used. There are three types of sample atomizer: atomization-vaporization method, the sample specimen on a
the flame type, the electrothermal type, and the cold-vapor quartz dish is heated electrically and the generated atomic
type. The first one is composed of a burner and a gas-flow mercury is vaporized and introduced into the absorption cell
regulator, the second one is composed of an electric furnace with a flow of inert gas. Thus, in both methods, the gener-
and a power source, and the third one is composed of a mer- ated atomic mercury is carried into the absorption cell as
cury generator and an absorption cell. The third one is fur- cold vapor and the intensity of the characteristic atomic ab-
ther classified into two subtypes, which differ in the atomiz- sorption of mercury is measured.
ing method for mercury containing-compounds: one utilizes
3. Determination
chemical reduction-vaporization and the other utilizes a ther-
Usually, proceed by any of the following methods. In the
mal reduction-vaporization method.
determination, the possibility of interference for various rea-
For the selection of an appropriate analytical wavelength
sons and the background effect must be considered and
in a spectroscope, a grating for light diffraction or an inter-
avoided if possible.
ference filter can be used. A recording system is composed of
3.1. Calibration curve method
a display and a recording device. A background compensa-
Prepare standard solutions at more than 3 concentration
tion system is employed for the correction of atmospheric
levels, measure the specific absorption due to these standard
effects on the measuring system. Several principles can be
solutions, and prepare the calibration curve of the atomic
utilized for background compensation, using continuous
absorption against the concentration. Then measure the
spectrum sources, the Zeeman splitted spectrum, the non-
atomic absorption due to the sample specimen, in which the
resonance spectrum, or self-inversion phenomena.
concentration of the element to be determined should be
Another special options such as a hydride generator and a
adjusted to be within the concentration range of the stand-
heating cell, can also be used for analyzing such as selenium.
ard solutions, and determine the amount or the concentra-
As a hydride generator, a batch method and/or a continuous
tion of the element to be examined using the calibration
flow method can be applied. While as a heating cell, there
curve.
are two kinds of cell: one for heating by flame and the other
3.2. Standard addition method
for heating by electric furnace.
To equal volumes of more than 3 sample solutions, pre-
2. Procedure pared as directed in the monograph, add a measured quan-
Unless otherwise specified, proceed by any of the follow- tity of the standard solutions to produce a series of solutions
ing methods. containing increasing amounts of the element to be exa-
2.1. Flame type mined, and further add a solvent to make up a constant
Fit the specific light source to the lamp housing and switch volume. Measure the atomic absorption for the respective
on the instrument. After lighting the lamp and selecting the solutions, and plot the obtained values on a graph with the
analytical wavelength specified in the monograph, set an ap- added amount or the concentration on the abscissa and the
propriate electric current and slit-width. Next, a mixture of a absorbance on the ordinate. Extrapolate the linear plot ob-
combustible gas and a supporting gas is ignited and the gas tained by linking the data points, and determine the amount
flow rate and/or pressure should be adjusted to optimum or the concentration of the element to be examined from the
conditions. The zero adjustment of the detecting system distance between the origin and the point where the plot in-
must be done through nebulizing the blank solvent into the tersects with the abscissa. This method is available only
flame. After setting up the measuring system, the sample so- when the calibration curve obtained by Method (1) is con-
lution prepared by the specified procedure is introduced into firmed to be linear and to pass through the origin.
the flame and the light absorption at the characteristic wave- 3.3. Internal standard method
length of the element to be determined is measured. Prepare a series of standard solutions of the element to be
2.2. Electrothermal type determined, each containing a definite amount of the inter-
Fit the specific light source to the lamp housing and switch nal standard element directed in the monograph. For these
on the instrument. After lighting the lamp and selecting the standard solutions, measure the atomic absorption due to
analytical wavelength specified in the monograph, set an ap- the standard element and the internal standard element sepa-
propriate electric current and slit-width. Further, set an elec- rately at the respective wavelengths under the same operating
tric furnace to the appropriate temperature, electric current, conditions, and obtain the ratio of absorbance by the stand-
and heating program, as directed separately in the mono- ard element to that by the internal standard element. Prepare
graph. When a suitable amount of sample is injected into the a calibration curve for the element to be determined, with
heated furnace with an appropriate stream of inert gas, the the amount or the concentration of the standard element on
sample is dried and ashed, simultaneously with atomization the abscissa and the above-mentioned ratio of the absor-
of the metallic compound included in the specimen. The bance on the ordinate. Then prepare sample solutions, add-
atomic absorption specified is observed and the intensity of ing the same amount of the internal standard element as
absorption is measured. Details of the sample preparation contained in the standard solutions. Measure the ratio of the
method are provided separately in the monograph. absorbance due to the element to be determined to that due
2.3. Cold vapor type to the internal standard element under the same conditions
Fit the mercury lamp to the lamp housing and switch on as employed for preparing the calibration curve, and deter-
the instrument. After lighting the lamp and selecting the ana- mine the amount or the concentration of the element being
lytical wavelength specified in the monograph, set an ap- examined by using the calibration curve.
propriate electric current and a slit-width. In the chemical

4. Note length which shows minimal transmission rate, the differ-

Reagents, test solutions, and gases used in this test should ence between the measured wavelength and the standard
not interfere in any process of the measurement. wavelength value should be within ± 0.5 nm. When the
measurement is repeated three times, each value obtained
should be within the mean ± 0.2 nm. It is also possible to
carry out the test using a low-pressure mercury lamp at
2.24 Ultraviolet-visible bright line wavelengths of 253.65 nm, 365.02 nm, 435.84 nm
Spectrophotometry and 546.07 nm, or a deuterium discharge lamp at bright line
wavelengths of 486.00 nm and 656.10 nm. In the case of
Ultraviolet-visible Spectrophotometry is a method to these tests, the difference between the measured wavelength
measure the degree of absorption of light between the wave- and the wavelength of the bright line should be within ± 0.3
lengths of 200 nm and 800 nm by substances for the tests of nm. When the measurement is repeated three times, each
their identity and purity, and for assay. When an atomic value obtained should be within the mean ± 0.2 nm.
absorption spectrophotometer is used for these purposes, The calibration of transmission rate or absorbance should
proceed as directed under Atomic Absorption Spectropho- be carried out as follows. Using an optical filter for trans-
tometry <2.23>. When monochromatic light passes through a mission rate calibration, determine the transmission rate at
substance in the solution, the ratio of transmitted light inten- the standard wavelength value under the test conditions
sity I to incident light intensity I0 is called transmittance t; given in the test results form attached to each of the filters.
transmittance expressed in the percentage is called percent The difference between the measured transmission rate and
transmission T, and common logarithm of the reciprocal of the standard transmission rate value should be within the
transmittance is called absorbance A. range of from 1z larger of the upper limit to 1z smaller of
the lower limit for the relative accuracy shown in the test
I I I0
t＝ T＝ × 100 ＝ 100 t A ＝ log results form. When the measurement is repeated three times,
I0 I0 I
each absorbance obtained (or calculated from the transmis-
The absorbance A is proportional to the concentration c sion rate) should be within the mean ± 0.002 when the ab-
of a substance in the solution and the length l of the layer of sorbance is not more than 0.500, and within the mean
the solution through which light passes. ± 0.004 when the absorbance is more than 0.500. In addi-
tion, it will be desirable to confirm the linearity of transmis-
A ＝ kcl ( k: constant)
sion rate at the same wavelength using several optical filters
The constant, calculated on the basis that l is 1 cm and c is for calibration of transmission rate with different transmis-
1 mol/L, is called molar absorption coefficient e. The molar sion rates.
absorption coefficient at the wavelength of maximum ab-
2. Procedure
sorption is expressed as emax.
After adjusting the apparatus as directed in the Apparatus
When a light beam passes through a substance in the solu-
and adjustment, select and set the light source, detector,
tion, the absorbance by the sample differs depending on the
mode of measurement, measuring wavelength or wavelength
wavelength of the light. So, an absorption spectrum is ob-
range, spectrum width and scanning speed.
tained by determining the absorbances of a light beam at
Subsequently, allow the apparatus to stand for a certain
various wavelengths and by graphically plotting the relation
time to confirm its stability. Then, usually adjust the appa-
between absorbance and wavelength. From the absorption
ratus so that the transmittance is 0z at measuring wave-
spectrum, it is possible to determine the wavelength of maxi-
length or over measuring wavelength range after shutting the
mum absorption lmax and that of minimum absorption lmin.
sample side of light path. Then open the shutter and adjust
The absorption spectrum of a substance in the solution is
the transmittance to 100z (the absorbance is zero).
characteristic, depending on its chemical structure. There-
Adjusting the transmittance to 100z is usually done by put-
fore, it is possible to identify a substance by comparing the
ting cells containing the control solution in both light paths.
spectrum of a sample within the specified wavelength range
For the control solution, unless otherwise specified, blank
with the Reference Spectrum or the spectrum of Reference
solvent is used.
Standard, by determining the wavelengths of maximum ab-
Then perform the measurement with the cell containing
sorption, or by measuring the ratio of absorbances at two
the sample solution, and read the absorbance at measuring
specified wavelengths. For the purpose of assay, the absor-
wavelength, or measure the spectrum over measuring wave-
bance by a sample solution with a certain concentration is
length range. Unless otherwise specified, a cell with a path
measured at the wavelength of the maximum absorption lmax
length of 1 cm, made of quartz for ultraviolet range and of
and compared it with the absorbance of a standard solution
quartz or glass for visible range, is used. Special considera-
with a certain concentration.
tion is needed with the absorption of solvents in the ultravio-
1. Apparatus and adjustment let range; use a solvent which does not disturb accurate
A spectrophotometer or a photoelectric photometer is measurement.
used for the measurement of absorbance.
3. Specific absorbance
After adjusting the spectrophotometer or photoelectric
In the Japanese Pharmacopoeia, the absorbance, calcu-
photometer based on the operation manual of the apparatus,
lated on the basis that l is 1 cm and c (concentration of a
it should be confirmed that the wavelength and the transmis-
medicament) is 1 w/vz, is called specific absorbance, and is
sion rate meet the specifications of the tests described below.
expressed as E 11zcm.
The calibration of wavelength should be carried out as
follows. Using an optical filter for wavelength calibration, A
E 11zcm ＝
measure the transmission rate in the vicinity of the standard c×l
wavelength value shown in the test results form, under the
l: Length of the layer of the solution (cm)
test conditions given in the test results form attached to each
A: Absorbance value
of the filters. When performing a test to determine the wave-
c: Concentration of the sample in the solution (w/vz)

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
52 2.25 Infrared Spectrophotometry / General Tests JP XVII
The description of, for example, ``E 11zcm (241 nm): 500 – stance. In the graphic representation of infrared spectra, the
530 (after drying, 2 mg, methanol, 200 mL)'' in the mono- plot usually shows units of wave numbers as the abscissa and
graph, indicates that observed E 11zcm value is between 500 and units of transmittance or absorbance as the ordinate. Wave
530, when the test is performed in the following manner: The number and transmittance or absorbance at each absorption
sample is dried under the conditions specified in the Test for maximum may be read graphically on an absorption spec-
Loss on Drying, and about 2 mg of the sample is weighed ac- trum and/or obtained by a data-processor. Since the wave
curately with a microbalance, and dissolved in methanol to number and the respective intensity of an absorption
make exactly 200 mL, then the absorbance of the solution is maximum depend on the chemical structure of a substance,
measured as directed in the Procedure at a wavelength of 241 this measurement can be used to identify or determine a
nm using a cell with a path length of 1 cm. substance.
4. Identification 1. Instrument and adjustment
Prepare the sample solution as directed in the monograph, Several models of dispersive infrared spectrophotometers
and test as directed in the Procedure. Usually, the test is per- or Fourier-transform infrared spectrophotometers are
formed by a single method or in a combination of a few available.
methods in the following methods using the absorbance or The instruments, adjusted according to the instruction
absorption spectrum obtained from the sample solution. manual of each individual instrument, should comply with
Subtle differences in the absorption spectrum arising from the following test for resolving power, transmittance repro-
differences in the apparatus used may be neglected. ducibility and wave number reproducibility. When the spec-
4.1. Identification using Reference Spectrum trum of a polystyrene film about 0.04 mm thick is recorded,
When the absorption spectrum obtained from the sample the depth of the trough from the maximum absorption at
solution exhibits similar intensities of absorption at the same about 2850 cm－1 to the minimum at about 2870 cm－1 should
wavelengths as those of the Reference Spectrum, the identity be not less than 18z transmittance and that from the
of the sample and the reference may be confirmed. In this maximum at about 1583 cm－1 to the minimum at about 1589
case, the range of the wavelength to be compared is the cm－1 should be not less than 12z transmittance.
range shown on the Reference Spectrum. The wave number (cm－1) scale is usually calibrated by the
Reference spectrum: Reference spectra are specified under use of several characteristic absorption wave numbers (cm－1)
the Ultraviolet-visual Reference Spectra, which are used as of a polystyrene film shown below. The number in paren-
the reference for the test of identification specified in the theses indicates the permissible range.
monograph.
3060.0 (±1.5) 2849.5 (±1.5) 1942.9 (±1.5)
4.2. Identification using Reference Standard
1601.2 (±1.0) 1583.0 (±1.0) 1154.5 (±1.0)
When the absorption spectrum obtained from the sample
1028.3 (±1.0)
solution exhibits similar intensities of absorption at the same
wavelengths as those of the spectrum obtained from the Ref- When the dispersive infrared spectrophotometer is used,
erence Standard, the identity of the sample and the reference the permissible range of the absorption wave numbers at
may be confirmed. In this case, the range of the wavelength 1601.2 cm－1 and at 1028.3 cm－1 should be both within ±2.0
to be compared is the range shown on the Reference Spec- cm－1.
trum. When the relevant Reference Spectrum is not availa- As the repeatability of transmittance and wave number,
ble, the range is that specified in the monograph. the difference of transmittance should be within 0.5z when
4.3. Identification using absorption wavelength the spectrum of a polystyrene film is measured twice at
When maximum absorption wavelengths of the spectrum several wave numbers from 3000 to 1000 cm－1, and the
obtained from the sample solution match the wavelengths difference of wave number should be within 5 cm－1 at about
specified in the monograph, the identity of the substance 3000 cm－1 and within 1 cm－1 at about 1000 cm－1.
may be confirmed. In this case, the range of the wavelength
2. Preparation of samples and measurement
to be compared is the range shown on the Reference Spec-
Unless otherwise specified, when it is directed to perform
trum.
the test ``after drying the sample'', use a sample dried under
4.4. Identification using the ratio of the absorbances ob-
the conditions specified in the monograph. Prepare the
tained at two or more wavelengths
specimen for the measurement according to one of the
When the ratios of absorbances at the specified wave-
following procedures. Because the amount of specimen or
lengths in the spectrum obtained from the sample solution
mixture described is as an example and that depends on the
meet the specifications in the monograph, the identity of the
measurement conditions, prepare it so that the transmittance
substance may be confirmed.
of most of the absorption bands is in the range of 5z to
5. Assay 80z. If the sample is a salt it should be noted that the salt
Prepare the control solution, the sample solution and the exchange can be occurred between added potassium bromide
standard solution as directed in the monograph, measure the or potassium chloride. As a general rule in the disk method
absorbances of the sample solution and the standard solu- or the diffuse reflectance method, potassium chloride is used
tion according to the method described in the Procedure, for a hydrochloride sample. For other salts, correspondence
and determine the amount of the substance to be assayed in such as to try the paste method is needed.
the sample by comparing the absorbances. Single crystals of sodium chloride, potassium bromide,
etc. are available for the optical plate.
Generally, the reference cell or material is placed in the
reference beam for double-beam instruments, while for
2.25 Infrared Spectrophotometry single-beam instruments, it is placed in the same optical path
in place of the specimen and measured separately under the
Infrared Spectrophotometry is a method of measurement
same operating conditions. The composition and prepara-
of the extent, at various wave numbers, of absorption of
tion of the reference depend on the sample preparation
infrared radiation when it passes through a layer of a sub-
methods, and sometimes the background absorption of the

atmosphere can be utilized. 3. Identification

Unless otherwise specified in the monograph, the spec- When the spectrum of a specimen and the Reference
trum is usually recorded between 4000 cm－1 and 400 cm－1. Spectrum of the substance expected to be found or the spec-
The spectrum should be scanned using the same instrumental trum of the Reference Standard exhibit similar intensities of
conditions as were used to ensure compliance with the absorption at the same wave numbers, the specimen can be
requirements for the resolving power and for the precision of identified as being the substance expected to be found. Fur-
wave number scale and of wave numbers. thermore, when several specific absorption wave numbers
2.1. Potassium bromide disk or potassium chloride disk are specified in the monograph, the identification of a
method specimen with the substance expected to be found can be
Powder 1 to 2 mg of a solid sample in an agate mortar, confirmed by the appearance of absorption bands at the
triturate rapidly with 0.10 to 0.20 g of potassium bromide specified wave numbers.
for infrared spectrophotometry or potassium chloride for in- 3.1. Identification by the use of a Reference Standard
frared spectrophotometry with precautions against moisture When the spectra of a specimen and the Reference Stand-
absorption, and compress the mixture with a press in a suita- ard exhibit similar intensities of absorption at the same wave
ble die (disk-forming container) to make the sample disk. numbers, the specimen can be identified as being the same
Adjust the amount of sample, potassium bromide or potas- substance as the Reference Standard. When a sample treat-
sium chloride according to the size of the disk. Prepare a ment method for a solid specimen is indicated in the mono-
potassium bromide reference disk or a potassium chloride graph in the case of nonconformity of the spectrum with that
reference disk in the same manner as the sample disk. If nec- of the Reference Standard, treat the specimen being exam-
essary to obtain a transparent disk, press the mixture under ined and the Reference Standard in the same manner as di-
reduced pressure not exceeding 0.67 kPa in a die with pres- rected in the monograph, then repeat the measurement.
sure applied to the die of 50 to 100 kN (5000 – 10,000 kg) per 3.2. Identification by the use of a Reference Spectrum
cm2 for 5 to 8 minutes. When the spectra of a specimen and the Reference Spec-
2.2. Solution method trum exhibit similar intensities of absorption at the same
Place the sample solution prepared by the method directed wave numbers, the specimen can be identified as being the
in each monograph in a fixed cell for liquid, and usually same substance associated with the Reference Spectrum.
measure the spectrum against the reference solvent used for When a sample treatment method for a solid specimen is in-
preparing the sample solution. The solvent used in this dicated in the monograph in the case of nonconformity of
method should not show any interaction or chemical reac- the spectrum with the Reference Spectrum, treat the speci-
tion with the specimen to be examined and should not men being examined as directed in the monograph, then
damage the optical plate. The thickness of the fixed cell is repeat the measurement. Infrared Reference Spectra, in the
usually 0.1 mm or 0.5 mm. range between 4000 cm－1 and 400 cm－1, are shown in the
2.3. Paste method section ``Infrared Reference Spectra'' for the monographs
Powder 5 to 10 mg of a solid specimen in an agate mortar, requiring the identification test by Infrared Spectrophotome-
and, unless otherwise specified, triturate the specimen with 1 try, except for monographs in which ``Identification by ab-
to 2 drops of liquid paraffin to give a homogeneous paste. sorption wave number'' is specified.
After spreading the paste to make a thin film in the center of 3.3. Identification by the use of absorption wave number
an optical plate, place the plate upon another optical plate When several specific absorption wave numbers of the
with precautions against intrusion of air, bubbles in the film, substance being examined are specified in the monograph, a
and examine its absorption spectrum. specimen can be identified as being the same substance as the
2.4. Liquid film method expected substance by confirmation of clear appearance of
Examine 1 to 2 drops of a liquid specimen as a thin film the absorption bands at all the specified wave numbers.
held between two optical plates. When the absorption inten-
sity is not sufficient, place spacers of aluminum foil, etc., be-
tween the two optical plates to make a thicker liquid film.
2.5. Film method Other Physical Methods
Examine a thin film just as it is or a prepared thin film as
directed in each monograph.
2.6. Gas sampling method 2.41 Loss on Drying Test
Put a sample gas in a gas cell previously evacuated under
Loss on Drying Test is a method to measure the loss in
the pressure directed in the monograph, and examine its ab-
mass of the sample, when dried under the conditions speci-
sorption spectrum. The path length of the gas cell is usually
fied in each monograph. This method is applied to determine
5 cm or 10 cm, but, if necessary, may exceed 1 m.
the amount of water, all or a part of water of crystallization,
2.7. ATR method
or volatile matter in the sample, which is removed during the
Place a specimen in close contact with an attenuated total
drying.
reflectance (ATR) prism, and examine its reflectance spec-
The description, for example, ``not more than 1.0z (1 g,
trum.
1059C, 4 hours)'' in a monograph, indicates that the loss in
2.8. Diffuse reflectance method
mass is not more than 10 mg per 1 g of the substance in the
Powder 1 to 3 mg of a solid specimen into a fine powder
test in which about 1 g of the substance is accurately weighed
of not more than about 50 mm particle size in an agate mor-
and dried at 1059 C for 4 hours, and ``not more than 0.5z
tar, and triturate rapidly with 0.05 to 0.10 g of potassium
(1 g, in vacuum, phosphorus (V) oxide, 4 hours),'' indicates
bromide for infrared spectrophotometry or potassium
that the loss in mass is not more than 5 mg per 1 g of the sub-
chloride for infrared spectrophotometry with precautions
stance in the test in which about 1 g of the substance is accu-
against moisture absorption. Place the mixture in a sample
rately weighed, transferred into a desiccator (phosphorus (V)
cup, and examine its reflectance spectrum.
oxide), and dried in vacuum for 4 hours.

2.42 Congealing Point

Determination
The congealing point is the temperature measured by the
following method.
1. Apparatus
Use the apparatus illustrated in Fig. 2.42-1.
2. Procedure
Transfer the sample into sample container B up to the
marked line C. When the sample is solid, melt the sample by
heating to a temperature not higher than 209C above the ex-
pected congealing point, and transfer to B. Fill the glass or
plastic bath D with water at a temperature about 59C below Fig. 2.42-1
the expected congealing point. When the sample is liquid at
room temperature, fill bath D with water at a temperature
between 109C and 159C lower than the expected congealing
point. 2.43 Loss on Ignition Test
Insert the sample container B containing the sample into
cylinder A. Adjust the immersion line H of thermometer F Loss on Ignition Test is a method to measure the loss in
to the same level of the meniscus of the sample. After cool- mass when the sample is ignited under the conditions speci-
ing the sample to about 59 C above the expected congealing fied in each monograph. This method is usually applied to
point, move vertically the stirrer E at the rate of about 60 to inorganic drugs which lose a part of the components or im-
80 strokes per minute, and observe the thermometer readings purities during ignition.
at 30-second intervals. The temperature falls gradually. Dis- The description, for example, ``40.0 – 52.0z (1 g, 450 –
continue stirring, when an appreciable amount of crystals 5509C, 3 hours)'' in a monograph, indicates that the loss in
has formed and the temperature is constant or has begun to mass is 400 to 520 mg per g of the substance in the test in
rise. Usually, read the maximum temperature (reading of F), which about 1 g of the substance is weighed accurately and
that is constant for a while after a rise of temperature. If no ignited between 4509 C and 5509C for 3 hours.
rise of temperature occurs, read the temperature that is con-
1. Procedure
stant for a while. The average of not less than four consecu-
Previously ignite a crucible or a dish of platinum, quartz
tive readings that lie within a range of 0.29C constitutes the
or porcelain to constant mass, at the temperature directed in
congealing point.
the monograph, and weigh accurately after cooling.
3. Note Take the sample within the range of ±10z of the amount
If a state of super cooling is anticipated, rub the inner wall directed in the monograph, transfer into the above ignited
of bath B or put a small fragment of the solid sample into container, and weigh it accurately. Ignite under the condi-
bath B for promoting the congealment, when the tempera- tions directed in the monograph, and, after cooling, reweigh
ture approaches near the expected congealing point. accurately. Use a desiccator (silica gel) for the cooling.

second medium.
2.44 Residue on Ignition Test In isotropic substances, the refractive index is a character-
istic constant at a definite wavelength, temperature, and
This test is harmonized with the European Pharmacopoeia pressure. Therefore, this measurement is applied to purity
and the U. S. Pharmacopeia. The parts of the text that are test of substances, or to determination of the composition of
not harmonized are marked with symbols ( ). homogeneous mixtures of two substances.
The The measurement is usually carried out at 209C, and the D
Residue on Ignition Test is a method to measure the
line of the sodium spectrum is used for irradiation. This
amount of residual substance not volatilized from a sample
value is expressed as n 20
D.
when the sample is ignited in the presence of sulfuric acid ac-
cording to the procedure described below. This test is usually 1. Procedure
used for determining the content of inorganic impurities in For the measurement of refractive index, usually the Abb áe
an organic substance. refractometer is used at a temperature in the range of
The description, for example, ``not more than 0.1z ±0.29 C of that directed in the monograph. Use of the Abb áe
(1 g)'', in a monograph, indicates that the mass of the refractometer permits direct reading of nD under incandes-
residue is not more than 1 mg per 1 g of the substance in cent light, with a measurable range from 1.3 to 1.7, and an
the test in which about 1 g of the substance is weighed accu- attainable precision of 0.0002.
rately and ignited by the procedure described below, and
``after drying'' indicates that the sample is tested after being
dried under the conditions specified in the test for Loss on
drying. 2.46 Residual Solvents
1. Procedure The chapter of residual solvents describes the control,
Ignite a suitable crucible (for example, silica, platinum, identification and quantification of organic solvents remain-
quartz or porcelain) at 600 ± 509C for 30 minutes, cool the ing in drug substances, excipients and drug products.
crucible in a desiccator (silica gel or other suitable desiccant)
I. Control of residual solvents
and weigh it accurately.
1. Introduction
Take the amount of test sample specified in the individual
Residual solvents in pharmaceuticals (except for crude
monograph in the crucible and weigh the crucible accurately.
drugs and their preparations) are defined here as organic
Moisten the sample with a small amount (usually 1 mL) of
volatile chemicals that are used or produced in the manufac-
sulfuric acid, then heat gently at a temperature as low as
ture of drug substances or excipients, or in the preparation
practicable until the sample is thoroughly charred. After
of drug products. The solvents are not completely removed
cooling, moisten the residue with a small amount (usually
by practical manufacturing techniques. Appropriate selec-
1 mL) of sulfuric acid, heat gently until white fumes are no
tion of the solvent for the synthesis of drug substance may
longer evolved, and ignite at 600 ± 509C until the residue is
enhance the yield, or determine characteristics such as crystal
completely incinerated. Ensure that flames are not produced
form, purity, and solubility. Therefore, the solvent may
at any time during the procedure. Cool the crucible in a
sometimes be a critical parameter in the synthetic process.
desiccator (silica gel or other suitable desiccant), weigh accu-
The guideline described in this chapter does not address sol-
rately and calculate the percentage of residue.
vents deliberately used as excipients nor does it address sol-
Unless otherwise specified, if the amount of the residue so
vates. However, the content of solvents in such products
obtained exceeds the limit specified in the individual mono-
should be evaluated and justified.
graph, repeat the moistening with sulfuric acid, heating and
Since there is no therapeutic benefit from residual sol-
ignition as before, using a 30-minute ignition period, until
vents, all residual solvents should be reduced to the extent
two consecutive weighings of the residue do not differ by
possible to meet product specifications, good manufacturing
more than 0.5 mg or until the percentage of residue complies
practices, or other quality-based requirements. Drug prod-
with the limit in the individual monograph.
ucts should contain no higher levels of residual solvents than
can be supported by safety data. Some solvents that are
known to cause unacceptable toxicities (Class 1, Table
2.45 Refractive Index 2.46-1) should be avoided in the production of drug sub-
stances, excipients, or drug products unless their use can be
Determination strongly justified in a risk-benefit assessment. Some solvents
associated with less severe toxicity (Class 2, Table 2.46-2)
Refractive Index Determination is a method to measure
should be limited in order to protect patients from potential
the refractive index of the sample to air. Generally, when
adverse effects. Ideally, less toxic solvents (Class 3, Table
light proceeds from one medium into another, the direction
2.46-3) should be used where practical.
is changed at the boundary surface. This phenomenon is
Testing should be performed for residual solvents when
called refraction. When light passes from the first isotropic
production or purification processes are known to result in
medium into the second, the ratio of the sine of the angle of
the presence of such solvents. It is only necessary to test for
incidence, i, to that of the angle of refraction, r, is constant
solvents that are used or produced in the manufacture or
with regard to these two media and has no relation to the an-
purification of drug substances, excipients, or drug prod-
gle of incidence. This ratio is called the refractive index of
ucts. Although manufacturers may choose to test the drug
the second medium with respect to the first, or the relative
product, a cumulative method may be used to calculate the
refractive index, n.
residual solvent levels in the drug product from the levels in
sin i the ingredients used to produce the drug product. If the cal-
n＝
sin r culation results in a level equal to or below that recommend-
ed in this guideline, no testing of the drug product for resid-
The refractive index obtained when the first medium is a
ual solvents needs be considered. If, however, the calculated
vacuum is called the absolute refractive index, N, of the

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
56 2.46 Residual Solvents / General Tests JP XVII
level is above the recommended level, the drug product The limits should be realistic in relation to analytical preci-
should be tested to ascertain whether the formulation proc- sion, manufacturing capability, reasonable variation in the
ess has reduced the relevant solvent level to within the ac- manufacturing process, and the limits should reflect contem-
ceptable amount. Drug product should also be tested if a sol- porary manufacturing standards.
vent is used during its manufacture. Option 2 may be applied by adding the amounts of a resid-
The limit applies to all dosage forms and routes of admin- ual solvent present in each of the components of the drug
istration. Higher levels of residual solvents may be accepta- product. The sum of the amounts of solvent per day should
ble in certain cases such as short term (30 days or less) or be less than that given by the PDE.
topical application. Justification for these levels should be
4. Analytical procedures
made on a case by case basis.
Residual solvents are typically determined using chro-
2. Application matographic techniques such as gas chromatography. The
Application of the rules shown in this chapter to control analytical method should be validated adequately. If only
Class 2 and Class 3 solvents will be specified separately. Class 3 solvents are present, a nonspecific method such as
loss on drying may be used.
3. General principles
3.1. Classification of residual solvents by risk assessment 5. Reporting levels of residual solvents
The term ``PDE'' (Permitted Daily Exposure) is defined in Manufacturers of drug products need certain information
the guideline as a pharmaceutically acceptable daily intake of about the content of residual solvents in excipients or drug
residual solvents. Residual solvents assessed in this guideline substances. The following statements are given as acceptable
were evaluated for their possible risk to human health and examples of the information.
placed into one of three classes as follows: (i) Only Class 3 solvents are likely to be present.
(i) Class 1 solvents: Solvents to be avoided in manufac- Loss on drying is not more than 0.5z.
ture of pharmaceuticals (ii) Only Class 2 solvents are likely to be present. Name
Known human carcinogens, strongly suspected human the class 2 solvents that are present. All are not more than
carcinogens, and environmental hazards. Class 1 solvents are the Option 1 limit.
listed in Table 2.46-1. (iii) Only Class 2 solvents and Class 3 solvents are likely
(ii) Class 2 solvents: Solvents to be limited in pharmaceu- to be present.
ticals Residual Class 2 solvents are not more than the Option 1
Non-genotoxic animal carcinogens or possible causative limit and residual Class 3 solvents are not more than 0.5z.
agents of other irreversible toxicity such as neurotoxicity or If Class 1 solvents are likely to be present, they should be
teratogenicity. Solvents suspected of other significant but identified and quantified. ``Likely to be present'' refers to
reversible toxicities. Class 2 solvents are listed in Table the solvents that were used in the final manufacturing step
2.46-2. and to the solvents that were used in earlier manufacturing
(iii) Class 3 solvents: Solvents with low toxic potential steps and not always possible to be excluded even in a vali-
Solvents with low toxic potential to man; no health-based dated process.
exposure limit is needed. Class 3 solvents are listed in Table If solvents of Class 2 or Class 3 are present at greater than
2.46-3 and have PDEs of 50 mg or more per day. their Option 1 limits or 0.5z, respectively, they should be
3.2. Option for describing limits of Class 2 solvents identified and quantified.
Two options are available when setting limits for Class 2
6. Limits of residual solvents
solvents.
6.1. Solvents to be avoided in manufacture of pharmaceu-
3.2.1. Option 1
ticals
The concentration limits in ppm can be calculated using
Solvents in Class 1 should not be employed in the
equation (1) below by assuming a product mass of 10 g ad-
manufacture of drug substances, excipients, and drug prod-
ministered daily.
ucts because of their unacceptable toxicity or their deleteri-
1000 × PDE ous environmental effect. However, if their use is unavoida-
Concentration limit (ppm) ＝ (1)
dose ble in order to produce a drug product with a significant
therapeutic advance, then their levels should be restricted as
Here, PDE is given in terms of mg per day and dose is
shown in Table 2.46-1, unless otherwise justified. 1,1,1-
given in g per day.
Trichloroethane is included in Table 2.46-1 because it is an
These limits are considered acceptable for all substances,
environmental hazard. The stated limit of 1500 ppm shown
excipients, or products. Therefore this option may be ap-
in Table 2.46-1 is based on a review of the safety data.
plied if the daily dose is not known or fixed. If all excipients
6.2. Solvents to be limited in pharmaceuticals
and drug substances in a formulation meet the limits given in
Solvents in Table 2.46-2 should be limited in drug prod-
Option 1, then these components may be used in any propor-
ucts because of their inherent toxicity. PDEs are given to the
tion. No further calculation is necessary provided the daily
nearest 0.1 mg per day, and concentrations are given to the
dose does not exceed 10 g. Products that are administered in
nearest 10 ppm. The stated values do not reflect the neces-
doses greater than 10 g per day should be considered under
sary analytical precision of determination. Precision should
Option 2.
be determined as part of the validation of the method.
3.2.2. Option 2
6.3. Solvents with low toxic potential
It is not considered necessary for each component of the
Solvents in Class 3 shown in Table 2.46-3 may be regarded
drug product to comply with the limits given in Option 1.
as less toxic and of lower risk to human health. Class 3 in-
The PDE in terms of mg per day as stated in Table 2.46-2
cludes no solvent known as a human health hazard at levels
can be used with the known maximum daily dose and equa-
normally accepted in pharmaceuticals. The amounts of these
tion (1) above to determine the concentration of residual sol-
residual solvents of 50 mg per day or less (corresponding to
vent allowed in drug product. Such limits are considered ac-
5000 ppm or 0.5z under Option 1) would be acceptable
ceptable provided that it has been demonstrated that the
without justification. Higher amounts may also be accepta-
residual solvent has been reduced to the practical minimum.

Table 2.46-1 Class 1 solvents in drug products (solvents Table 2.46-3 Class 3 solvents which should be limited by
that should be avoided). GMP or other quality- based requirements.

Concentration Acetic acid Heptane

Solvent Concern
Limit (ppm) Acetone Isobutyl acetate
Anisole Isopropyl acetate
Benzene 2 Carcinogen 1-Butanol Methyl acetate
Carbon tetrachloride 4 Toxic and environ- 2-Butanol 3-Methyl-1-butanol
mental hazard n-Butyl acetate Methyl ethyl ketone
1,2-Dichloroethane 5 Toxic tert-Butyl methyl ether Methyl isobutyl ketone
1,1-Dichloroethene 8 Toxic Dimethylsulfoxide 2-Methyl-1-propanol
1,1,1-Trichloroethane 1500 Environmental Ethanol Pentane
hazard Ethyl acetate 1-Pentanol
Diethyl ether 1-Propanol
Ethyl formate 2-Propanol
Table 2.46-2 Class 2 Solvents which residual amount Formic acid Propyl acetate
should be limited in drug products

PDE Concentration
Solvent Table 2.46-4 Solvents for which no adequate toxicological
(mg/day) limit (ppm)
data was found.
Acetonitrile 4.1 410
Chlorobenzene 3.6 360 1,1-Diethoxypropane Methyl isopropyl ketone
Chloroform 0.6 60 1,1-Dimethoxymethane Methyltetrahydrofuran
Cumene 0.7 70 2,2-Dimethoxypropane Petroleum ether
Cyclohexane 38.8 3880 Isooctane Trichloroacetic acid
1,2-Dichloroethene 18.7 1870 Isopropyl ether Trifluoroacetic acid
Dichloromethane 6.0 600
1,2-Dimethoxyethane 1.0 100
N, N-Dimethylacetamide 10.9 1090 ucts, as well as active ingredients and excipients are treated,
N, N-Dimethylformamide 8.8 880 it may be acceptable that in some cases, some of the compo-
1,4-Dioxane 3.8 380 nents of the formulation will not dissolve completely. In
2-Ethoxyethanol 1.6 160 those cases, the drug product may first need to be pulverized
Ethylene glycol 6.2 620 into a fine powder so that any residual solvent that may be
Formamide 2.2 220 present can be released. This operation should be performed
Hexane 2.9 290 as fast as possible to prevent the loss of volatile solvents dur-
Methanol 30.0 3000 ing the procedure.
2-Methoxyethanol 0.5 50
1. Class 1 and Class 2 residual solvents
Methyl butyl ketone 0.5 50
The following procedures are useful to identify and quan-
Methylcyclohexane 11.8 1180
tify residual solvents when the information regarding which
N-Methylpyrrolidone 5.3 530
solvents are likely to be present in the material is not availa-
Nitromethane 0.5 50
ble. When the information about the presence of specific
Pyridine 2.0 200
residual solvents are available, it is not necessary to perform
Sulfolane 1.6 160
Procedure A and Procedure B, and only Procedure C or
Tetrahydrofuran 7.2 720
other appropriate procedure is needed to quantify the
Tetralin 1.0 100
amount of residual solvents.
Toluene 8.9 890
A flow chart for the identification of residual solvents and
1,1,2-Trichloroethene 0.8 80
the application of limit and quantitative tests is shown in
Xylene* 21.7 2170
Fig. 2.46-1.
* Usually 60z m-xylene, 14z p-xylene, 9z o-xylene with 1.1. Water-soluble articles
17z ethylbenzene 1.1.1. Procedure A
The test is performed by gas chromatography <2.02> ac-
cording to the following conditions.
ble provided they are realistic in relation to manufacturing Class 1 standard stock solution: Pipet 1 mL of Residual
capability and good manufacturing practice. Solvents Class 1 RS, dissolve in about 9 mL of dimethylsul-
6.4 Solvents for which no adequate toxicological data was foxide, and add water to make exactly 100 mL. Pipet 1 mL
found of this solution in a volumetric flask, previously filled with
The following solvents (Table 2.46-4) may also be of in- about 50 mL of water and add water to make exactly 100
terest to manufacturers of excipients, drug substances, or mL. Pipet 10 mL of this solution in a volumetric flask, pre-
drug products. However, no adequate toxicological data on viously filled with about 50 mL of water and add water to
which to base a PDE was found. Manufacturers should make exactly 100 mL.
supply justification for residual levels of these solvents in Class 1 standard solution: Pipet 1 mL of Class 1 standard
drug products. stock solution in an appropriate head space vial containing
exactly 5 mL of water, apply the stopper, cap, and mix.
II. Identification and quantification of residual solvents
Class 2 standard stock solution A: Pipet 1 mL of Residual
Whenever possible, the substance under test needs to be
Solvents Class 2A RS, add water to make exactly 100 mL.
dissolved to release the residual solvent. Because drug prod-
Class 2 standard stock solution B: Pipet 1 mL of Residual

Class 1 system suitability solution is not less than 3. tion, and spiked test solution into the chromatograph,
System performance: When the procedure is run with record the chromatograms, and measure the responses for
Class 2 standard solution A or the solution for system the major peaks. Calculate the amount of each residual sol-
suitability under the above operating conditions, the resolu- vent found in the article under test by the formula:
tion between acetonitrile and cis-1,2-dichloroethene is not
Residual solvent (ppm) ＝ 5 (C/M ){AT/(AS － AT)}
less than 1.0‚ Pipet 1 mL of a solution of Residual Solvents
RS for System Suitability (1 in 100) in an appropriate head- C: Concentration (mg/mL) of the appropriate Reference
space vial, add exactly 5 mL of water, and apply the stopper, Standard in the standard stock solution
cap, and mix, and use this solution as the solution for system M: Amount (g) of the article under test taken to prepare
suitability. the test stock solution
System repeatability: When the test is repeated 6 times AT: Peak responses of each residual solvent obtained from
with Class 1 standard solution under the above operating the test solution
conditions, the relative standard deviation of each peak area AS: Peak responses of each residual solvent obtained from
is not more than 15z. the spiked test solution
Separately inject (following one of the headspace operat-
1.2. Water-insoluble article
ing parameter sets described in Table 2.46-5) equal volumes
1.2.1. Procedure A
of headspace (about 1.0 mL) of the Class 1 standard solu-
The test is performed by gas chromatography <2.02> ac-
tion, Class 2 standard solution A, Class 2 standard solution
cording to the following conditions. Dimethylsulfoxide may
B, and test solution into the chromatograph, record the
be substituted as an alternative solvent to N,N-dimethylfor-
chromatograms, and measure the responses for the major
mamide.
peaks. If the peak response(s) in the test solution of the
Class 1 standard stock solution: Pipet 1 mL of Residual
peak(s) identified in Procedure A is/are greater than or
Solvents Class 1 RS, dissolve in about 80 mL of N,N-
equal to a corresponding peak(s) in either the Class 1 stand-
dimethylformamide, and add N,N-dimethylformamide to
ard solution, Class 2 standard solution A or Class 2 standard
make exactly 100 mL. Pipet 1 mL of this solution in a volu-
solution B, proceed to Procedure C to quantify the peak(s);
metric flask, previously filled with about 80 mL of N,N-
otherwise the article meets the requirements of this test.
dimethylformamide and add N,N-dimethylformamide to
1.1.3. Procedure C
make exactly 100 mL (this solution is the intermediate dilu-
The test is performed by gas chromatography <2.02> ac-
tion prepared from Residual Solvents Class 1 RS and use it
cording to the following conditions.
for preparation of Class 1 system suitability solution). Pipet
Class 1 standard stock solution, Class 1 standard solution,
1 mL of this solution, and add N,N-dimethylformamide to
Class 2 standard stock solution A, Class 2 standard solution
make exactly 10 mL.
A and Class 1 system suitability solution: Prepare as directed
Class 1 standard solution: Pipet 1 mL of Class 1 standard
for Procedure A.
stock solution in an appropriate head space vial containing
Standard stock solution (Note: Prepare a separate stand-
exactly 5 mL of water, apply the stopper, cap, and mix.
ard stock solution for each peak identified and verified by
Class 2 standard stock solution A: Pipet 1 mL of Residual
Procedures A and B. For the Class 1 solvents other than 1,
Solvents Class 2A RS, dissolve in about 80 mL of N,N-
1,1-trichloroethane, prepare the first dilution as directed for
dimethylformamide, and add N,N-dimethylformamide to
the first dilution under Class 1 standard stock solution in
make exactly 100 mL.
Procedure A.): Transfer an accurately measured volume of
Class 2 standard stock solution B: Pipet 0.5 mL of Resid-
each individual solvent corresponding to each residual sol-
ual Solvents Class 2B RS, add N,N-dimethylformamide to
vent peak identified and verified by Procedures A and B to a
make exactly 10 mL.
suitable container, and dilute quantitatively and stepwise if
Class 2 standard solution A: Pipet 1 mL of Class 2 stand-
necessary, with water to obtain a solution having a final con-
ard stock solution A in an appropriate headspace vial, add
centration of 1/20 of the value stated in Table 2.46-1 or
exactly 5 mL of water, and apply the stopper, cap, and mix.
Table 2.46-2.
Class 2 standard solution B: Pipet 1 mL of Class 2 stand-
Standard solution: Pipet 1 mL of standard stock solution
ard stock solution B in an appropriate headspace vial, add
in an appropriate headspace vial, add exactly 5 mL of water,
exactly 5 mL of water, and apply the stopper, cap, and mix.
and apply the stopper, cap, and mix.
Test stock solution: Dissolve 0.5 g of the article under test
Test stock solution: Weigh accurately about 0.25 g of the
in N,N-dimethylformamide, and add N,N-dimethylfor-
article under test, dissolve in water, and add water to make
mamide to make exactly 10 mL.
exactly 25 mL.
Test solution: Pipet 1 mL of test stock solution in an
Test solution: Pipet 5 mL of test stock solution in an
appropriate headspace vial, add exactly 5 mL of water, and
appropriate headspace vial, add exactly 1 mL of water, and
apply the stopper, cap, and mix.
apply the stopper, cap, and mix.
Class 1 system suitability solution: Pipet 5 mL of test
Spiked test solution (Note: prepare a separate spiked test
stock solution and 0.5 mL of the intermediate dilution
solution for each peak identified and verified by Procedure
prepared from Residual Solvents Class 1 RS, and mix. Pipet
A and B): Pipet 5 mL of test stock solution in an appropriate
1 mL of this solution in an appropriate headspace vial, add
headspace vial, add exactly 1 mL of standard stock solution,
exactly 5 mL of test stock solution, and apply the stopper,
and apply the stopper, cap, and mix.
cap, and mix.
Chromatographic conditions fundamentally follow the
Operating conditions—
procedure A. If the results of the chromatography from
Detector: Hydrogen flame-ionization detector.
Procedure A are found to be inferior to those found with
Column: Coat the inside wall of a fused silica tube, 0.53
Procedure B, the chromatographic system from Procedure B
mm in inside diameter and 30 m in length, to 3.0 mm thick-
may be substituted.
ness with 6z cyanopropylphenylmethyl silicon polymer for
Separately inject (following one of the headspace operat-
gas chromatography.
ing parameters described in Table 2.46-5) equal volumes of
Column temperature: Maintain at 409C for 20 minutes,
headspace (about 1.0 mL) of the standard solution, test solu-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
60 2.46 Residual Solvents / General Tests JP XVII
then raise to 2409 C at 109 C per minute and maintain at meets the requirements of this test.
2409C for 20 minutes. 1.2.3 Procedure C
Injection port temperature: 1409 C. The test is performed by gas chromatography <2.02> ac-
Detector temperature: 2509C. cording to the following conditions.
Carrier gas: Helium. Class 1 standard stock solution, Class 1 standard solution,
Flow rate: About 35 cm per second. Class 1 system suitability solution, Class 2 standard stock so-
Split ratio: 1:3. (Note: The split ratio can be modified in lution A, and Class 2 standard solution A: Proceed as di-
order to optimize sensitivity.) rected for Procedure A.
System suitability— Standard stock solution (Note: Prepare a separate stand-
Test for required detectability: When the procedure is run ard stock solution for each peak identified and verified by
with Class 1 standard solution and Class 1 system suitability Procedures A and B. For the Class 1 solvents other than
solution under the above operating conditions, the signal to 1,1,1-trichloroethane, prepare the first dilution as directed
noise ratio of 1,1,1-trichloroethane in the Class 1 standard for the first dilution under Class 1 standard stock solution in
solution is not less than 5, and the signal to noise ratio of Procedure A.): Transfer an accurately measured volume of
each peak in the Class 1 system suitability solution is not less each individual solvent corresponding to each residual sol-
than 3. vent peak identified and verified by Procedures A and B to a
System performance: When the procedure is run with suitable container, and dilute quantitatively and stepwise if
Class 2 standard solution A or the solution for system necessary, with water to obtain a solution having a final con-
suitability under the above operating conditions, the resolu- centration of 1/20 of the value stated in Table 2.46-1 or
tion between acetonitrile and dichloromethane is not less Table 2.46-2.
than 1.0. Pipet 1 mL of the N,N-dimethylformamide solu- Standard solution: Pipet 1 mL of standard stock solution
tion of Residual Solvents RS for System Suitability (1 in 100) in an appropriate headspace vial, add exactly 5 mL of water,
in an appropriate headspace vial, add exactly 5 mL of water, and apply the stopper, cap, and mix.
and apply the stopper, cap, and mix, and use this solution as Test stock solution: Weigh accurately about 0.5 g of the
the solution for system suitability. article under test, and add N,N-dimethylformamide to make
System repeatability: When the test is repeated 6 times exactly 10 mL.
with Class 1 standard solution under the above operating Test solution: Pipet 1 mL of test stock solution in an
conditions, the relative standard deviation of each peak appropriate headspace vial, add exactly 5 mL of water, and
areas is not more than 15z. apply the stopper, cap, and mix.
Separately inject (use headspace operating parameters in Spiked test solution (Note: prepare a separate spiked test
column 3 of Table 2.46-5) equal volumes of headspace solution for each peak identified and verified by Procedure
(about 1.0 mL) of Class 1 standard solution, Class 2 stand- A and B): Pipet 1 mL of test stock solution in an appropriate
ard solution A, Class 2 standard solution B, and test solution headspace vial, add exactly 4 mL of water, and apply the
into the chromatograph, record the chromatograms, and stopper, cap, and mix.
measure the responses for the major peaks. If a peak Chromatographic conditions fundamentally follow the
response of any peak, other than a peak for 1,1,1- procedure A. If the results of the chromatography from
trichloroethane, in the test solution is greater than or equal Procedure A are found to be inferior to those found with
to a corresponding peak in either the Class 1 standard solu- Procedure B, the chromatographic system from Procedure B
tion, Class 2 standard solution A or Class 2 standard solu- may be substituted.
tion B, or a peak response of 1,1,1-trichloroethane is greater Separately inject (use headspace operating parameters in
than or equal to 150 times the peak response corresponding column 3 of Table 2.46-5) equal volumes of headspace
to 1,1,1-trichloroethane in the Class 1 standard solution, (about 1.0 mL) of the standard solution, test solution, and
proceed to Procedure B to verify the identity of the peak; spiked test solution into the chromatograph, record the chro-
otherwise, the article meets the requirements of this test. matograms, and measure the responses for the major peaks.
1.2.2. Procedure B Calculate the amount of each residual solvent found in the
The test is performed by gas chromatography <2.02> ac- article under test by the formula:
cording to the following conditions.
Residual solvent (ppm) ＝ 10 (C/M ){AT/(AS － AT)}
Class 1 standard stock solution, Class 1 standard solution,
Class 1 system suitability solution, Class 2 standard stock so- C: Concentration (mg/mL) of the appropriate Reference
lutions A and B, Class 2 standard solutions A and B, test Standard in the standard stock solution
stock solution, and test solution: Proceed as directed for M: Amount (g) of the article under test taken to prepare
Procedure A. the test stock solution
Proceed as directed for Procedure B under Water-Soluble AT: Peak responses of each residual solvent obtained from
Articles with a split ratio of 1:3. (Note: The split ratio can be the test solution
modified in order to optimize sensitivity.) The solution for AS: Peak responses of each residual solvent obtained from
system suitability: Proceed as directed for Procedure A. the spiked test solution
Separately inject (use headspace operating parameters in
1.3. Headspace operating parameters and other considera-
column 3 of Table 2.46-5) equal volumes of headspace
tions
(about 1.0 mL) of the Class 1 standard solution, Class 2
These test methods describe the analytical methods using
standard solution A, Class 2 standard solution B, and test
the headspace gas chromatography. The following Class 2
solution into the chromatograph, record the chromato-
residual solvents are not readily detected by the headspace
grams, and measure the responses for the major peaks. If the
injection conditions, 2-ethoxyethanol, ethylene glycol, for-
peak response(s) in test solution of the peak(s) identified in
mamide, 2-methoxyethanol, N-methylpyrrolidone, and sul-
Procedure A is/are greater than or equal to a corresponding
folane. Other appropriate validated procedures are to be em-
peak(s) in either the Class 1 standard solution, Class 2 stand-
ployed for the quantification of these residual solvents. In
ard solution A or Class 2 standard solution B, proceed to
the headspace methods, N,N-dimethylformamide and N,N-
Procedure C to quantify the peak; otherwise, the article

Table 2.46-5 Headspace operating parameters passes into the solution compartment through the mem-
brane. The pressure difference produced between the two
Headspace Operating compartments concomitantly with the solvent migration
Parameter Sets through the membrane, is defined as the osmotic pressure P
1 2 3 (Pa). The osmotic pressure is a physical quantity depending
on the total of the molecular species present, including neu-
Equilibration temperature (9C) 80 105 80 tral molecules and ions, and does not depend on the kind of
Equilibration time (min.) 60 45 45 solute. A solution property, such as osmotic pressure,
C)
Transfer-line temperature (9 85 110 105 freezing-point depression, boiling-point elevation etc., which
Syringe temperature(9C) 80 – 90 105 – 115 80 – 90 depends not on the kind of solute, but on the total number
of all molecular species, is called a colligative property of a
Carrier gas: nitrogen or helium at an appropriate pressure
solution.
Pressurization time (s) ≧ 60 ≧ 60 ≧ 60 The osmotic pressure of a polymer solution can be meas-
Injection volume (mL)* 1 1 1 ured directly as the hydrostatic pressure difference between
two compartments separated by a semipermeable membrane,
* Or follow the instrument manufacture's recommenda- such as a cellulose membrane. However, this is not applica-
tions, as long as the method criteria are met. Injecting less ble to a solution containing low molecular species, which can
than this amount is allowed as long as adequate sensitivity pass through a semipermeable membrane. Though the os-
is achieved. motic pressure of such a solution cannot be measured di-
rectly, the direction and extent of solvent migration through
biological membranes can be predicted from the total num-
dimethylacetoamide are often used as solvents. As not only 6 ber of all molecular species present when the solution is
solvents described above but these two solvents are not in- placed under physiological conditions. Other colligative
cluded in the Residual Solvents Class 2A RS and/or the properties of a solution such as freezing-point depression,
Residual Solvents Class 2B RS, appropriate validated proce- boiling-point elevation, vapor-pressure depression, etc. can
dures are to be employed for these residual solvents as neces- be directly obtained by observing changes of temperature
sary. and/or pressure, etc. These solution properties depend on
the total number of ionic and neutral species in the solution
2. Class 3 residual solvents
in the same way as the osmotic pressure, and the molecular
Perform the test according to 1. Otherwise an appropriate
particle concentration is defined as the osmotic concentra-
validated procedure is to be employed. Prepare appropriate-
tion. The osmotic concentration can be defined in two ways,
ly standard solutions, etc. according to the residual solvent
one being mass-based concentration (osmolality, mol/kg)
under test.
and the other, volume-based concentration (osmolarity,
If only Class 3 solvents are present, the level of residual
mol/L). In practice, the latter is more convenient.
solvents may be determined by loss on drying test <2.41>.
Unless otherwise specified, the freezing-point depression
However when the value of the loss on drying is more than
method is used for measuring the osmotic concentration.
0.5z, or other solvents exist, the individual Class 3 residual
The method is based on the linear dependency of the
solvent or solvents present in the article under test should be
freezing-point depression DT (9 C) upon the osmolality m
identified using the procedures as described above or other
(mol/kg), as expressed in the following equation,
appropriate procedure, and quantified as necessary.
DT ＝ K・m
3. Standards
(i) Residual Solvents Class 1 RS (A mixture of benzene, In this equation, K is the molal freezing-point depression
carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethene constant, and it is known to be 1.869 C kg/mol for water.
and 1,1,1-trichloroethane) Since the constant K is defined on the basis of molarity, the
(ii) Residual Solvents Class 2A RS (A mixture of aceto- molar osmotic concentration can be obtained from the above
nitrile, chlorobenzene, cumene, cyclohexane, 1,2-dichloro- equation. In the dilute osmotic concentration range, os-
ethene (cis-1,2-dichloroethene, trans-1,2-dichloroethene), molality m (mol/kg) can be assumed to be numerically equal
dichloromethane, 1,4-dioxane, methanol, methylcyclo- to osmolarity c (mol/L). Thus, the conventional osmolarity
hexane, tetrahydrofuran, toluene and xylene (m-xylene, p- (mol/L) and the unit of osmole (Osm) are adopted in this
xylene, o-xylene, ethylbenzene)) test method. One Osm means that the Avogadro number
(iii) Residual Solvents Class 2B RS (A mixture of chlo- (6.022 × 1023/mol) of species is contained in 1 L of solution.
roform, 1,2-dimethoxyethane, hexane, methyl butyl ketone, Usually the osmotic concentration is expressed as the sub-
nitromethane, pyridine, tetralin and 1,1,2-trichloroethene) multiple milliosmole (mOsm, mosmol/L) in the Pharmaco-
(iv) Residual Solvents RS for system suitability (A mix- poeia.
ture of acetonitrile, cis-1,2-dichloroethene and dichloro-
1. Apparatus
methane)
Usually, the osmotic concentration of a solution can be
obtained by measuring the extent of the freezing-point
depression. The apparatus (osmometer) is composed of a
2.47 Osmolarity Determination sample cell for a fixed volume of sample solution and a cell
holder, a cooling unit and bath with a temperature regulator,
Osmolarity Determination is a method for measuring the and a thermistor for detecting temperature.
osmotic concentration of the sample solution from the extent
2. Procedure
of the freezing-point depression.
A fixed volume of the test solution is introduced into the
When a solution and a pure solvent are separated by a
sample cell, as indicated for the individual apparatus.
semipermeable membrane, through which the solvent can
The apparatus must first be calibrated by the two-point
pass freely, but the solute cannot, a part of the solvent
calibration method by using osmolal standard solutions. For

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
62 2.48 Water Determination (Karl Fischer Method) / General Tests JP XVII
the calibration, select two different standard solutions just (mOsm), can be calculated by means of the following equa-
covering the expected osmolar concentration of a sample so- tion,
lution. Other than the indicated osmolal standard solutions
Osmolar ratio ＝ cT/cS
in the Table 2.47-1, water can also be used as a standard so-
lution (0 mOsm) for measuring low osmolar sample solu- cS: 286 mOsm
tions (0 – 100 mOsm). Next, after washing the sample cell
When the measurement is done by the dilution method,
and the thermistor as indicated for the individual apparatus,
because the sample has an osmolarity over 1000 mOsm, the
measure the degree of the freezing-point depression caused
apparent osmolarity of the sample solution cT can be calcu-
by a sample solution. Using the above-mentioned relation of
lated as n?/n･c?T ＝ cT, in which n?/n is the dilution number
osmolar concentration m and DT, the osmolarity of a sam-
and c?T is the measured osmolarity for the diluted solution.
ple solution can be obtained, and it is assumed to be numeri-
In this calculation, a linear relation between osmolarity and
cally equal to the osmolarity.
solute concentration is assumed. The dilution must be made
In the case of higher osmolar solutions over 1000 mOsm,
in one step. Thus when the dilution measurement is per-
dilute the sample with water and prepare n?/n times diluted
formed, the dilution number is stated as (n in n?).
sample solution (n in n?). Measure the osmolarity of the
diluted solution, as described above. In this case, it is neces-
sary to state that the calculated osmolarity for the sample
(see below) is an apparent osmolarity obtained by the dilu- 2.48 Water Determination
tion method. When the determination is performed using
n?/n times diluted solution, the dilution number should be (Karl Fischer Method)
selected so that the osmolar concentration is near but not
Water Determination is a method to determine water con-
exceeding 1000 mOsm, and dilute in one step.
tent in sample materials, utilizing the fact that water reacts
In the case of solid samples, such as freeze-dried medi-
with iodine and sulfur dioxide quantitatively in the presence
cines, prepare a sample solution by dissolving the solid using
of a lower alcohol such as methanol, and an organic base
the indicated solution for dissolution.
such as pyridine. The reaction proceeds in the manner shown
3. Suitability of the apparatus in the following equation:
After the calibration of the apparatus, a suitability test
I2 ＋ SO2 ＋ 3C5H5N ＋ CH3OH ＋ H2O
must be done by repeating the measurement of osmolarity
→ 2(C5H5N＋H)I－＋ (C5H5N＋H)－OSO2OCH3
for one of the standard solutions not less than 6 times. In
performing the test, it is advisable that the osmolarity of a In this measurement there are two methods different in
sample solution and the selected standard solution are simi- iodine-providing principle: one is the volumetric titration
lar to each other. In this test, the repeatability of measured method and the other, the coulometric titration method. In
values and the deviation of the average from the indicated the former, iodine is previously dissolved in a reagent for
value should be less than 2.0z and 3.0z, respectively. water determination, and water content is determined by
When the requirement is not met, calibrate the apparatus measuring the amount of iodine consumed as a result of
again by the two-point calibration method, and repeat the reaction with water. In the latter, iodine is produced by elec-
test. trolysis of reagent for water determination containing iodide
ion. Based on the quantitative reaction of the generated
4. Preparation of the osmolar standard solutions
iodine with water, the water content in a sample specimen
Weigh exactly an amount indicated in Table 2.47-1 of so-
can be determined by measuring the quantity of electricity
dium chloride (standard reagent), previously dried between
which is required for the production of iodine during the
5009C and 6509C for 40 to 50 minutes and allowed to cool in
titration.
a desiccator (silica gel). Dissolve the weighed sodium chlo-
ride in exactly 100 g of water to make the corresponding os- 2I－ → I2 ＋ 2e－
molar standard solution.
1. Volumetric titration
5. Osmolar ratio 1.1. Apparatus
In this test method the osmolar ratio is defined as the ratio Generally, the apparatus consists of automatic burettes, a
of osmolarity of a sample solution to that of the isotonic so- titration flask, a stirrer, and equipment for amperometric
dium chloride solution. The ratio can be used as a measure titration at constant voltage or potentiometric titration at
of isotonicity of sample solution. Since the osmolarity of the constant current.
isotonic sodium chloride solution (NaCl 0.900 g/100 mL) cS The Karl Fischer TS for water determination is extremely
(mOsm) is assumed to be constant (286 mOsm), the osmolar hygroscopic, so the apparatus should be designed to be pro-
ratio of a sample solution, of which the osmolarity is cT tected from atmospheric moisture. Desiccants such as silica
gel or calcium chloride for water determination are used for
moisture protection.
1.2. Reagents
Table 2.47-1 (i) Chloroform for water determination—To 1000 mL of
chloroform add 30 g of synthetic zeolite for drying, stopper
Standard solution for osmometer Amount of
calibration (milliosmoles) sodium chloride (g) tightly, allow to stand for about 8 hours with occasional gen-
tle shaking, then allow to stand for about 16 hours, and col-
100 0.309 lect the clear layer of chloroform. Preserve the chloroform,
200 0.626
protecting it from moisture. The water content of this chlo-
300 0.946
400 1.270
roform should not be more than 0.1 mg per mL.
500 1.593 (ii) Methanol for water determination—To 1000 mL of
700 2.238 methanol add 30 g of synthetic zeolite for drying, stopper
1000 3.223 tightly, allow to stand for about 8 hours with occasional gen-

tle shaking, then allow to stand for about 16 hours, and col- in this solution, and allow to stand for more than 24 hours
lect the clear layer of methanol. Preserve the methanol, pro- before use.
tecting it from moisture. The water content of this methanol 1.3.1.2. Standardization
should not be more than 0.1 mg per mL. According to the procedure described below, take a suita-
(iii) Propylene carbonate for water determination—To ble quantity of methanol for water determination in a dried
1000 mL of propylene carbonate add 30 g of synthetic zeolite titration flask, and titrate the solvent with a Karl Fischer TS
for drying, stopper tightly, allow to stand for about 8 hours for water determination to make the inside of the flask anhy-
with occasional gentle shaking, then allow to stand for about drous. Then, weigh about 30 mg of water accurately and put
16 hours, and collect the clear propylene carbonate layer. it in the titration flask quickly, and titrate the water dis-
Preserve this protecting from moisture. The water content solved in the solvent with a Karl Fischer TS for water deter-
should not be more than 0.3 mg per mL. mination to the end point, under vigorous stirring. Calculate
(iv) Diethylene glycol monoethyl ether for water determi- the water equivalence factor, f (mg/mL), corresponding to
nation—To 1000 mL of diethylene glycol monoethyl ether the amount of water (H2O) in mg per mL of the Karl Fischer
add 30 g of synthetic zeolite for drying, stopper tightly, TS for water determination by using the following equation:
allow to stand for about 8 hours with occasional gentle shak- Amount of water taken (H2O) (mg)
ing, then allow to stand for about 16 hours, and collect the f (mg/mL) ＝
clear layer of diethylene glycol monoethyl ether. Preserve the Volume of Karl Fischer TS for water
determination consumed for titration
diethylene glycol monoethyl ether, protecting it from mois- of water (H2O) (mL)
ture. The water content of this diethylene glycol monoethyl
1.3.2. Standard water-methanol solution
ether should not be more than 0.3 mg per mL.
Standard water-methanol solution is preserved in a cold
(v) Pyridine for water determination—Add potassium
place, protecting it from light and moisture.
hydroxide or barium oxide to pyridine, stopper tightly, and
1.3.2.1. Preparation
allow to stand for several days. Distill and preserve the puri-
Take 500 mL of methanol for water determination in a
fied and dried pyridine, protecting it from moisture. The
dried 1000-mL volumetric flask, add 2.0 mL of water, and
water content of this pyridine should not be more than 1 mg
adjust with the methanol for water determination to make
per mL.
1000 mL.
(vi) Imidazole for water determination—Use imidazole
1.3.2.2. Standardization
for thin-layer chromatography, of which the water content
According to the procedure described below, take a suita-
should not be more than 1 mg per g.
ble quantity of methanol for water determination in a dried
(vii) 2-Methylaminopyridine for water determination—
titration flask, and titrate the water contaminated with Karl
Distill and preserve 2-methylaminopyridine, protecting it
Fischer TS for water determination to make the content of
from moisture. The water content of this 2-methylamino-
the flask anhydrous. Then, add exactly 10 mL of Karl
pyridine should not be more than 1 mg per mL.
Fischer TS for water determination to this solution in the
1.3. Preparation of test solutions and standard solutions
flask, and titrate it with the prepared standard water-
1.3.1. Karl Fischer TS for water determination
methanol solution to the end point. Calculate the water
The Karl Fischer TS for water determination is preserved
concentration in the standard water-methanol solution,
in a cold place, protecting it from light and moisture.
f?(mg/mL), by using the following equation:
1.3.1.1. Preparation
Prepare according to the following method (i), (ii) or (iii). f (mg/mL) × 10 (mL)
f?(mg/mL) ＝
Additives may be added for the purpose of improving the Volume of the standard water-methanol
stability or other performances if it is confirmed that they solution consumed for titration (mL)
give almost the same results as those obtained from the spe- 1.4. Procedure
cified method. As a rule, the titration of water with a Karl Fischer TS for
(i) Preparation 1 water determination should be performed at the same tem-
Dissolve 63 g of iodine in 100 mL of pyridine for water de- perature as that at which the standardization was done, with
termination, cool the solution in ice bath, and pass dried sul- protection from moisture. The apparatus is equipped with a
fur dioxide gas through this solution until the mass increase variable resistor in the circuit, and this resistor is manipulat-
of the solution reaches 32 g. Then make up to 500 mL by ed so as to maintain a constant voltage (mV) between two
adding chloroform for water determination or methanol for platinum electrodes immersed in the solution to be titrated.
water determination, and allow to stand for more than 24 The variable current ( mA) can be measured (Amperometric
hours before use. titration at constant voltage). During titration with Karl
(ii) Preparation 2 Fischer TS for water determination, the current in the circuit
Dissolve 102 g of imidazole for water determination in 350 varies noticeably, but returns to the original value within
mL of diethylene glycol monoethyl ether for water determi- several seconds. At the end of a titration, the current stops
nation, cool the solution in ice bath, and pass dried sulfur di- changing and persists for a certain time (usually, longer than
oxide gas through this solution until the mass increase of the 30 seconds). When this electric state has been attained, it is
solution reaches 64 g, keeping the temperature between 259C designated as the end point of titration.
and 309C. Then dissolve 50 g of iodine in this solution, and Otherwise, the manipulation of the resistor serves to pass
allow to stand for more than 24 hours before use. a definite current between two platinum electrodes. The vari-
(iii) Preparation 3 able potential (mV) can be measured (Potentiometric titra-
Pass dried sulfur dioxide gas through 220 mL of propylene tion at constant current). With the progress of titration of
carbonate for water determination until the mass increase of water with a Karl Fischer TS for water determination, the
the solvent reaches 32 g. To this solution, add 180 mL of value indicated by the potentiometer in the circuit decreases
propylene carbonate for water determination, or diethylene suddenly from a polarization state of several hundreds (mV)
glycol monoethyl ether for water determination, in which to the non-polarization state, but it returns to the original
81 g of 2-methylaminopyridine for water determination is value within several seconds. At the end of titration, the
dissolved and cooled in ice bath. Then dissolve 36 g of iodine non-polarization state persists for a certain time (usually,

Karl Fischer TS for water determination, and it returns  for water determination × f (mg/mL)
 added (mL)


rapidly to the original position when the titration system has

reached the end point. In the case of the potentiometric titra-
« $
Volume of the standard water- 
× f?(mg/mL)


tion method at constant current in the back titration mode, － methanol solution consumed
for titration (mL)


the needle of the millivoltmeter is at the original position 

× 100
during excessive presence of Karl Fischer TS for water deter- Amount of sample (mg)
mination. Finally a definite voltage is indicated when the
titration system has reached the end point. 2. Coulometric titration
Unless otherwise specified, the titration of water with Karl 2.1. Apparatus
Fischer TS for water determination can be performed by Usually, the apparatus is comprised of a titration flask
either one of the following methods. Usually, the end point equipped with an electrolytic cell for iodine production, a
of the titration can be observed more clearly in the back stirrer, and a potentiometric titration system at constant cur-
titration method, compared with the direct titration method. rent. The iodine production system is composed of an anode
1.4.1. Direct titration and a cathode, separated by a diaphragm. The anode is im-
Unless otherwise specified, proceed by the following mersed in the anolyte solution for water determination and
method. Take a suitable quantity of methanol for water de- the cathode is immersed in the catholyte solution for water
termination in a dried titration flask, and titrate the water determination. Both electrodes are usually made of plati-
contaminated with Karl Fischer TS for water determination num-mesh.
to make the content of the flask anhydrous. Take a quantity Because both the anolyte and the catholyte solutions for
of sample specimen containing 5 to 30 mg of water, transfer water determination are strongly hygroscopic, the titration
it quickly into the titration flask, dissolve by stirring, and system should be protected from atmospheric moisture. For
titrate the solution to be examined with Karl Fischer TS for this purpose, silica gel or calcium chloride for water determi-
water determination to the end point under vigorous stirring. nation can be used.
In the case of an insoluble sample specimen, powder the 2.2. Preparation of anolyte and catholyte solutions for
sample quickly, weigh a suitable amount of the sample con- water determination
taining 5 to 30 mg of water, and transfer it quickly into the Electrolytic solutions shall consist of an anolyte solution
titration vessel, stir the mixture for 5 – 30 minutes, protect- for water determination and a catholyte solution for water
ing it from moisture, and perform a titration under vigorous determination, the preparations of which are described
stirring. Alternatively, in the case of a sample specimen below.
which is insoluble in the solvent for water determination or 2.2.1. Preparation 1
which interfere with the Karl Fisher reaction, water in the (i) Anolyte for water determination—Dissolve 102 g of
sample can be removed by heating under a stream of nitroimidazole for water determination in 900 mL of methanol
gen gas, and introduced into the titration vessel by using a for water determination, cool the solution in ice bath, and
water evaporation technique. pass dried sulfur dioxide gas through the solution, which is
Though the titration procedure should be performed kept below 309 C. When the mass increase of the solution has
under atmospheric conditions at low humidity, if the effect reached 64 g, the gas flow is stopped and 12 g of iodine is
of atmospheric moisture cannot be avoided, for instance, if dissolved by stirring. Then drop a suitable amount of water
a long time is required for extraction and titration of water, into the solution until the color of liquid is changed from
a blank test must be done under the same conditions as used brown to yellow, and add methanol for water determination
for the sample test, and the data must be corrected, accord- to make up 1000 mL.
ingly. (ii) Catholyte for water determination—Dissolve 24 g of
diethanolamine hydrochloride in 100 mL of methanol for
Volume of Karl Fischer water determination.
TS for water determination × f (mg/mL)
consumed for titration (mL) 2.2.2. Preparation 2
Water (H2O) z ＝ × 100 (i) Anolyte for water determination—Dissolve 40 g of
Amount of sample (mg) 1,3-di(4-pyridyl)propane and 30 g of diethanolamine in
1.4.2. Back titration about 200 mL of methanol for water determination, and
Unless otherwise specified, proceed by the following pass dried sulfur dioxide gas through the solution. When the
method. Take a suitable quantity of methanol for water de- mass increase of the solution has reached 25 g, the gas flow
termination in the dried titration vessel, and titrate the water is stopped. Add 50 mL of propylene carbonate, and dissolve
contaminated with Karl Fischer TS for water determination 6 g of iodine in the solution. Then make up the solution to
to make the content of the flask anhydrous. Take a suitable 500 mL by addition of methanol for water determination
quantity of sample specimen having 5 – 30 mg of water, and drop in a suitable amount of water until the color of liq-
transfer the sample quickly into the titration vessel, dissolve uid is changed from brown to yellow.
it in the solution by stirring, add an excessive and definite (ii) Catholyte for water determination—Dissolve 30 g of
volume of Karl Fischer TS for water determination, and then choline hydrochloride into methanol for water determination
titrate the solution with the standard water-methanol solu- and adjust the volume to 100 mL by adding the methanol.
tion to the end point under vigorous stirring. 2.2.3. Preparation 3
In the case of an insoluble sample specimen, powder the (i) Anolyte for water determination—Dissolve 100 g of
sample quickly, weigh a suitable amount accurately, transfer diethanolamine in 900 mL of methanol for water determina-
it quickly into the titration vessel, and add an excessive and tion or a mixture of methanol for water determination and
definite volume of Karl Fischer TS for water determination. chloroform for water determination (3:1), and pass dried

sulfur dioxide gas through the solution. When the mass in- the substance.
crease of the solution has reached 64 g, the gas flow is The optical rotation is a degree of rotation of polarized
stopped. Dissolve 20 g of iodine in the solution, and drop in plane, caused by the optically active substance or its solu-
a suitable amount of water until the color of liquid is tion, and it is measured by the polarimeter. The optical rota-
changed from brown to yellow. tion is proportional to the length of the polarimeter tube,
(ii) Catholyte for water determination—Dissolve 25 g of and is also related to the solution concentration, the temper-
lithium chloride in 1000 mL of a mixture of methanol for ature and the measurement wavelength. The character of the
water determination and nitroethane (4:1). rotation is indicated by the direction of the rotation, when
2.3. Procedure facing to the advancing direction of the polarized light. Thus
Take a suitable volume of an anolyte for water determina- in case of rotation to the right, it is called dextrorotatory and
tion in the titration vessel, immerse in this solution a pair of expressed by placing plus sign (＋), while in case of rotation
platinum electrodes for potentiometric titration at constant to the left, it is called levorotatory and expressed by placing
current. Then immerse the iodine production system filled minus sign (－) before the figure of the angular rotation. For
with a catholyte for water determination in the anolyte for example, ＋209means 209of rotation to the right, while
water determination. Switch on the electrolytic system and －209means 209of rotation to the left.
make the content of the titration vessel anhydrous. Next take The optical rotation a tx (9) means degree of rotation of the
an accurately weighed amount of a sample specimen contain- plate of polarization, when it is measured at t9C by using
ing 0.2 – 5 mg of water, add it quickly to the vessel, dissolve specific monochromatic light x (expressed by wavelength of
by stirring, and perform the titration to the end point under light source or the specific beam name).
vigorous stirring.
2. Apparatus and measurement
When a sample specimen cannot be dissolved in the
Polarimeter consists of a light source, a polarizer, a
anolyte, powder it quickly, and add an accurately weighed
polarimeter tube and an analyzer. The measurement is gener-
amount of the sample estimated to contain 0.2 – 5 mg of
ally performed at 209 C or 259 C, using a 100-mm tube and
water to the vessel. After stirring the mixture for 5 – 30
the D line of sodium lamp as the light source. The bright line
minutes, with protection from atmospheric moisture, per-
spectrum of mercury lamp can be used as the monochromat-
form the titration under vigorous stirring. Alternatively, in
ic light source.
the case of an insoluble solid or a sample containing a com-
If a light beam close to the sodium D line can be obtained
ponent which interferes with the Karl Fisher reaction, water
by the use of an appropriate interference filter, a continuous
in the sample can be removed by heating, and carried by a
beam such as of xenon lamp may be used alternatively.
nitrogen gas flow into the titration vessel, by using a water
2.1. Verification for accuracy of apparatus
evaporation technique.
Accuracy of the scale of the apparatus is verified by meas-
Determine the quantity of electricity (C) [＝ electric cur-
uring the optical rotation of a solution of sucrose for optical
rent (A) × time (s)] required for the production of iodine
rotation if the reading indicates the value of its known spe-
during the titration, and calculate the water content (z) in
cific optical rotation. For daily verification an optical rota-
the sample specimen by use of the following equation.
tion known quartz plate may be used for this purpose.
Though the titration procedure should be performed
under atmospheric conditions at low humidity, if the effect 3. Characteristic evaluation by optical rotation
of atmospheric moisture cannot be avoided, for instance, if Generally, when the optical rotation is settled as the speci-
a long time is required for extraction and titration of water, fication to express the quality characteristic of a drug,
a blank test must be done under the same conditions as used Specific optical rotation [a]tx (9), i.e., the optical rotation
for the sample test, and the data must be corrected, accord- equivalent of 1 g/mL in sample concentration and 1 mm in
ingly. path length of sample solution, is used. For the evaluation of
the quality characteristic of drugs such as crude drugs, when
Quantity of electricity required
for iodine production (C) it is not possible to determine the concentration of an opti-
Water (H2O) z ＝ × 100 cally active drug, the optical rotation a tx (9) is used as the
10.72 (C/mg) specification or to specify the amount of optically active im-
× Amount of sample (mg)
purities.
10.72: quantity of electricity corresponding to 1 mg of The specific optical rotation and the optical rotation can
water (C/mg) also be used for the description, purity and assay of drugs.
The specific optical rotation, [a]tx, is calculated from the
measured rotation angle of the polarization plane, a tx, by the
following equation. Though, the degree (9) is expediently
2.49 Optical Rotation used as the unit of the specific optical rotation in the phar-
Determination maceutical monographs, accurately it is stated as (9 ･mm－1･
(g/mL)－1).
1. Principle
a
Generally, the vibrations of light take place on planes per- [a]tx ＝ × 100
lc
pendicular to the direction of the beam. In case of the ordi-
nary light, the directions of the planes are unrestricted, while t: The temperature (9C) of measurement.
in case of the plane polarized light, commonly called as pola- x: The wavelength (nm) of the specific monochromatic
rized light, the vibrations take place on only one plane that light. In the case of the sodium D line, it is described as
includes the advancing direction of the beam. And it is called D.
that these beams have plane of polarization. Some drugs in a: The angle, in degrees, of rotation of the plane of the
the liquid state or in solution have a property of rotating the polarized light.
plane of the polarized light either to the right or to the left. l: The thickness of the layer of sample solution, i.e., the
This property is referred to as optical activity or optical rota- length of the polarimeter tube (mm).
tion, and is inherently related to the chemical constitution of c: Drug concentration in g/mL. When an intact liquid

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
66 2.50 Endpoint Detection Methods in Titrimetry / General Tests JP XVII
drug is used for the direct measurement without dilu- endpoint detection method, equivalent amount of substance
tion by an appropriate solvent, c equals to its density to be examined (mg)/standard solution (mL), should be spe-
(g/mL). However, unless otherwise specified, the specified in the individual monograph. Standardization of the
cific gravity may be used in stead of the density. standard solution and titration of a specimen are recom-
mended to be done at the same temperature. When there is a
The description in the monograph, for example, ``[a]20 D:
marked difference in the temperatures at which the former
－33.0 – －36.09 (after drying, 1 g, water, 20 mL, 100
and the latter are performed, it is necessary to make an ap-
mm),'' means the measured specific optical rotation [a]20 D
propriate correction for the volume change of the standard
should be in the range of －33.09and －36.09, when 1 g of
solution due to the temperature difference.
accurately weighed sample dried under the conditions, speci-
fied in the test item of Loss on drying, is taken, and dis- 1. Indicator Method
solved in water to make exactly 20 mL, then put in the Weigh an amount of a specimen in a flask or a suitable
polarimeter tube of 100 mm length, of which temperature is vessel as directed in the monograph or in ``Standard Solu-
kept at 209 C. And the description ``a 20
D : －33.0 – －36.09 tions for Volumetric Analysis'', and add a specified quantity
(100 mm)'' means the measured optical rotation, a 20
D , should of solvent to dissolve the specimen. After adding a defined
be in the range of －33.09and －36.09, when sample or a indicator to the solution to prepare the titrate, titrate by add-
solution of sample is put in the tube of 100 mm length, at ing a standard solution for volumetric analysis by using a
209 C. buret. In the vicinity of the endpoint, observe the color
change induced by the cautious addition of 0.1 mL or less of
the titrant. Calculate the quantity of titrant added from the
readings on the scale of the buret used for the titration at the
2.50 Endpoint Detection Methods starting point and at the endpoint at which the specified
in Titrimetry color change appears, as directed in the individual mono-
graph or in the ``Standard Solutions for Volumetric Analy-
Titrimetry is a method or a procedure for volumetric sis''. Although addition of the volumetric standard solution
analysis, which is usually classified into acid-base titration by buret is usually done manually, an automatic buret can
(neutralization titration or pH titration), precipitation titra- also be used.
tion, complexation titration, oxidation-reduction titration, Unless otherwise specified, perform a blank determination
etc., according to the kind of reaction or the nature of the according to the following method, and make any necessary
phenomenon occurring between the titrate and the titrant correction.
(standard solution for volumetric analysis). Furthermore, Measure a specified quantity of solvent, as directed in the
titration performed in a nonaqueous solvent is generally monograph or in the ``Standard Solutions for Volumetric
called nonaqueous titration, which is frequently used for Analysis'', and titrate as directed. The required quantity of
volumetric analysis of weak acids, weak bases, and their the standard solution added to reach a specified color
salts. The endpoint in titrimetry can be detected by color change, is assumed to be the blank quantity for the titration
changes of indicators and/or by changes of electrical signals system. However, when the blank quantity is too small to
such as electrical potential or electrical current. evaluate accurately, the quantity can be assumed to be zero.
The indicator method is one of the endpoint detection
2. Electrical Endpoint Detection Methods
methods in titrimetry. In this method the color of an indica-
2.1. Potentiometric titration
tor dye, dissolved in the titrate, changes dramatically in the
2.1.1. Apparatus
vicinity of the equivalence point due to its physico-chemical
The apparatus consists of a beaker to contain the speci-
character, and this property is used for visual endpoint de-
men, a buret for adding a standard solution, an indicator
tection. Selection of an indicator and specification of the
electrode and a reference electrode, a potentiometer for
color change induced in the respective titration system,
measuring potential difference between the electrodes or an
should be described in the individual monograph. An ap-
adequate pH meter, a recorder, and a stirrer for gentle stir-
propriate indicator should change color clearly, in response
ring of the solution to be examined. Separately, an auto-
to a slight change in physico-chemical properties of the
matic titration apparatus assembled from suitable units
titrate, such as pH, etc., in the vicinity of the equivalence
and/or parts, including a data processing system, can also be
point.
used.
Regarding the electrical endpoint detection methods, there
In this titration method, unless otherwise specified, indica-
are an electrical potential method and an electrical current
tor electrodes designated in Table 2.50-1 are used according
method, which are called potentiometric and amperometric
to the kind of titration. As a reference electrode, usually a
titration methods, respectively. They are generically named
silver-silver chloride electrode is used. Besides the single indi-
electrometric titration. In the potentiometric titration
cator electrodes as seen in Table 2.50-1, a combined refer-
method, the endpoint of a titration is usually determined to
ence electrode and indicator electrode can also be used.
be the point at which the differential potential change
When the potentiometric titration is carried out by the pH
becomes maximum or minimum as a function of the quan-
measurement method, the pH meter should be adjusted ac-
tity of titrant added. In the amperometric titration method,
cording to the pH Determination <2.54>.
unless otherwise specified, a bi-amperometric titration
2.1.2. Procedure
method is used, and the endpoint is determined by following
Weigh a defined amount of a specimen in a beaker, and
the change of microcurrent during the course of a titration.
add an indicated quantity of solvent to dissolve the speci-
Furthermore, the quantity of electricity (electrical current
men, as directed in the monograph. After the potential
× time) is often used as another electrochemical signal to
difference E (mV) or the pH value of the solvent to be used
follow a chemical reaction, as described in Coulometric
for titration has reached a stable value, immerse both refer-
Titration under Water Determination <2.48>.
ence and indicator electrodes, which have previously been
The composition of a titration system, such as amount of
washed with the solvent being used, in the solution to be exa-
specimen, solvent, standard solution for volumetric analysis,
mined, and titrate with a standard solution for volumetric

Table 2.50-1 Kind of titration and indicator electrode 2.2. Amperometric titration
2.2.1. Apparatus
Kind of titration Indicator electrode The apparatus consists of a beaker for holding a speci-
Acid-base titration Glass electrode men, a buret for adding a standard solution for volumetric
(Neutralization titra- analysis, two small platinum plates or wires of the same
tion, pH titration) shape as the indicator electrode, a device to load direct cur-
Precipitation titration Silver electrode. A silver- rent microvoltage between two electrodes, a microammeter
(Titration of halogen silver chloride electrode to measure the indicator current between the two electrodes,
ion by silver nitrate) is used as a reference a recorder, and a stirrer which can gently stir the solution in
electrode, which is con- a beaker. Separately, an automatic titration apparatus as-
nected with the titrate sembled from suitable units and/or parts, including a data
by a salt bridge of satu- processing system, can also be used.
rated potassium nitrate 2.2.2. Procedure
solution. Weigh a defined amount of a specimen in a beaker, and
Oxidation-reduction titra- Platinum electrode add an indicated quantity of solvent to dissolve the speci-
tion men, as directed in the individual monograph. Next, after
(Diazo titration, etc.) washing the two indicator electrodes with water, immerse
Complexation titration Mercury-mercury chloride both electrodes in the solution to be examined, apply a con-
(Chelometric titration) (II) electrode stant voltage suitable for measurement across two electrodes
Nonaqueous titration Glass electrode by using an appropriate device, and titrate the solution with
(Perchloric acid titra- a standard solution for volumetric analysis. During the titra-
tion, Tetramethylam- tion, the tip of the buret should be dipped into the solution
monium hydroxide to be examined. The endpoint of titration is determined by
titration) following the changes of microcurrent between the two elec-
trodes as a function of the quantity of titrant added. In the
vicinity of the endpoint, the amounts of the titrant added
should be 0.1 mL or less for adequate titrimetry. Plot the ob-
analysis with gentle stirring of the solution. During the titra- tained current values along the ordinate and the quantity of
tion, the tip of the buret should be dipped into the solution, the titrant added V (mL) along the abscissa to draw a titra-
to be examined. The endpoint of titration is determined by tion curve, and usually take the inflection point of the titra-
following the variation of the potential difference between tion curve (the point of intersection given by the extrapola-
two electrodes as a function of the quantity of titrant added. tion of two straight lines before and after the inflection) as
In the vicinity of the endpoint, the amounts of a titrant ad- the endpoint in amperometric titration.
ded should be 0.1 mL or less for adequate titrimetry. Plot The blank test in this titration is usually performed as
the obtained potential values along the ordinate and the follows: Take a volume of the solvent specified in the in-
quantity of a titrant added V (mL) along the abscissa to dividual monograph or in the ``Standard Solution for Volu-
draw a titration curve, and obtain the endpoint from the metric Analysis'', and use this as the sample solution. Deter-
maximum or the minimum value of DE/DV or from the mine the amount of the volumetric standard solution needed
value of electromotive force or pH corresponding to the for giving the endpoint, and use this volume as the blank. If
equivalence point. this volume is too small to determine accurately, the blank
Unless otherwise specified, the decision of the endpoint in may be considered as 0 (mL).
this method is usually made by either of the following Unless otherwise specified, the endpoint in this titration is
methods. decided by either of the following methods.
(i) Drawing method (i) Drawing method
Usually, draw two parallel tangent lines with a slope of Usually, extrapolate the two straight lines before and after
about 459to the obtained titration curve. Next, draw a 3rd the inflection, and obtain the inflection point of the titration
parallel line at the same distance from the previously drawn curve. Next, read the quantity of titrant added at the inflec-
two parallel lines, and decide the intersection point of this tion point, and assume this point to be the endpoint.
line with the titration curve. Further, from the intersection (ii) Automatic detection method
point, draw a vertical line to the abscissa, and read the quan- In the case of amperometric titration using an automatic
tity of titrant added as the endpoint of the titration. titration system, the endpoint can be determined by follow-
Separately, the endpoint of the titration can also be ing the instrumental indications. The endpoint is decided by
otained from the maximum or the minimum of the differen- following the variation of the indicator current during the
tial titration curve (DE/DV vs. V ). course of a titration, and the quantity of titrant added is
(ii) Automatic detection method assumed to be that at which the current has reached the
In the case of potentiometric titration using an automatic endpoint current set previously for the individual titration
titiration system, the endpoint can be determined by follow- system.
ing the respective instrumental indications. The endpoint is When atmospheric carbon dioxide or oxygen is expected
decided either by following the variation of the differential to influence the titration, a beaker with a lid should be used,
potential change or the absolute potential difference as a and the procedure should be carried out in a stream of an
function of the quantity of titrant added: in the former case inert gas, such as nitrogen gas. Further, when a specimen is
the quantity given by the maximum or the minimum of the expected to be influenced by light, use a light-resistant con-
differential values, and in the latter the quantity given by the tainer to avoid exposure of the specimen to direct sunlight.
indicator reaching the endpoint potential previously set for
the individual titration system, are assumed to be the
endpoint volumes, respectively.

Conductivity Measurement is a method for the measuring Concentration Conductivity k Resistivity r

(g/1000.0 g) ( mS・cm－1) (Q・cm)
the flowability of electric current in an aqueous solution.
The measurement is made with a conductivity meter or a re- 0.7455 1330 752
sistivity meter, and is used, for example, in the purity tests in 0.0746 133.0 7519
monographs. The method is applied to evaluate the test item 0.0149 26.6 37594
``Conductivity (Electrical Conductivity)'' specified in the
monographs. Further it is also used for monitoring the quali-
ty of water in the preparation of highly purified water.
However, when applying this method for monitoring the 2. Standard Solution of Potassium Chloride
quality of water, the details of measurement should be speci- After pulverizing an appropriate amount of potassium
fied by the user, based on the principles described here. chloride for conductivity measurement, dry it at 500 – 6009C
Conductivity of a solution k (S・m－1) is defined as the for 4 hours. For the preparation of the standard solutions,
reciprocal of resistivity r (Q・m), which is an indicator of the take the amount of the dried potassium chloride indicated
strength of ionic conductivity for a fluid conductor. Resis- in Table 2.51-1, dissolve it in distilled water previously
tivity is defined as the product of electrical resistance per boiled and cooled, or water with a conductivity less than
unit length and cross-sectional area of a conductor. When 2 m S・cm－1, and adjust to make 1000.0 g. The conductivity
resistivity is r, cross-section area A (m2), and length l (m), and the resistivity of the respective standard solutions at
resistance R (Q) can be expressed by the following equation. 209C are shown in Table 2.51-1. These standard solutions
should be kept in tightly closed polyethylene or hard glass
R ＝ r (l/A)
bottles.
Thus, conductivity k is expressed as follows, When measurement at 209 C can not be performed, the
value of conductivity for the respective standard solution
k ＝ 1/r ＝ (1/R)(l/A)
(shown in Table 2.51-1), can be corrected by using the fol-
If l/A is known, the conductivity k can be obtained by lowing equation. However, this equation is valid only within
measuring resistance R or conductance G(＝R－1). the range of 15 – 309C.
In the International System (SI), the unit of conductivity is
kT ＝ k20[1 ＋ 0.021(T － 20)]
the Siemens per meter (S・m－1). In practice, conductivity of a
solution is generally expressed by m S・cm－1, and resistivity T: Measuring temperature specified in the monograph
by Q・cm. kT: Calculated conductivity of the KCl standard solution
Unless otherwise specified, the reference temperature for at T9C
the expression of conductivity or resistivity is 209C. k20: Conductivity of the KCl standard solution at 209C
Items such as the sample preparation method, the necessi-
3. Operating Procedure
ty of blank correction, the calculation method, the spe-
3.1. Cell Constant
cification value, and the measuring temperature should be
An appropriate conductivity cell should be chosen accord-
described in the monograph, if necessary.
ing to the expected conductivity of the sample solution. The
1. Apparatus higher the expected conductivity, the larger the cell constant
A conductivity meter or a resistivity meter is composed of required for the conductivity cell, so that the electrical
an indicator part (operating panel, display, recording unit) resistance is within the measuring range of the apparatus
and a detector part, the latter of which includes a conduc- being used. Conductivity cells with a cell constant of the
tivity cell. In the conductivity cell a pair of platinum elec- order of 0.1 cm－1, 1 cm－1, or 10 cm－1, are generally used.
trodes is embedded. The cell is immersed in a solution, and For determination or confirmation of the cell constant, an
the resistance or the resistivity of the liquid column between appropriate KCl standard solution should be chosen and pre-
the electrodes is measured. Alternating current is supplied to pared, taking account of the expected conductivity of the
this apparatus to avoid the effects of electrode polarization. sample solution to be measured. Rinse the cell several times
Further, a temperature compensation system is generally with distilled water. Next, after rinsing the cell 2 – 3 times
contained in the apparatus. with the standard solution used for the cell constant determi-
Conductivity measurement is generally performed by nation, immerse the cell in the standard solution contained
using an immersion-type cell. A pair of platinum electrodes, in a measuring vessel. After confirming that the temperature
the surfaces of which are coated with platinum black, is of the standard solution is maintained at 20 ± 0.19C or at
fixed in parallel. Both electrodes are generally protected by a the temperature specified in the monograph, measure the
glass tube to prevent physical shocks. resistance RKCl or the conductance GKCl of the standard solu-
When the surface area of the electrode is A (cm2), and the tion, and calculate the cell constant C (cm－1) by use of the
separation distance of the two electrodes is l (cm), the cell following equation.
constant C (cm－1) is given by the following equation.
C ＝ RKCl・kKCl or C ＝ kKCl/GKCl
C ＝ a・(l/A)
RKCl: Measured resistance (MQ)
a is a dimensionless numerical coefficient, and it is charac- GKCl: Measured conductance ( mS)
teristic of the cell design. kKCl: Conductivity of the standard solution being used
In addition to the immersion-type cell, there are flow- ( mS・cm－1)
through-type and insert-in-pipe-type cells. These cells are set
The measured cell constant should be consistent with the
or inserted in an appropriate position in the flow system for
given value within 5z. If it is not consistent, coat the elec-
monitoring the quality of water continuously or intermit-
trodes with platinum black again, or replace the cell with a
tently, during the preparation of highly purified water.
new one.

3.2. Suitability Test for the Apparatus romagnetic substance such as nickel. In the case of an instru-
Using an appropriate KCl standard solution according to ment capable of simultaneously conducting TG/TGA and
the expected conductivity of the sample solution, perform Differential Thermal Analysis (DTA), the same certified ref-
the suitability test for the apparatus. Rinse the conductivity erence materials as those for Differential Scanning Calori-
cell several times with distilled water, and rinse again 2 – 3 metry (DSC) and DTA may be used, such as indium for ther-
times with the selected standard solution. Fill the standard mal analysis, tin for thermal analysis and/or zinc (standard
solution in the measuring vessel. After confirming that the reagent).
temperature of the measuring system is maintained at 20 1.3. Calibration of the electrobalance
± 0.19C, measure the conductivity of the standard solution. Place an appropriate quantity of Calcium Oxalate
When this measuring procedure is repeated several times, the Monohydrate RS for Calibration of Apparatus or a suita-
average conductivity should be consistent with an indicated ble certified reference material in the sample holder and
value in Table 1 within 5z. Further, the relative standard record the mass. Set the heating rate according to the manu-
deviation should be less than 2z. facturer's instructions (e.g. 59C/min) and start the tempera-
3.3. Measurement ture increase. Record the thermogravimetric curve as a graph
After confirmation of the suitability of the apparatus, per- with temperature, or time, on the abscissa, increasing from
form the conductivity measurement for the sample solution. left to right, and mass on the ordinate, decreasing down-
Unless otherwise specified, the preparation method for sam- wards. Stop the temperature increase at about 2509C. Meas-
ple solution should be as specified in the respective mono- ure the difference on the graph between the initial and final
graph. Rinse the conductivity cell several times with distilled mass-temperature plateaux, or mass-time plateaux, which
water, and rinse again 2 – 3 times with sample solution. Im- corresponds to the loss of mass. The declared loss of mass
merse the cell in the sample solution placed in a measuring for the certified reference material is stated on the label.
vessel. If necessary, agitate gently the sample solution. After 1.4. Method
confirming that the temperature of the sample solution is Apply the same procedure to the substance to be exam-
maintained at 20 ± 0.19C or at the temperature specified in ined, using the conditions prescribed in the monograph. Cal-
the monograph, measure the resistance RT (MQ) or conduc- culate the loss of mass of the substance to be examined from
tance GT ( mS) of the sample solution, and calculate the con- the difference measured in the graph obtained. Express the
ductivity kT by using the following equation. loss of mass as Dm/m (z). If the instrument is in frequent
use, carry out temperature calibration regularly. Otherwise,
kT ＝ CGT or k T ＝ C/RT
carry out such checks before each measurement.
Since the conditions are critical, the following parameters
are noted for each measurement: pressure or flow rate, com-
2.52 Thermal Analysis position of the gas, mass of the sample, heating rate, temper-
ature range, sample pre-treatment including any isothermal
This test is harmonized with the European Pharmacopoeia period.
and the U. S. Pharmacopeia. The parts of the text that are
2. Differential Scanning Calorimetry
not harmonized are marked with symbols ( ).
Differential Scanning Calorimetry (DSC) is a technique
Thermal analysis is a group of techniques in which the that can be used to demonstrate the energy phenomena pro-
variation of a physical property of a substance is measured duced during heating (or cooling) of a substance (or a mix-
as a function of temperature. The most commonly used tech- ture of substances) and to determine the changes in enthalpy
niques are those which measure changes of mass or changes and specific heat and the temperatures at which these occur.
in energy of a sample of a substance. The technique is used to determine the difference in heat
These techniques have different applications: flow (with reference to the temperature) evolved or absorbed
—determination of phase changes, by the test sample compared with the reference cell, as a
—determination of changes in chemical composition, function of the temperature. Two types of DSC instruments
—determination of purity. are available, those using power compensation to maintain a
Among the below methods, Thermogravimetry can be null temperature difference between sample and reference
used as an alternative method for ``Loss on Drying <2.41>'' and those that apply a constant rate of heating and detect
or ``Water Determination <2.48>''. However, it must be temperature differential as a difference in heat flow between
confirmed beforehand that no volatile component except for sample and reference.
water is included in the test specimen when Thermogravi- 2.1. Instrument
metry is used as an alternative method for ``Water Determi- The instrument for the power compensation DSC consists
nation''. of a furnace containing a sample holder with a reference cell
and a test cell. The instrument for the heat flow DSC con-
1. Thermogravimetry
sists of a furnace containing a single cell with a sample hol-
Thermogravimetry (TG) or Thermogravimetric Analysis
der for the reference crucible and the test crucible.
(TGA) is a technique in which the mass of a sample of a sub-
A temperature-programming device, thermal detector(s)
stance is recorded as a function of temperature according to
and a recording system which can be connected to a com-
a controlled temperature programme.
puter are attached. The measurements are carried out under
1.1. Instrument
a controlled atmosphere.
The essential components of a thermobalance are a device
2.2. Calibration of the instrument
for heating or cooling the substance according to a given
Calibrate the instrument for temperature and enthalpy
temperature program, a sample holder in a controlled at-
change, using suitable certified materials or reference stand-
mosphere, an electrobalance and an electronic output of the
ards.
signal to a recorder or a computer.
2.2.1. Temperature calibration
1.2. Temperature calibration
It can be performed using certified reference materials
The temperature sensor close to or in contact with the
having an intrinsic thermal property, such as melting point
sample is calibrated using the Curie temperature of a fer-

Fig‚ 2.52-1 Thermogram Fig‚ 2.52-2 Thermal diagrams according to purity

For low amounts of impurities x2 ≪ 1 and for tempera- ment. This flow property of a liquid is expressed in terms of
tures close to the melting point T0 the equation can be writ- viscosity. The internal friction per unit area on the parallel
ten as follows, in which T and x2 are variables: plane is called slip stress or shear stress, and the velocity
gradient with respect to the direction rectangular to that of
RT 20
T = T0 － × x2 (1) flow is called slip velocity or shear velocity. A liquid of
D Hf
which the slip velocity is proportional to its slip stress is
T: temperature of the sample, in kelvins, called a Newtonian liquid. The proportionality constant, h,
T0: melting point of the chemically pure substance, in kel- is a characteristic of a liquid at a certain temperature and is
vins, called viscosity. The viscosity is expressed in the unit of Pas-
R: gas constant for ideal gases, in joules-kelvin－1･mole－1, cal second (Pa・s), and usually milli-Pascal second (mPa・s).
DHf: molar heat of fusion of the pure substance, in A liquid whose slip velocity is not proportional to its slip
joules・mole－1, stress is called a non-Newtonian liquid. Since the viscosity
x2: mole fraction of the impurity i.e. the number of molec- for a sample of a non-Newtonian liquid changes with its slip
ules of the impurity divided by the total number of velocity, the viscosity measured at a certain slip velocity is
molecules in the liquid phase (or molten phase) at tem- called an apparent viscosity. In that case, the value of slip
perature T (expressed in kelvins), stress divided by the corresponding slip velocity is called an
apparent viscosity. Thus, the relationship between apparent
Hence, the determination of purity by DSC is limited to
viscosity and slip velocity will permit characterization of the
the detection of impurities forming a eutectic mixture with
flow properties of a given non-Newtonian liquid.
the principal compound and present at a mole fraction of
The value of the viscosity, h, divided by the density, r, at
typically less than 2 per cent in the substance to be examined.
the same temperature is defined as a kinematic viscosity, n,
This method cannot be applied to:
which is expressed in the unit of meters squared per second
—amorphous substances,
(m2/s), and usually millimeters squared per second (mm2/s).
—solvates or polymorphic compounds that are unstable
The viscosity of a liquid is determined either by the follow-
within the experimental temperature range,
ing Method I or Method II.
—impurities forming solid solutions with the principal sub-
stance, 1. Method I Viscosity measurement by capillary tube vis-
—impurities that are insoluble in the liquid phase or in the cometer
melt of the principal substance. For measuring the viscosity of a Newtonian liquid, a capil-
During the heating of the substance to be examined, the lary tube viscometer is usually used, in which the downflow-
impurity melts completely at the eutectic temperature. ing time of a liquid, t (s), required for a definite volume of
Above this temperature, the solid phase contains only the the liquid to flow through a capillary tube is measured and
pure substance. As the temperature increases progressively the kinematic viscosity, n, is calculated according to the fol-
from the eutectic temperature to the melting point of the lowing equation.
pure substance, the mole fraction of impurity in the liquid
n ＝ Kt
decreases, since the quantity of liquefied pure substance in-
creases. For all temperatures above the eutectic point: Further, the viscosity, h, is calculated from the next equa-
tion:
1
x2 = × x2 (2)
F h ＝ nr ＝ Ktr
F: molten fraction of the analyzed sample, where r (g/mL) is the density of the liquid measured at the
x2*: mole fraction of the impurity in the analyzed sample. same temperature, t (9C).
The parameter K (mm2/s2) represents the viscometer con-
When the entire sample has melted, F = 1 and x2 = x2*.
stant and is previously determined by using the Standard
If equation (2) is combined with equation (1), the following
Liquids for Calibrating Viscometers with known kinematic
equation is obtained:
viscosity. In the case of a liquid having a similar viscosity to
RT 20 1 water, water itself can be used as a reference standard liquid
T = T0 － × × x2*
DH f F for the calibration. The kinematic viscosity of water is
1.0038 mm2/s at 209C. In the cases of liquids having a
The value of the heat of fusion of the pure substance is ob-
slightly higher viscosity than water, the Standard Liquids
tained by integrating the melting peak. The melting point T0
for Calibrating Viscometers should be used for the calibra-
of the pure substance is extrapolated from the plot of tem-
tion.
perature T, expressed in kelvins versus 1/F. The slope a of
The intrinsic viscosity, [h] (dL/g), of a polymer solution is
the curve, obtained after linearization, if necessary, corre-
obtained by plotting the relation of viscosity versus concen-
sponding to RT 20 x2*/DHf allows x2* to be evaluated. The
tration and extrapolating the obtained straight line to zero
fraction x2* multiplied by 100 gives the mole fraction in per
concentration. Intrinsic viscosity shows the degree of mo-
cent for the total eutectic impurities.
lecular expansion of a polymer substance in a given solvent
(sample solution) and is also a measure of the average mo-
lecular mass of the polymer substance.
2.53 Viscosity Determination The downflowing time t (s) for a polymer solution, whose
concentration is c (g/dL), and t0 (s) for the solvent used for
Viscosity Determination is a method to determine the vis- dissolving the polymer, are measured by using the same vis-
cosity of liquid samples using a viscometer. cometer, and then the intrinsic viscosity of a given polymer
When a liquid moves in a definite direction, and the liquid substance, [h], is calculated according to the following equa-
velocity has a gradient with respect to the direction rectangu- tion:
lar to that of flow, a force of internal friction is generated
along both sides of a hypothetical plane parallel to the move-

Ø »
t t sample solution flow down through the capillary tube.
－1 ln Record the time, t (s), required for the meniscus of the sam-
t0 t0
[h] ＝ lim or [h] ＝ lim ple solution to fall from the upper to the lower marked line
c→0 c c→0 c of bulb B.
When the concentration dependency of {(t/t0) － 1}/c is Determine the viscometer constant K previously, using the
not large, the value of {(t/t0) － 1}/c at a concentration di- Standard Liquids for Calibrating Viscometers under the
rected in the respective monograph can be assumed to be the same conditions. The temperature at which the calibration is
intrinsic viscosity for a given substance. conducted must be identical with that specified in the mono-
Unless otherwise specified, the viscosity of a sample solu- graph.
tion is measured with the following apparatus and proce-
2. Method II Viscosity measurement by rotational vis-
dure.
cometer
1.1. Apparatus
A rotational viscometer is usually used for measuring the
For measurement of the kinematic viscosity in the range of
viscosity of Newtonian or non-Newtonian liquids. The meas-
1 to 100,000 mm2/s, the Ubbelohde-type viscometer illus-
uring principle of a rotational viscometer generally consists
trated in Fig. 2.53-1 can be used. The approximate relations
in the detection and determination of the force acting on a
between kinematic viscosity range and inside diameter of the
rotor (torque), when it rotates at a constant angular velocity
capillary tube suitable for the measurement of various
in a liquid. The extent of torque generated by the rotation
liquids with different viscosity, are given in Table 2.53-1.
can be detected in terms of the torsion of a spring and the
Although a capillary tube viscometer other than the Ub-
liquid viscosity is calculated from the scale-indicated value
belohde-type one specified in Table 2.53-1 can also be used,
corresponding to the degree of torsion.
a viscometer should be selected in which the downflowing
The viscosity of a sample solution is measured with the
time, t (s), of a sample solution to be determined would be
following apparatus and procedure.
between 200 s and 1000 s.
2.1. Apparatus
1.2. Procedure
Viscosity measurement is performed by using any one of
Place a sample solution in a viscometer from the upper
the following three types of rotational viscometers.
end of tube 1, so that the meniscus of the solution is at a
2.1.1. Coaxial double cylinder-type rotational viscometer
level between the two marked lines of bulb A. Place the vis-
(Couette type viscometer)
cometer vertically in a thermostatted bath maintained at a
In the coaxial double cylinder-type rotational viscometer,
specified temperature within 0.19C, until bulb C is fully im-
viscosity is determined by placing a liquid in the gap between
mersed, and let it stand for about 20 minutes to attain the
the inner and the outer cylinders, which share the same cen-
specified temperature. Close tube 3 with a finger and pull the
tral axis and rotate separately, and the generated torque act-
sample solution up to the middle part of bulb C by gentle
ing on one cylinder surface when the other cylinder is rotat-
suction from the top of tube 2, taking care not to introduce
ed, and the corresponding angular velocity, are measured.
any bubbles into tube 2, and stop the suction. Open the end
As shown in Fig. 2.53-2a, the inner cylinder is hung by a
of tube 3, and immediately close the end of tube 2. After
wire whose twist constant is designated as k. In Fig. 2.53-2a,
confirming that the liquid column is cut off at the lowest end
half the outer diameter of the inner cylinder and inner diam-
of the capillary tube, open the end of tube 2 to make the
eter of the outer cylinder are designated as Ri and Ro, respec-
tively, and the length of the inner cylinder immersed in a
liquid is designated as l. When a liquid is introduced into the
gap between the two cylinders, and the outer cylinder is
made to rotate at a constant angular velocity, v, the inner
cylinder is also forced to rotate due to the viscosity of the

Table 2.53-1 Specifications of the Ubbelohde-type

viscometer
Inner diameter Volume of
Measuring
Viscometer of capillary bulb B
range of
constant tube (mm) (mL)
kinematic
K Permissible Permissible
viscosity
(mm2/s2) tolerance tolerance
(mm2/s)
±10z ±10z

0.005 0.46 3.0 1– 5

0.01 0.58 4.0 2– 10
0.03 0.73 4.0 6– 30
0.05 0.88 4.0 10 – 50
0.1 1.03 4.0 20 – 100
0.3 1.36 4.0 60 – 300
0.5 1.55 4.0 100 – 500
1.0 1.83 4.0 200 – 1,000
3.0 2.43 4.0 600 – 3,000
5.0 2.75 4.0 1,000 – 5,000
10.0 3.27 4.0 2,000 – 10,000
30.0 4.32 4.0 6,000 – 30,000
50.0 5.20 5.0 10,000 – 50,000
Fig. 2.53-1 Ubbelohde-type viscometer 100 6.25 5.0 20,000 – 100,000

Fig. 2.53-2a Coaxial double cylinder-type rotational

viscometer
Fig. 2.53-2b Single cylinder-type rotational viscometer

liquid. Consequently, torque, T, is generated by the forced

rotation in a viscous liquid, and in the steady state the torque
is balanced by the torsion of the wire, as indicated by the
degree of rotation u. Then, the relationship can be expressed
by T ＝ ku and the viscosity of a liquid, h, is determined
from the following equation by measuring the relationship
between v and u. Conversely, viscosity measurement can
also be performed by rotating the inner cylinder, and the
same relationship holds.

h＝
100T
4plv « 1
Ri2
－
1
Ro2 $
h: Viscosity of a liquid (mPa・s)
p: Circumference/diameter ratio Fig. 2.53-2c Cone-flat plate-type rotational viscometer
l: Length of the inner cylinder (cm)
v: Angular velocity (rad/s)
T: Torque acting on cylinder surface (10－7 N・m)
Ri: 1/2 of outer diameter of the inner cylinder (cm) gap between a flat disc and a cone forming an angle a(rad).
Ro: 1/2 of inner diameter of the outer cylinder (cm) When either the flat disc or the cone is rotated at a constant
angular velocity or a constant torque, the torque acting on
2.1.2. Single cylinder-type rotational viscometer (Brook- the disc or cone surface rotated by the viscous flow and the
field type viscometer) corresponding angular velocity in the steady state, are meas-
In the single cylinder-type rotational viscometer, viscosity ured. The viscosity of the liquid, h, can be calculated from
is determined by measuring the torque acting on the cylinder the following equation.
surface when the cylinder immersed in a liquid is rotated at a
3a 100T
given angular velocity. Use an apparatus of the type illus- h＝・
2pR3 v
trated in Fig. 2.53-2b. If the apparatus constant KB is previ-
ously determined experimentally by using the Standard Liq- h: Viscosity of a liquid (mPa・s)
uids for Calibrating Viscometers, the viscosity of a liquid, h, p: Circumference/diameter ratio
can be obtained from the following equation. R: Radius of cone (cm)
a: Angle between flat disc and cone (rad)
T
h ＝ KB v: Angular velocity (rad/s)
v
T: Torque acting on flat disc or cone surface (10－7 N・m)
where, h: viscosity of a liquid (mPa・s)
2.2. Procedure
KB: Apparatus constant of viscometer (rad/cm3)
Set up the viscometer so that its rotational axis is perpen-
v: Angular velocity (rad/s)
dicular to the horizontal plane. Place a sufficient quantity of
T: Torque acting on cylinder surface (10－7 N・m)
a sample solution in the viscometer, and allow the measuring
2.1.3. Cone-flat plate-type rotational viscometer (Cone- system to stand until a specified temperature is attained, as
plate type viscometer) directed in the monograph. Where it is desired to measure
In the cone-flat plate-type rotational viscometer, viscosity the viscosity within a precision of 1z, measuring tempera-
is determined by placing a liquid in the gap between a flat ture should be controlled within 0.19C. Next, after confirm-
disc and a cone with a large vertical angle sharing the same ing that the sample solution is at the designated temperature,
rotational axis, and the torque and the corresponding angu- start operating the rotational viscometer. After the forced
lar velocity are measured, when either the disc or the cone is rotation induced by the viscous flow has reached a steady
rotated in a viscous liquid. state and the indicated value on the scale, which corresponds
As shown in Fig. 2.53-2c, a liquid is introduced to fill the to the rotational frequency or the torque, has become con-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
74 2.54 pH Determination / General Tests JP XVII
stant, read the value on the scale. Then, calculate the vis- Table 2.54-1 Temperature dependency of the electromotive
cosity h by using the respective equation appropriate to the force (V)
type of viscometer being used. Determination or confirma-
Temperature Temperature
tion of the apparatus constant should be conducted before- of solution 2.3026 RT/F of solution 2.3026 RT/F
hand by using the Standard Liquids for Calibrating Viscom- (9C) (V) (9C) (V)
eters, and the validation of the apparatus and operating
5 0.05519 35 0.06114
procedure should also be performed by using those standard 10 0.05618 40 0.06213
liquids. 15 0.05717 45 0.06313
In the case of a non-Newtonian liquid, repeat the proce- 20 0.05817 50 0.06412
dure for measuring the viscosity of the liquid with variation 25 0.05916 55 0.06511
of the rotation velocity or torque from one measurement to 30 0.06015 60 0.06610
another. From a series of such viscosity measurements, the
relationship between the slip velocity and the slip stress of a
non-Newtonian liquid, i.e., the flow characteristics of a non-
Newtonian liquid, can be obtained. Table 2.54-2 pH values of six pH standard solutions
Calibration of a rotational viscometer is conducted by
using water and the Standard Liquids for Calibrating Vis- Calcium
Oxalate Phthalate Phosphate Borate Carbonate hydroxide
Temper- pH pH pH pH pH
cometers. These standard liquids are used for the determina- ature pH
standard standard standard standard standard standard
tion or confirmation of the apparatus constant of the rota- (9C) solution solution solution solution solution solution
tional viscometer. They are also used for periodic recalibra-
tion of the viscometer to confirm maintenance of a specified 0 1.67 4.01 6.98 9.46 10.32 13.43
precision. 5 1.67 4.01 6.95 9.39 10.25 13.21
10 1.67 4.00 6.92 9.33 10.18 13.00
15 1.67 4.00 6.90 9.27 10.12 12.81
20 1.68 4.00 6.88 9.22 10.07 12.63
2.54 pH Determination 25 1.68 4.01 6.86 9.18 10.02 12.45
30 1.69 4.01 6.85 9.14 9.97 12.30
pH is defined as the reciprocal of the common logarithm 35 1.69 4.02 6.84 9.10 9.93 12.14
of hydrogen ion activity, which is the product of hydrogen 40 1.70 4.03 6.84 9.07 11.99
ion concentration and the activity coefficient. Conventional- 50 1.71 4.06 6.83 9.01 11.70
60 1.73 4.10 6.84 8.96 11.45
ly it is used as a scale of hydrogen ion concentration of a
sample solution.
pH of a sample solution is expressed by the following
equation in relation to the pH of a standard solution (pHs),
and can be measured by a pH meter using a glass electrode.
lene bottles. For storage of alkaline pH standard solutions, it
E － Es is preferable to use a bottle fitted with a carbon dioxide-
pH ＝ pHs ＋
2.3026 RT/F absorbing tube. Since the pH may change gradually during
storage over a long period, it is necessary to acertain whether
pHs: pH value of a pH standard solution.
the expected pH value is held or not by comparison with
E: Electromotive force (volt) induced on the following
newly prepared standard, when the solution is used after
galvanic cell composed of a glass electrode and suitable
long storage.
reference electrode in a sample solution:
(i) Oxalate pH standard solution—Pulverize potassium
Glass electrode|sample solution|reference electrode trihydrogen dioxalate dihydrate for pH determination, and
dry in a desiccator (silica gel). Weigh 12.71 g (0.05 mole) of
Es: Electromotive force (volt) induced on the following
it accurately, and dissolve in water to make exactly 1000 mL.
galvanic cell composed of a glass electrode and suita-
(ii) Phthalate pH standard solution—Pulverize potas-
ble reference electrode in a pH standard solution:
sium hydrogen phthalate for pH determination, and dry at
Glass electrode|standard pH solution|reference electrode 1109C to constant mass. Weigh 10.21 g (0.05 mole) of it ac-
curately, and dissolve in water to make exactly 1000 mL.
R: Gas constant
(iii) Phosphate pH standard solution—Pulverize potas-
T: Absolute temperature
sium dihydrogen phosphate for pH determination and diso-
F: Faraday's constant
dium hydrogen phosphate for pH determination, and dry at
The value of 2.3026 RT/F (V) in the above equation 1109C to constant mass. Weigh 3.40 g (0.025 mole) of potas-
means the degree of electromotive force (V) per one pH unit sium dihydrogen phosphate and 3.55 g (0.025 mole) of diso-
and it is dependent on the temperature as shown in Table dium hydrogen phosphate accurately, and dissolve in water
2.54-1: to make exactly 1000 mL.
(iv) Borate pH standard solution—Allow sodium
1. pH Standard solution
tetraborate for pH determination to stand in a desiccator
The pH standard solutions are used as a standard of pH,
(saturated sodium bromide aqueous solution) until it reaches
for standardization of a pH meter. To prepare the pH stand-
constant mass. Weigh 3.81 g (0.01 mole) of it accurately, and
ard solutions, use distilled water or water with a conductivity
dissolve in water to make exactly 1000 mL.
not more than 2 mS・cm－1 (259C) and an organic carbon not
(v) Carbonate pH standard solution—Dry sodium
more than 0.50 mg/L, boiled for not less than 15 minutes
hydrogen carbonate for pH determination in a desiccator
and cooled in a container fitted with a carbon dioxide-
(silica gel) to constant mass, and weigh 2.10 g (0.025 mole)
absorbing tube (soda lime). Next, prepare individually 6
of it accurately. Dry sodium carbonate for pH determination
kinds of pH standard solutions shown in Table 2.54-2.
between 3009 C and 5009 C to constant mass, and weigh
Store the pH standard solutions in hard glass or polyethy-
2.65 g (0.025 mole) of it accurately. Dissolve both reagents in

water to make exactly 1000 mL. alkali metal ions, an alkali error may be induced in the pH
(vi) Calcium hydroxide pH standard solution—Reduce measurement. Thus, in such a case, an electrode with less
calcium hydroxide for pH determination to a fine powder, alkali error should be used and an appropriate correction
transfer 5 g to a flask, add 1000 mL of water, shake well, should be applied to the measured value.
and allow the solution to become saturated at a temperature
4. Note
between 239C and 279C. Then filter the supernatant at the
Construction and treatment in detail are different for
same temperature and use the clear filtrate (about 0.02
different pH meters.
mol/L).
The pH values of these pH standard solutions at various
temperatures are shown in the Table 2.54-2. pH values at an
arbitrary temperature not indicated in Table 2.54-2 can be 2.55 Vitamin A Assay
calculated by the interpolation method.
Vitamin A Assay is a method to determine vitamin A in
2. Apparatus
Retinol Acetate, Retinol Palmitate, Vitamin A Oil, Cod
A pH meter generally consists of an electrode system of a
Liver Oil and other preparations. Method 1 is for the assay
glass electrode and a reference electrode, an amplifier and an
of synthetic vitamin A esters, using the ultraviolet-visible
indicating unit for controlling the apparatus and for dis-
spectrophotometry (Method 1-1) or the liquid chromatogra-
playing the measured value of electromotive force. The indi-
phy (Method 1-2). Method 2 is for the assay of vitamin A of
cating unit is usually fitted with dials for zero and span (sen-
natural origin, containing many geometrical isomers, using
sitivity) adjustment. Sometimes a temperature compensation
the ultraviolet-visible spectrophotometry to determine vita-
dial is included.
min A as vitamin A alcohol obtained by saponification in an
The reproducibility of a pH meter should be within 0.05
alkaline solution and extraction.
pH unit, when measurements for an arbitrary pH standard
One Vitamin A Unit (equal to 1 vitamin A I.U.) is equiva-
solution are repeated five times, following the procedure
lent to 0.300 mg of vitamin A (all-trans vitamin A alcohol).
described below. After each measurement it is necessary to
wash the detecting unit well with water. 1. Procedure
All procedures should be carried out quickly and care
3. Procedure
should be taken as far as possible to avoid exposure to light,
Immerse the glass electrode previously in water for more
air, oxidants, oxidizing catalysts (e.g. copper, iron), acids
than several hours. Start the measurement after confirming
and heat. If necessary, light-resistant vessels may be used.
stable running of the apparatus. Rinse well the detecting unit
Generally, for synthetic vitamin A esters apply Method 1-1
with water, and remove the remaining water gently with a
or Method 1-2, but if the assay conditions required for
piece of filter paper.
Method 1-1 are not suitable, apply Method 2.
To standardize the pH meter, two pH standard solutions
1.1. Method 1-1
are usually used as follows. Immerse the detection unit in the
Weigh accurately about 0.1 g of the sample, and dissolve
phosphate pH standard solution and adjust the indicated pH
in 2-propanol for vitamin A assay to make exactly 50 mL.
to the pH value shown in the Table. Next, immerse the de-
Dilute this solution with 2-propanol for vitamin A assay to
tection system in the second pH standard solution, which
make a solution so that each mL contains 10 to 15 vitamin A
should be selected so that the expected pH of the sample so-
Units, and use this solution as the sample solution. Deter-
lution to be determined is between the pH values of the two
mine the absorption spectrum of the sample solution be-
pH standard solutions, and measure the pH under the same
tween 220 nm and 400 nm as directed under Ultraviolet-
conditions as used for the first pH standard solution. Adjust
visible Spectrophotometry <2.24> to obtain the wavelength of
the indicated pH to the defined pH value using the span
the maximum absorption and the absorbances at 300 nm,
adjustment dial, when the observed pH is not identical with
310 nm, 320 nm, 326 nm, 330 nm, 340 nm and 350 nm.
that tabulated. Repeat the above standardization procedure
When the maximum absorption lies between 325 nm and 328
until both pH standard solutions give observed pH values
nm, and the ratios, Ali/A326, of each absorbance, Ali, at 300
within 0.02 pH unit of the tabulated value without further
nm, 310 nm, 320 nm, 330 nm, 340 nm and 350 nm to the ab-
adjustments. When a pH meter is fitted with a temperature
sorbance, A326, at 326 nm are within the range of ±0.030 of
compensation dial, the standardization procedure is done
the values in the Table 2.55-1, the potency of vitamin A in
after the setting of the temperature to that of the pH stand-
Units per g of the sample is calculated from the following
ard solution to be measured.
equation.
In the case of using an apparatus having an auto-calibra-
tion function, it is necessary to confirm periodically that the
pH values of two pH standard solutions are identical with
the tabulated values within 0.05 pH unit.
After finishing the standardization procedure described
above, rinse well the electrodes with water, remove the at- Table 2.55-1 Absorbance ratio, Ali/A326, of retinol acetate
tached water using a filter paper, immerse the electrode sys- and retinol palmitate
tem in the sample solution, and read the indicated pH value Ali/A326
after confirming the value is stable. If necessary, a sample li (nm)
solution can be agitated gently. Retinol acetate Retinol palmitate
In the pH determination, the temperature of a sample so-
300 0.578 0.590
lution must be controlled to be the same as that of the pH
standard solutions with which the pH meter was stand- 310 0.815 0.825
320 0.948 0.950
ardized (within 29 C). When a sample solution is alkaline, the
330 0.972 0.981
measurement should be done in a vessel with a cover and if
340 0.786 0.795
necessary, in a stream of inert gas such as nitrogen. Further-
350 0.523 0.527
more for a strongly alkaline solution above pH 11 containing

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
76 2.56 Determination of Specific Gravity and Density / General Tests JP XVII
A326 V Units of vitamin A in 1 g of the sample
Units of vitamin A in 1 g ＝ × × 1900
M 100 A325 V
＝ × × f × 1830
A326: Absorbance at 326 nm M 100
V: Total volume (mL) of the sample solution
A310 A334
M: Amount (g) of sample in V mL of the sample solution f ＝ 6.815 － 2.555 × － 4.260 ×
A325 A325
1900: Conversion factor from specific absorbance of
retinol ester to IU (Unit/g) A325: Absorbance at 325 nm
V: Total volume (mL) of the sample solution
This method is applied to drugs or preparations containing
M: Amount (g) of sample in V mL of the sample solution
vitamin A esters (retinol acetate or retinol palmitate) as the
f: Correction factor
main component. However, when the wavelength of maxi-
1830: Conversion factor from specific absorbance of
mum absorption does not lie between 325 nm and 328 nm, or
retinol alcohol to IU (Unit/g)
when the absorbance ratio Ali/A326 is not within the range of
±0.030 of the values in Table 2.55-1, apply Method 2.
1.2. Method 1-2
Proceed with an appropriate amount of sample as directed 2.56 Determination of Specific
under Liquid Chromatography <2.01>.
For the assay of retinol acetate and retinol palmitate use Gravity and Density
Retinol Acetate Reference Standard and Retinol Palmitate
The density r (g/mL or g/cm3) means the mass per unit
Reference Standard, respectively, and fix appropriately the
volume, and the relative density means the ratio of the mass
operating procedure, the operating conditions and the sys-
of a sample specimen to that of an equal volume of a stand-
tem suitability based on the characteristics of the substance
ard substance. The relative density is also called the specific
to be tested and the species and amount of coexisting sub-
gravity.
stances. ?
The specific gravity, d tt , means the ratio of the mass of the
1.3. Method 2
sample specimen at t?9C to that of an equal volume of water
Unless otherwise specified, weigh accurately a sample con-
(H2O) at t9C . Unless otherwise specified, the measurement
taining not less than 500 Units of vitamin A, and not more
is to be performed by Method 1, Method 2 or Method 4.
than 1 g of fat, transfer to a flask, and add 30 mL of alde-
When the specified value is accompanied with the term
hyde-free ethanol and 1 mL of a solution of pyrogallol in
``about'' in the monograph, Method 3 is also available.
ethanol (95) (1 in 10). Then add 3 mL of a solution of potas-
sium hydroxide (9 in 10), attach a reflux condenser, and heat 1. Method 1. Measurement using a pycnometer
on a water bath for 30 minutes to saponify. Cool quickly to A pycnometer is a glass vessel with a capacity of usually 10
ordinary temperature, add 30 mL of water, transfer to a mL to 100 mL, having a ground-glass stopper fitted with a
separator A, wash the flask with 10 mL of water and then 40 thermometer, and a side inlet-tube with a marked line and a
mL of diethyl ether, transfer the washings to the separator ground-glass cap.
A, shake well, and allow to stand. Transfer the water layer Weigh a pycnometer, previously cleaned and dried, to de-
so obtained to a separator B, wash the flask with 30 mL of termine its mass M. Remove the stopper and the cap. Fill the
diethyl ether, add the washing to the separator B, and extract pycnometer with the sample solution, keeping them at a
by shaking. Transfer the water layer to a flask, add the slightly lower temperature by 19C to 39C than the specified
diethyl ether layer to the separator A, transfer the water temperature t?9 C, and stopper them, taking care not to leave
layer in the flask to the separator B, add 30 mL of diethyl bubbles. Raise the temperature gradually, and when the ther-
ether, and extract by shaking. Transfer the diethyl ether mometer shows the specified temperature, remove the por-
layer so obtained to the separator A, add 10 mL of water, tion of the sample solution above the marked line through
allow the separator A to stand after gentle turning upside- the side tube, cap the side tube, and wipe the outside surface
down 2 or 3 times, and remove the water layer. Wash the thoroughly. Measure the mass M1 of the pycnometer filled
content of the separator A with three 50-mL portions of with the sample solution. Perform the same procedure, using
water with increasingly vigorous shaking as the washing the same pycnometer containing water, and note the mass
?
proceeds. Further wash with 50-mL portions of water until M2 at the specified temperature t9 C. The specific gravity d tt
the washing no longer shows a pink color with phenolphthal- can be calculated by use of the following equation.
ein TS, and allow to stand for 10 minutes. Remove remain- M1 － M
?
ing water as far as possible, transfer the diethyl ether to an d tt ＝
Erlenmeyer flask, wash the separator with two 10-mL por- M2 － M
tions of diethyl ether, add the washings to the flask, add 5 g Further, when measurements for a sample solution and
of anhydrous sodium sulfate to the flask, mix by shaking, water are performed at the same temperature (t9C ＝ t?9C),
and transfer the diethyl ether to a round-bottomed flask by the density of the sample solution at the temperature t?9C
? ?
decantation. Wash the remaining sodium sulfate in the flask ( r tT) can be calculated from the measured specific gravity d tt?
? t?
with two or more 10-mL portions of diethyl ether, and trans- and the density of water at the temperature t 9C ( r S1) indi-
fer the washings to the flask. Evaporate the diethyl ether in a cated in Table 2.56-1 by using the following equation.
water bath at 459C while swirling the flask, using an aspira- ? ? ?
r tT ＝ r tS1 d tt?
tor, to about 1 mL, immediately add an exactly measured
amount of 2-propanol for vitamin A assay to make a solu- 2. Method 2. Measurement using a Sprengel-Ostwald
tion containing 6 to 10 vitamin A Units per mL, and desig- pycnometer
nate the solution as the sample solution. Determine the ab- A Sprengel-Ostwald pycnometer is a glass vessel with a
sorbances, A310 at 310 nm, A325 at 325 nm, and A334 at 334 capacity of usually 1 mL to 10 mL. As shown in Fig. 2.56-1,
nm, of the sample solution as directed under Ultraviolet- both ends are thick-walled fine tubes (inside diameter: 1 –
visible Spectrophotometry. 1.5 mm, outside diameter: 3 – 4 mm), one of which, tube A,
has a line C marked on it. Determine the mass of a pycnome-

Table 2.56-1 Density of water

Temp. Density Temp. Density Temp. Density Temp. Density
9C g/mL 9C g/mL 9C g/mL 9C g/mL
0 0.999 84
1 0.999 90 11 0.999 61 21 0.997 99 31 0.995 34
2 0.999 94 12 0.999 50 22 0.997 77 32 0.995 03
3 0.999 96 13 0.999 38 23 0.997 54 33 0.994 70
4 0.999 97 14 0.999 24 24 0.997 30 34 0.994 37
5 0.999 96 15 0.999 10 25 0.997 04 35 0.994 03
6 0.999 94 16 0.998 94 26 0.996 78 36 0.993 68
7 0.999 90 17 0.998 77 27 0.996 51 37 0.993 33
8 0.999 85 18 0.998 60 28 0.996 23 38 0.992 97
9 0.999 78 19 0.998 41 29 0.995 94 39 0.992 59
10 0.999 70 20 0.998 20 30 0.995 65 40 0.992 22
 In this Table, although the unit of density is represented by g/mL
in order to harmonize with the unit expression in the text, it Fig. 2.56-1 Sprengel-Ostwald pycnometer
should be expressed in g/cm3 seriously.

trinsic vibration period T in proportion to the mass of the

ter, M, previously cleaned and dried, by hanging it on the sample specimen. If the volume of the vibrating part of the
arm of a chemical balance with a platinum or aluminum wire sample cell is fixed, the relation of the square of intrinsic os-
D. Immerse the fine tube B in the sample solution, which is cillation period and density of the sample specimen shall be
at a lower temperature by 39C to 59 C than the specified tem- linear.
perature t?9C. Attach rubber tubing or a ground-glass tube Before measuring a sample density, the respective intrinsic
to the end of A, and suck up the sample solution until the oscillation periods TS1 and TS2 for two reference substances
meniscus is above the marked line C, taking care to prevent (density: rS1, rS2) must be measured at a specified tempera-
bubble formation. Immerse the pycnometer in a water bath ture t?9C, and the cell constant Kt? (g・cm－3 s－2) must be de-
kept at the specified temperature t?9C for about 15 minutes, termined by using the following equation.
and then, by attaching a piece of filter paper to the end of B, ? ?
r tS1 － r tS2
adjust the level of the sample solution to the marked line C. Kt? ＝
TS12 － TS22
Take the pycnometer out of the water bath, wipe thoroughly
the outside surface and determine the mass M1. By use of the Usually, water and dried air are chosen as reference sub-
?
same pycnometer, perform the same procedure for the stances. Here the density of water at t?9C, r tS1, is taken from
t?
standard solution of water. Weigh the pycnometer contain- Table 2.56-1, and that of dried air r S2 is calculated by using
ing water at the specified temperature t9C, and note the mass the following equation, where the pressure of dried air is at p
?
M2. Calculate the specific gravity d tt , according to the equa- kPa.
tion described in Method 1. ?
r tS2 ＝ 0.0012932 × {273.15/(273.15 ＋ t?)} × ( p/101.325)
Further, when measurements of specific gravity for a sam-
ple solution and water are performed at the same tempera- Next, introduce a sample specimen into a sample cell hav-
ture (t?9C ＝ t9C), the density of sample solution at tempera- ing a cell constant Kt?, the intrinsic vibration period, TT, for
ture t?9C can be calculated by using the equation described in the sample under the same operation conditions as employed
Method 1. for the reference substances. The density of a sample speci-
?
men at t?9C, r tT, is calculated by use of the following equa-
3. Method 3. Masurement using a hydrometer
tion, by introducing the intrinsic oscillation period TS1 and
Clean a hydrometer with ethanol (95) or diethyl ether. Stir ?
the density of water at a specified temperature t?9C, r tS1, into
the sample well with a glass rod, and float the hydrometer in
the equation.
the well. When the temperature is adjusted to the specified
? ?
temperature t?9C and the hydrometer comes to a standstill, r tT ＝ r tS1 ＋ Kt? (TT2 － TS12 )
? ?
read the specific gravity d tt or the density r tT at the upper ?
Further, the specific gravity of a sample specimen d tt
brim of the meniscus. Here the temperature t9 C indicates the
against water at a temperature t9C can be obtained by using
temperature at which the hydrometer is calibrated. If specific
the equation below, by introducing the density of water at a
instructions for reading the meniscus are supplied with the
temperature t9 C, r tS1, indicated in Table 2.56-1.
hydrometer, the reading must be in accordance with the in-
?
structions. ? r tT
d tt ＝
Further, when measurement of the specific gravity for a r tS1
sample solution is performed at the same temperature (t?9C 4.1. Apparatus
＝ t9 C), at which the hydrometer is calibrated, the density of An oscillator-type density meter is usually composed of a
?
a sample solution at t?9C, r tT, can be calculated by using the glass tube cell of about 1 mL capacity, the curved end of
t?
specific gravity d t and the equation shown in Method 1. which is fixed to the vibration plate, an oscillator which ap-
plies an initial vibration to the cell, a detector for measuring
4. Method 4. Measurement using an oscillator-type den-
the intrinsic vibration period, and a temperature controlling
sity meter
system.
Density measurement with an oscillator-type density meter
A schematic illustration of the apparatus is depicted in
is a method for obtaining the density of liquid or gas by
Fig. 2.56-2.
measuring the intrinsic vibration period T (s) of a glass tube
4.2. Procedure
cell filled with sample specimen. When a glass tube contain-
A sample cell, water, and a sample specimen are previ-
ing a sample is vibrated, it undergoes a vibration with an in-

Fig. 2.56-2 Oscillator-type density meter

ously adjusted to a specified temperature t?9C. Wash the

sample cell with water or an appropriate solvent, and dry it
thoroughly with a flow of dried air. Stop the flow of dried Fig. 2.57-1
air, confirm that the temperature is at the specified value,
and then measure the intrinsic oscillation period TS2 given by
the dried air. Separately, the atmospheric pressure p (kPa)
must be measured at the time and place of the examination. mL, and transfer it to a distilling flask A of 50- to 60-mL
Next, introduce water into the sample cell and measure the capacity. Use this cylinder as the receiver for the distillate
intrinsic oscillation period TS1 given by water. Using these without rinsing out any of the adhering liquid. Put boiling
values of the intrinsic oscillation period and the atmospheric chips into the distilling flask A, insert a thermometer B with
pressure, the sample cell constant Kt? can be determined by an immersion line so that its immersion line C is on a level
use of the above-mentioned equation. with the lower end of cork stopper D and the upper end of
Next, introduce a sample specimen into the glass cell, con- its mercury bulb is located in the center of the delivery tube,
firm the specified temperature, and measure the intrinsic os- and connect condenser E with the distilling flask A and
cillation period TT given by the sample specimen. Using the adapter F with the condenser E. Insert the open end of F into
intrinsic oscillation periods for water and the sample speci- the mouth of cylinder G (receiver) so that air can pass
?
men, the density of water r tS1, and the cell constant Kt?, the through slightly. Use a hood with a height sufficient to shield
?
density of the sample specimen r tT can be obtained by use of A, and heat A with a suitable heat source. When direct flame
the above equation. If necessary, the specific gravity of the is applied as the heat source, put A on a hole of a fire-
?
sample specimen d tt against water at a temperature t9C, can resistant, heat-insulating board [a board consisting of a fire-
be calculated by using the density of water r tS1 shown in resistant, heat-insulating material, 150 mm square and about
Table 2.56-1. 6 mm thick (or a wire gauge of 150 mm square bonded to
In this measurement, avoid the occurrence of bubble for- fire-resistant, heat-insulation materials in about 6 mm thick-
mation in the sample cell, when a sample specimen or water ness), having an its center a round hole 30 mm in diameter].
is introduced into the cell. Unless otherwise specified, distil the liquid sample by the
application of heat, at a rate of 4 to 5 mL per minute of dis-
tillate in the case of liquids whose boiling temperature to be
determined is lower than 2009 C and at a rate of 3 to 4 mL
2.57 Boiling Point and per minute in the case of liquids whose boiling temperature is
Distilling Range Test 2009C or over, and read the boiling point. For the distilling
range test, bring the temperature of distillate to the tempera-
The boiling point and distilling range are determined by ture at which the volume was originally measured, and meas-
Method 1 or Method 2 as described herein, unless otherwise ure the volume of distillate.
specified. Boiling point is the temperature shown between Liquids that begin to distil below 809 C are cooled to be-
when the first 5 drops of distillate leave the tip of the con- tween 109C and 159C before measuring the volume, and the
denser and when the last liquid evaporates from the bottom receiving cylinder is kept immersed in ice up to a point 25
of the flask. Distilling range test is done to determine the mm from the top during the distillation.
volume of the distillate which has been collected in the range Correct the observed temperature for any variation in the
of temperature directed in the monograph. barometric pressure from the normal (101.3 kPa), by
allowing 0.1 degree for each 0.36 kPa of variation, adding if
1. Method 1 This method is applied to a sample for
the pressure is lower, or subtracting if higher than 101.3
which the permissible range of boiling temperature is smaller
kPa.
than 59C.
1.1. Apparatus 2. Method 2 This method is applied to the sample for
Use the apparatus illustrated in Fig. 2.57-1. which the permissible range of boiling temperature is 59C or
1.2. Procedure more.
Measure 25 mL of the sample, whose temperature is previ- 2.1. Apparatus
ously noted, using a volumetric cylinder G graduated in 0.1 The same apparatus as described in Method 1 is used.

However, use a 200-mL distilling flask A with a neck 18 to

24 mm in inside diameter having a delivery tube 5 to 6 mm in
inside diameter. The fire-resistant, heat-insulating board
used for direct flame heating should have in its center a
round hole 50 mm in diameter.
2.2. Procedure
Measure 100 mL of the sample, whose temperature is pre-
viously noted, using a volumetric cylinder graduated in 1
mL, and carry out the distillation in the same manner as in
Method 1.

2.58 X-Ray Powder Diffraction

Method
This test is harmonized with the European Pharmacopoeia Fig. 2.58-1 Diffraction of X-rays by a crystal according to
and the U. S. Pharmacopeia. The parts of the text that are Bragg's law
not harmonized are marked with symbols ( ).

X-Ray Powder Diffraction Method is a method for
integer, of the intercepts that a plane makes with the unit cell
measuring characteristic X-ray diffraction angles and inten-
axes. The unit cell dimensions are given by the spacings, a, b
sities from randomly oriented powder crystallites irradiated
and c and the angles between them, a, b and g. The inter-
by a monochromated X-ray beam.
planar spacing for a specified set of parallel hkl planes is
Every crystalline phase of a given substance produces a
denoted by dhkl. Each such family of planes may show higher
characteristic X-ray diffraction pattern. Diffraction patterns
orders of diffraction where the d values for the related
can be obtained from a randomly oriented crystalline pow-
families of planes, nh, nk, nl are diminished by the factor
der composed of crystallites or crystal fragments of finite
1/n (n being an integer: 2,3,4, etc.). Every set of planes
size. Essentially 3 types of information can be derived from a
throughout a crystal has a corresponding Bragg diffraction
powder diffraction pattern: angular position of diffraction
angle, hkl, associated with it (for a specific wavelength l).
lines (depending on geometry and size of the unit cell); inten-
A powder specimen is assumed to be polycrystalline so
sities of diffraction lines (depending mainly on atom type
that at any angle hkl there are always crystallites in an orien-
and arrangement, and particle orientation within the sam-
tation allowing diffraction according to Bragg's law(2). For a
ple); and diffraction line profiles (depending on instrumental
given X-ray wavelength, the positions of the diffraction
resolution, crystallite size, strain and specimen thickness).
peaks (also referred to as `lines', `reflections' or `Bragg
Experiments giving angular positions and intensities of
reflections') are characteristic of the crystal lattice (d-spac-
lines can be used for applications such as qualitative phase
ings), their theoretical intensities depend on the crystallo-
analysis (for example, identification of crystalline phases)
graphic unit cell content (nature and positions of atoms),
and quantitative phase analysis of crystalline materials. An
and the line profiles on the perfection and extent of the crys-
estimate of the amorphous and crystalline fractions(1) can
tal lattice. Under these conditions the diffraction peak has a
also be made. The X-ray powder diffraction (XRPD)
finite intensity arising from atomic arrangement, type of
method provides an advantage over other means of analysis
atoms, thermal motion and structural imperfections, as well
in that it is usually non-destructive in nature (specimen
as from instrument characteristics. The intensity is depen-
preparation is usually limited to grinding to ensure a ran-
dent upon many factors such as structure factor, tempera-
domly oriented sample). XRPD investigations can also be
ture factor, crystallinity, polarization factor, multiplicity
carried out under in situ conditions on specimens exposed to
and Lorentz factor. The main characteristics of diffraction
non-ambient conditions, such as low or high temperature
line profiles are 2 position, peak height, peak area and
and humidity.
shape (characterized by, for example, peak width or asym-
1. Principle metry, analytical function, empirical representation). An
X-ray diffraction results from the interaction between X- example of the type of powder patterns obtained for 5 differ-
rays and electron clouds of atoms. Depending on the atomic ent solid phases of a substance are shown in Fig. 2.58-2.
arrangement, interferences arise from the scattered X-rays. In addition to the diffraction peaks, an X-ray diffraction
These interferences are constructive when the path difference experiment also generates a more-or-less uniform back-
between 2 diffracted X-ray waves differs by an integral num- ground, upon which the peaks are superimposed. Besides
ber of wavelengths. This selective condition is described by specimen preparation, other factors contribute to the back-
the Bragg equation, also called Bragg's law (see Fig. 2.58-1) ground, for instance the sample holder, diffuse scattering
from air and equipment, other instrumental parameters such
2dhkl sin hkl ＝ nl
as detector noise, general radiation from the X-ray tube, etc.
The wavelength l of the X-rays is of the same order of The peak to background ratio can be increased by minimiz-
magnitude as the distance between successive crystal lattice ing background and by choosing prolonged exposure times.
planes, or dhkl (also called `d-spacings'). hkl is the angle be-
2. Instrument
tween the incident ray and the family of lattice planes, and
2.1. Instrument set-up
sin hkl is inversely proportional to the distance between suc-
X-ray diffraction experiments are usually performed using
cessive crystal planes or d-spacings.
powder diffractometers or powder cameras. A powder dif-
The direction and spacing of the planes with reference to
fractometer generally comprises 5 main parts: an X-ray
the unit cell axes are defined by the Miller indices {hkl}.
source; incident beam optics, which may perform mono-
These indices are the reciprocals, reduced to the next-lower

A. X-ray tube
B. Divergence slit
C. Sample
D. Anti-diffusion slit
E. Receiving slit
Fig. 2.58-2 X-ray powder diffraction patterns collected for F. Monochromator
5 different solid phases of a substance (the intensities are G. Detector receiving slit
normalized) H. Detector
J. Diffractometer circle
K. Focusing circle

Fig. 2.58-3 Geometric arrangement of the Bragg-Brentano

chromatization, filtering, collimation and/or focusing of the parafocusing geometry
beam; a goniometer; diffraction beam optics, which may
perform monochromatization, filtering, collimation and
focusing or parallelising of the beam; and a detector. Data
collection and data processing systems are also required and detector, usually a scintillation counter, a sealed-gas propor-
are generally included in current diffraction measurement tional counter, or a position-sensitive solid-state detector
equipment. such as imaging plate or CCD detector. The receiving slit as-
Depending on the type of analysis to be performed (phase sembly and the detector are coupled together and move tan-
identification, quantitative analysis, lattice parameters deter- gentially to the focusing circle. For /2 scans the goniome-
mination, etc.), different XRPD instrument configurations ter rotates the specimen about the same axis as that of the
and performance levels are required. The simplest instru- detector, but at half the rotational speed, in /2 motion.
ments used to measure powder patterns are powder cameras. The surface of the specimen thus remains tangential to the
Replacement of photographic film as the detection method focusing circle. The parallel plate collimator limits the axial
by photon detectors has led to the design of diffractometers divergence of the beam and hence partially controls the
in which the geometric arrangement of the optics is not truly shape of the diffracted line profile.
focusing but parafocusing, such as in the Bragg-Brentano A diffractometer may also be used in transmission mode.
geometry. The Bragg-Brentano parafocusing configuration The advantage with this technology is to lessen the effects
is currently the most widely used and is therefore briefly due to preferred orientation. A capillary of about 0.5 – 2
described here. mm thickness can also be used for small sample amounts.
A given instrument may provide a horizontal or vertical 2.2. X-ray radiation
/2 geometry or a vertical / geometry. For both geo- In the laboratory, X-rays are obtained by bombarding a
metries, the incident X-ray beam forms an angle with the metal anode with electrons emitted by the thermionic effect
specimen surface plane and the diffracted X-ray beam forms and accelerated in a strong electric field (using a high-voltage
an angle 2 with the direction of the incident X-ray beam (an generator). Most of the kinetic energy of the electrons is
angle with the specimen surface plane). The basic geo- converted to heat, which limits the powder of the tubes and
metric arrangement represented in Fig. 2.58-3. The divergent requires efficient anode cooling. A 20- to 30-fold increase in
beam of radiation from the X-ray tube (the so-called `pri- brilliance can be obtained using rotating anodes and by using
mary beam') passes through the parallel plate collimators X-ray optics. Alternatively, X-ray photons may be produced
and a divergence slit assembly and illuminates the flat sur- in a large-scale facility (synchrotron).
face of the specimen. All the rays diffracted by suitably The spectrum emitted by an X-ray tube operating at
oriented crystallites in the specimen at an angle 2 converge sufficient voltage consists of a continuous background of
to a line at the receiving slit. A second set of parallel plate polychromatic radiation and additional characteristic radia-
collimators and a scatter slit may be placed either behind or tion that depends on the type of anode. Only this character-
before the receiving slit. The axes of the line focus and of the istic radiation is used in X-ray diffraction experiments. The
receiving slit are at equal distances from the axis of the principal radiation sources utilized for X-ray diffraction are
goniometer. The X-ray quanta are counted by a radiation vacuum tubes utilizing copper, molybdenum, iron, cobalt or

chromium as anodes; copper, molybdenum or cobalt X-rays (crystallite sizes less than approximately 0.5 mm) may cause
are employed most commonly for organic substances (the line broadening and significant changes to the sample itself
use of cobalt anodes can be especially preferred to separate such as:
distinct X-ray lines). The choice of radiation to be used de- (i) specimen contamination by particles abraded from
pends on the absorption characteristics of the specimen and the milling instruments (mortar, pestle, balls, etc.);
possible fluorescence by atoms present in the specimen. The (ii) reduced degree of crystallinity;
wavelengths used in powder diffraction generally correspond (iii) solid-state transition to another polymorph;
to the Ka radiation from the anode. Consequently, it is ad- (iv) chemical decomposition;
vantageous to make the X-ray beam `monochromatic' by (v) introduction of internal stress;
eliminating all the other components of the emission spec- (vi) solid-state reactions.
trum. This can be partly obtained using Kb filters, i.e. metal Therefore, it is advisable to compare the diffraction pat-
filters selected as having an absorption edge between the Ka tern of the non-ground specimen with that corresponding to
and Kb wavelengths emitted by the tube. a specimen of smaller particle size (e.g. a milled specimen).
Such a filter is usually inserted between the X-ray tube and If the X-ray powder diffraction pattern obtained is of ade-
the specimen. Another, more-and-more-commonly used way quate quality considering its intended use, then grinding may
to obtain a monochromatic X-ray beam is via a large not be required. It should be noted that if a sample contains
monochromator crystal (usually referred to as a `monochro- more than one phase and if sieving is used to isolate particles
mator'). This crystal is placed before or behind the specimen to a specific size, the initial composition may be altered.
and diffracts the different characteristic peaks of the X-ray
4. Control of the instrument performance
beam (i.e. Ka and Kb) at different angels, so that only one of
Goniometers and the corresponding incident and dif-
them may be selected to enter into the detector. It is even
fracted X-ray beam optics have many mechanical parts that
possible to separate Ka1 and Ka2 radiations by using a special-
need adjustment. The degree of alignment or misalignment
ized monochromator. Unfortunately, the gain in getting a
directly influences the quality of the results of an XRPD in-
monochromatic beam by using a filter or a monochromator
vestigation. Therefore, the different components of the dif-
is counteracted by a loss in intensity. Another way of
fractometer must be carefully adjusted (optical and mechani-
separating Ka and Kb wavelengths is by using curved X-rays
cal systems, etc.) to adequately minimize systematic errors,
mirrors that can simultaneously monochromate and focus or
while optimizing the intensities received by the detector. The
parallelize the X-ray beam.
search for maximum intensity and maximum resolution is
2.3. Radiation protection
always antagonistic when aligning a diffractometer. Hence,
Exposure of any part of the human body to X-rays can be
the best compromise must be sought whilst performing the
injurious to health. It is therefore essential that whenever X-
alignment procedure. There are many different configura-
ray equipment is used, adequate precautions are taken to
tions and each supplier's equipment requires specific align-
protect the operator and any other person in the vicinity.
ment procedures.
Recommended practice for radiation protection as well as
The overall diffractometer performance must be tested
limits for the levels of X-radiation exposure are those estab-
and monitored periodically using suitable certified reference
lished by national legislation in each country. If there are no
materials. Depending on the type of analysis, other well-
official regulations or recommendations in a country, the
defined reference materials may also be employed, although
latest recommendations of the International Commission on
the use of certified reference materials is preferred.
Radiological Protection should be applied.
5. Qualitative phase analysis (Identification of phases)
3. Specimen preparation and mounting
The identification of the phase composition of an
The preparation of the powdered material and mounting
unknown sample by XRPD is usually based on the visual or
of the specimen in a suitable holder are critical steps in many
computer-assisted comparison of a portion of its X-ray
analytical methods, and are particularly so for X-ray powder
diffraction powder pattern to the experimental or calculated
diffraction analysis, since they can greatly affect the quality
pattern of a reference material. Ideally, these reference
of the data to be collected(3). The main sources of error due
patterns are collected on well-characterized single-phase
to specimen preparation and mounting are briefly discussed
specimens. This approach makes it possible in most cases to
here for instruments in Bragg-Brentano parafocusing geo-
identify a crystalline substance by its 2 diffraction angles or
metry.
d-spacings and by its relative intensities. The computer-aided
3.1. Specimen preparation
comparison of the diffraction pattern of the unknown sam-
In general, the morphology of many crystalline particles
ple to the comparison data can be based either on a more-or-
tends to give a specimen that exhibits some degree of
less extended 2 -range of the whole diffraction pattern or on
preferred orientation in the specimen holder. This is particu-
a set of reduced data derived from the pattern. For example,
larly evident for needle-like or plate-like crystals when size
the list of d-spacings and normalized intensities Inorm, a so-
reduction yields finer needles or platelets. Preferred orienta-
called (d, Inorm)-list extracted from the pattern, is the crystal-
tion in the specimen influences the intensities of various
lographic fingerprint of the material, and can be compared
reflections, so that some are more intense and others are less
to (d, Inorm)-lists of single-phase samples complied in data-
intense, compared to what would be expected from a com-
bases.
pletely random specimen. Several techniques can be em-
For most organic crystals, when using CuKa radiation, it is
ployed to improve randomness in the orientation of crystal-
appropriate to record the diffraction pattern in a 2 -range
lites (and therefore to minimize preferred orientation), but
from as near 09as possible to at least 409. The agreement in
further reduction of particle size is often the best and sim-
the 2 -diffraction angles between specimen and reference is
plest approach. The optimum number of crystallites depends
within 0.29for the same crystal form, while relative intensi-
on the diffractometer geometry, the required resolution and
ties between specimen and reference may vary considerably
the specimen attenuation of the X-ray beam. In some cases,
due to preferred orientation effects. By their very nature,
particle sizes as large as 50 mm will provide satisfactory
variable hydrates and solvates are recognized to have varying
results in phase identification. However, excessive milling
unit cell dimensions and as such shifting occurs in peak posi-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
82 2.58 X-Ray Powder Diffraction Method / General Tests JP XVII
tions of the measured XRPD patterns for these materials. In material with crystallite size and X-ray absorption coe-
these unique materials, variance in 2- positions of greater fficient comparable to those of the components of the sam-
than 0.29is not unexpected. As such, peak position vari- ple, and with a diffraction pattern that does not overlap at
ances such as 0.29are not applicable to these materials. For all that of the sample to be analyzed, can be used. A known
other types of samples (e.g. inorganic salts), it may be neces- quantity of this reference material is added to the sample to
sary to extend the 2 -region scanned to well beyond 409 . It is be analyzed and to each of the reference mixtures. Under
generally sufficient to scan past the 10 strongest reflections these conditions, a linear relationship between line intensity
identified in single phase X-ray powder diffraction database and concentration exists. This application, called the ìnter-
files. nal standard method', requires a precise measurement of
It is sometimes difficult or even impossible to identify diffraction intensities.
phases in the following cases: In the `spiking method' (or `standard addition method'),
(i) non-crystallized or amorphous substances; some of the pure phase a is added to the mixture containing
(ii) the components to be identified are present in low the unknown concentration of a. Multiple additions are
mass fractions of the analyte amounts (generally less than 10 made to prepare an intensity-versus-concentration plot in
per cent m/m); which the negative x intercept is the concentration of the
(iii) pronounced preferred orientation effects; phase a in the original sample.
(iv) the phase has not been filed in the database used;
7. Estimate of the amorphous and crystalline fractions
(v) formation of solid solutions;
In a mixture of crystalline and amorphous phases, the
(vi) presence of disordered structures that alter the unit
crystalline and amorphous fractions can be estimated in
cell;
several ways. The choice of the method used depends on the
(vii) the specimen comprises too many phases;
nature of the sample:
(viii) presence of lattice deformations;
(i) if the sample consists of crystalline fractions and an
(ix) structural similarity of different phases.
amorphous fraction of different chemical compositions, the
6. Quantitative phase analysis amounts of each of the individual crystalline phases may be
If the sample under investigation is a mixture of 2 or more estimated using appropriate standard substances as described
known phases, of which not more than 1 is amorphous, the above; the amorphous fraction is then deduced indirectly by
percentage (by volume or by mass) of each crystalline phase subtraction;
and of the amorphous phase can, in many cases, be deter- (ii) if the sample consists of one amorphous and one
mined. Quantitative phase analysis can be based on the inte- crystalline fraction, either as a 1-phase or a 2-phase mixture,
grated intensities, on the peak heights of several individual with the same elemental composition, the amount of the
diffraction lines(4), or on the full pattern. These integrated crystalline phase (`the degree of crystallinity') can be estimat-
intensities, peak heights or full-pattern data points are com- ed by measuring 3 areas of the diffractogram:
pared to the corresponding values of reference materials.
A ＝ total area of the peaks arising from diffraction from
These reference materials shall be single-phase or a mixture
the crystalline fraction of the sample:
of known phases. The difficulties encountered during quan-
B ＝ total area below area A;
titative analysis are due to specimen preparation (the ac-
C ＝ background area (due to air scattering, fluorescence,
curacy and precision of the results require in particular
equipment, etc.)
homogeneity of all phases and a suitable particle size distri-
bution in each phase) and to matrix effects. In favorable When these areas have been measured, the degree of
cases, amounts of crystalline phases as small as 10 per cent crystallinity can be roughly estimated using the following
may be determined in solid matrices. formula:
6.1. Polymorphic samples
z crystallinity ＝ 100A/(A ＋ B － C )
For a sample composed of 2 polymorphic phases a and b,
the following expression may be used to quantify the frac- It is noteworthy that this method does not yield absolute
tion Fa of phase a: degree-of-crystallinity values and hence is generally used for
comparative purposes only. More sophisticated methods are
1
Fa ＝ also available, such as the Ruland method.
1 ＋ K (Ib/Ia)
8. Single crystal structure
The fraction is derived by measuring the intensity ratio
In general, the determination of crystal structures is per-
between the 2 phases, knowing the value of the constant K.
formed from X-ray diffraction data obtained using single
K is the ratio of the absolute intensities of the 2 pure poly-
crystals. However, crystal structure analysis of organic crys-
morphic phases Ioa/Iob. Its value can be determined by meas-
tals is a challenging task, since the lattice parameters are
uring standard samples.
comparatively large, the symmetry is low and the scattering
6.2. Methods using a standard
properties are normally very low. For any given crystalline
The most commonly used methods for quantitative analy-
form of a substance, knowledge of the crystal structure
sis are:
allows the calculation of the corresponding XRPD pattern,
–the èxternal standard method';
thereby providing a `preferred-orientation-free' reference
–the ìnternal standard method';
XRPD pattern, which may be used for phase identification.
–the `spiking method' (often also called the `standard ad-
dition method'). (1) There are many other applications of the X-ray powder diffrac-
The èxternal standard method' is the most general
tion technique that can be applied to crystalline pharmaceutical
method and consists of comparing the X-ray diffraction pat-
substances such as: determination of crystal structures, refine-
tern of the mixture, or the respective line intensities, with
ment of crystal structures, determination of crystallographic
those measured in a reference mixture or with the theoretical
purity of crystalline phases, characterization of crystallographic
intensities of a structural model, if it is fully known.
texture, etc. These applications are not described in this chapter.
To limit errors due to matrix effects, an internal reference (2) An ìdeal' powder for diffraction experiments consists of a large

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
JP XVII General Tests / 2.59 Test for Total Organic Carbon 83
number of small, randomly oriented spherical crystallites 2. Reagents and standard solutions
(coherently diffracting crystalline domains). If this number is (i) Water used for measuring organic carbon (water for
sufficiently large, there are always enough crystallites in any measurement): This water is used for preparing standard so-
diffracting orientation to give reproducible diffraction patterns. lutions or decomposition aid or for rinsing the instrument.
(3) Similarly, changes in the specimen can occur during data collec- The amount of organic carbon in this water, when collected
tion in the case of a non-equilibrium specimen (temperature, hu- into a sample container, should be not more than 0.250
midity). mg/L.
(4) If the crystal structures of all components are known, the Riet- (ii) Standard potassium hydrogen phthalate solution:
veld method can be used to quantify them with good accuracy. The concentration of this standard solution is determined as
If the crystal structures of the components care not known, the specified for the instrument. Dry potassium hydrogen phtha-
Pawley or least squares methods can be used. late (standard reagent) at 1059C for 4 hours, and allow it to
cool in a desiccator (silica gel). Weigh accurately a
prescribed amount of dried potassium hydrogen phthalate,
and dissolve it in the water for measurement to prepare the
2.59 Test for standard solution.
Total Organic Carbon (iii) Standard solution for measuring inorganic carbon:
The concentration of this standard solution is determined as
Test for Total Organic Carbon is a method for measuring specified for the instrument. Dry sodium hydrogen carbon-
the amount of organic carbon, which forms organic com- ate in a desiccator (sulfuric acid) for not less than 18 hours.
pounds, in water. Normally, organic carbon can be oxidized Dry sodium carbonate decahydrate separately between
to carbon dioxide by a dry decomposition method, where 5009C and 6009 C for 30 minutes, and allow to cool in a
organic compounds are oxidized by combustion, or by a wet desiccator (silica gel). Weigh accurately prescribed amounts
decomposition method, where organic compounds are oxi- of these compounds so that the ratio of their carbon content
dized by applying ultraviolet rays or by adding oxidizing is 1:1, and dissolve them in the water for measurement to
agent. The amount of carbon dioxide generated in the prepare the standard solution.
decomposition process is measured using an appropriate (iv) Decomposition aid: Dissolve a prescribed amount of
method such as infrared gas analysis, electric conductivity potassium peroxodisulfate or other substances that can be
measurement, or resistivity measurement. The amount of used for the same purpose, in the water for measurement up
organic carbon in water can be calculated from the amount to the concentration as specified for the instrument.
of carbon dioxide measured in one of the above methods. (v) Gas for removing inorganic carbon or carrier gas:
There are two types of carbon in water: organic carbon Nitrogen, oxygen, or other gases that can be used for the
and inorganic carbon. For measuring the amount of organic same purpose.
carbon, two approaches can be taken. One method is to (vi) Acid for removing inorganic carbon: Dilute hydro-
measure the amount of total carbon in water, then to sub- chloric acid, phosphoric acid or other acids that can be used
tract the amount of inorganic carbon from that of total car- for the same purpose, with the water for measurement down
bon. The other method is to remove inorganic carbon from to the concentration as specified for the instrument.
the test water, then to measure the amount of remaining
3. Apparatus
organic carbon.
(i) Sample container and reagent container: Use a con-
1. Instrument tainer made of the material which does not release organic
The instrument consists of a sample injection port, a carbon from its surface, such as hard glass. Soak the con-
decomposition device, a carbon dioxide separation block, a tainer before use in a mixture of diluted hydrogen peroxide
detector, and a data processor or a recorder. The instrument solution (1 in 3) and dilute nitric acid (1:1), and wash well
should be capable of measuring the amount of organic car- with the water for measurement.
bon down to 0.050 mg/L. (ii) Microsyringe: Wash a microsyringe with a mixture of
The sample injection port is designed to be able to accept a a solution of sodium hydroxide (1 in 20) and ethanol (99.5)
specific amount of sample injected by a microsyringe or (1:1), or diluted hydrochloric acid (1 in 4), and rinse well
other appropriate sampling devices. The decomposition with the water for measurement.
device for the dry decomposition method consists of a com-
4. Procedure
bustion tube and an electric furnace to heat the sample. Both
Employ an analytical method suitable for the instrument
devices are adjusted to operate at specified temperatures.
used. Calibrate the instruments using the standard potassium
The decomposition device for the wet decomposition method
hydrogen phthalate solution with the test procedure specified
consists of an oxidizing reaction box, an ultraviolet ray
for the instrument.
lamp, a decomposition aid injector, and a heater. The de-
It is recommended that this instrument be incorporated
composition device for either method should be capable of
into the manufacturing line of the water to be tested.
generating not less than 0.450 mg/L of organic carbon when
Otherwise, this test should be performed in a clean cir-
using a solution of 0.806 mg/L sodium dodecylbenzenesul-
cumstance where the use of organic solvents or other sub-
fonate as the sample. The carbon dioxide separation block
stances that may affect the result of this test is prohibited,
removes water from carbon dioxide formed in the decompo-
using a large sample container to collect a large volume of
sition process or separates carbon dioxide from the decom-
the water to be tested. The measurement should be done im-
posed gas. An infrared gas analyzer, electric conductivity
mediately after the sample collection.
meter or specific resistance meter is used as the detector
4.1. Measurement of organic carbon by subtracting inor-
which converts the concentration of carbon dioxide into elec-
ganic carbon from total carbon
tric signal. The data processor calculates the concentration
According to the test procedure specified for the instru-
of the total organic carbon in the sample based on the elec-
ment used, inject a suitable volume of the sample for meas-
tric signal converted by the detector. The recorder records
uring the expected amount of total carbon into the instru-
the electric signal intensity converted by the detector.
ment from sample injection port, and decompose organic

2.60 Melting Point Determination

The melting point is defined to be the temperature at
which a crystalline substance melts during heating, when the
solid phase and the liquid phase are in an equilibrium.
However, in this Pharmacopoeia it is conventionally defined Fig. 2.60-1 Melting point determination apparatus
to be the temperature at which the remaining solid sample
melts completely when it is subjected to continuous heating
and the change of the sample state that accompanies heating
is accurately observed. Since a pure substance has an intrin-
types of thermometers, Type 1—Type 6, which are specified
sic melting point, it is used for the identification and/or
by an appropriate measuring temperature range. For melting
confirmation of a substance and also as an indicator of the
points lower than 509C, use a thermometer Type 1; for 409 C
purity of a substance.
to 1009 C, Type 2; for 909C to 1509C, Type 3; for 1409 C to
The melting point is determined by either of the following
2009C, Type 4; for 1909 C to 2509 C, Type 5; for 2409C to
methods: Method 1 is applied to those substances of which
3209C, Type 6.
the purity is comparably high and which can be pulverized,
(iii) Capillary tube: Use a hard glass capillary tube 120
Method 2 to those substances which are insoluble in water
mm long, 0.8 to 1.2 mm in inner diameter and 0.2 to 0.3 mm
and can not be readily pulverized, and Method 3 to petrola-
thick, with one end closed.
tums.
1.2. Procedure
Unless otherwise specified, measurement is performed by
Pulverize the sample to a fine powder, and, unless other-
Method 1.
wise specified, dry in a desiccator (silica gel) for 24 hours.
1. Method 1 When it is specified to do the test after drying, dry the sam-
This method is applied to those substances of which the ple under the conditions specified in the monograph before
purity is comparably high and which can be pulverized. measurement. Place the sample in a dried capillary tube H,
1.1. Apparatus and pack it tightly so as to form a layer about 2.5 – 3.5 mm
Use the apparatus illustrated in the Fig. 2.60-1. high by dropping the capillary repeatedly, with the closed
Alternatively, apparatus in which some of the procedures, end of H down, through a glass tube, about 70 cm long, held
such as stirring, heating, and cooling are automated, can be vertically on a glass or porous plate.
used. Heat the bath fluid B until the temperature rises to about
(i) Bath fluid: Usually use clear silicone oil having a vis- 109C below the expected melting point, place the thermome-
cosity of 50 to 100 mm2/s at an ordinary temperature. ter D in the bath with the immersion line at the same level as
(ii) Thermometer with an immersion line: There are six the meniscus of the bath fluid, and insert capillary tube H

into a coil spring G so that the packed sample is placed in a capillary tube in a water-containing beaker to such a position
position corresponding to the center of the mercury bulb of that the lower edge of the sample is located 30 mm below the
the thermometer D. Continue heating to raise the tempera- water surface. Heat the beaker with constant stirring until
ture at a rate of approximately 39C per minute until the tem- the temperature rises to 59C below the expected melting
perature rises to 59C below the expected melting point, then point. Then regulate the rate of temperature increase to 19C
carefully regulate the rate of temperature increase to 19 C per per minute. The temperature at which the sample begins
minute. floating in the capillary is taken as the melting point of the
Read the thermometer indication of the instantaneous sample specimen.
temperature at which the sample liquefies completely and no
3. Method 3
solid is detectable in the capillary, and designate the indi-
This method is applied to petrolatums.
cated temperature as the melting point of the sample speci-
3.1. Apparatus
men.
Instead of the apparatus specified in Method 1, use a
1.2.1. System suitability test
water-containing beaker as a bath fluid and a heating vessel.
Confirmation of the system suitability of the apparatus
In this measurement, total immersion mercury-filled ther-
should be done periodically by using Reference Standards
mometers can also be used in place of the thermometer with
for Apparatus Suitability. The Reference Standard is pre-
an immersion line.
pared for the suitability test of the apparatus when it is used
3.2. Procedure
with Type 2—Type 5 thermometers, and consists of 6 highly
Melt the sample slowly by heating, with thorough stirring,
purified substances: acetanilide, acetophenetidine, caffeine,
until the temperature reaches 90 – 929C. Discontinue the
sulfanilamide, sulfapyridine, and vanillin. The label shows
heating, and allow the sample to cool to 8 – 109C above the
the certified melting points of the respective substances (the
expected melting point. Chill the bulb of the thermometer to
end point of the melting change), MPf.
59 C, wipe and dry, and, while still cold, stick half of the
After selecting one of the thermometers and the appropri-
thermometer bulb into the melted sample. Withdraw it im-
ate Melting Point Standard based upon the expected melting
mediately, hold vertically, cool until the attached sample
point of a sample specimen, perform a melting point meas-
becomes turbid, then dip the sample-bearing bulb for 5
urement of the selected Reference Standard, according to the
minutes in water having a temperature below 169C. Next, fix
above procedure. When the value of the obtained melting
the thermometer securely in a test tube by means of a cork
point of the Reference Standard is within MPf ± 0.59 C in
stopper so that the lower end is located 15 mm above the
the case of vanillin and acetanilide, within MPf ± 0.89 C in
bottom. Suspend the test tube in a water-containing beaker
the case of acetophenetidine and sulfanilamide, and within
held at a temperature about 169C, and raise the temperature
MPf ± 1.09C in the case of sulfapyridine and caffeine, the
of the water bath to 309 C at a rate of 29C per minute, then
apparatus is assumed to be suitable.
continue heating carefully at a rate of 19C per minute until it
The above-mentioned measurement is repeated 3 times
reaches the melting point. Read the thermometer indication
and the average is determined to be the melting point of the
of the instantaneous temperature at which the first drop of
Reference Standard tested. When the above suitability test
the sample leaves the thermometer. If the variations between
criteria are not met in a certain melting point measurement
three repeated determinations are not more than 19C, take
system of an apparatus and a Reference Standard, do the
the average of the three as the melting point. If any variation
test again, after checking the packing of the sample specimen
is greater than 19
C, make two additional measurements, and
into the capillary tube, the locations and positioning of the
take the average of the five as the melting point.
thermometer and the capillary tube, the heating and stirring
of the bath fluid, and the control of the temperature increas-
ing rate. When a melting point measurement system does not
meet the suitability test criteria again after checking these 2.61 Turbidity Measurement
measuring conditions, the thermometer with an immersion
line should be calibrated again or replaced with a new one. Turbidity measurement is used to determine the turbidity
(degree of opalescence) for the decision whether the article to
2. Method 2
be examined complies with the clarity requirement stated in
This method is applied to substances such as fats, fatty
the Purity.
acids, paraffins or waxes.
As a rule, the visual method is specified for the
2.1. Apparatus
requirement in individual monograph.
Instead of the apparatus specified in Method 1, use a
water-containing beaker as a bath fluid and a heating vessel. 1. Visual method
In this measurement, total immersion mercury-filled ther- This is used to determine the degree of opalescence with
mometers can also be used in place of the thermometer with white (or faintly-colored) fine particles. So the degree of
an immersion line. Furthermore, the capillary tube should be opalescence of a colored sample is liable to be determined
the same as specified in Method 1, except that both ends of lower that it is difficult to compare the degree correctly with-
the tube are open. out using similarly colored reference suspension.
2.2. Procedure 1.1. Reference suspensions
Carefully melt the sample at as low a temperature as possi- Pipet 5 mL, 10 mL, 30 mL and 50 mL of formazin opales-
ble, and, taking care to prevent bubbles, introduce it into a cence standard solution, dilute them separately to exactly
capillary tube to a height of about 10 mm. Allow the capil- 100 mL with water, and use these solutions so obtained as
lary containing the sample to stand for 24 hours at below Reference suspensions I, II, III and IV, respectively. Shake
109 C, or for at least 1 hour in contact with ice, holding the before use. Degrees of opalescence of Reference suspensions
capillary so that the sample can not flow out. Then attach I, II, III and IV are equivalent to 3 NTU, 6 NTU, 18 NTU
the capillary to the thermometer by means of a rubber band and 30 NTU, respectively.
so that the absorbed sample is located at a position cor- 1.2. Procedure
responding to the center of the mercury bulb. Adjust the Place sufficient of the test solution, water or the solvent to

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
86 2.62 Mass Spectrometry / General Tests JP XVII
prepare the test solution and, where necessary, newly pre- ple, and the measurement is usually performed at around
pared Reference suspensions in separate flat-bottomed test 660 nm to avoid possible disturbance occurred from the ab-
tubes, 15 – 25 mm in inside diameter and of colorless and sorption by the color.
transparent, to a depth of 40 mm, and compare the contents 2.2. Nephelometry
of the tubes against a black background by viewing in When a suspension is viewed at right angles to the direc-
diffused light down the vertical axes of the tubes. The tion of the incident light, it appears opalescent due to the
diffused light must be such that Reference suspension I can refraction of light from the particles of the suspension
be readily distinguished from water, and that Reference sus- (Tyndall effect). A certain portion of the light entering a tur-
pension II can readily be distinguished from Reference sus- bid liquid is transmitted, another portion is absorbed and the
pension I. remaining portion is scattered by the suspended particles.
In this test Reference suspensions are used when the clarity The scattered light measuring method shows the linear
of the test solution is obscurely and it is not easy to deter- relationship between the nephelometric turbidity units
mine that its degree of opalescence is similar or not similar to (NTU) values and relative detector signals in a low turbidity
water or to the solvent used to prepare the test solution. range. As the degree of turbidity increases, not all the parti-
1.3. Interpretation cles are exposed to the incident light and the scattered radia-
A liquid is considered ``clear'' when its clarity is the same tion of other particles is hindered on its way to the detector.
as that of water or of the solvent used to prepare the liquid 2.3. Ratio Turbidimetry
or its turbidity is not more pronounced than that of Refer- This method measures both scattered and transmitted light
ence suspension I. If the turbidity of the liquid is more than values at the same time, and the turbidity is determined from
that of Reference suspension I, consider as follows: When the ratio of the scattered light value to the transmitted light
the turbidity is more than that of Reference suspension I but value. This procedure compensates for the light that is di-
not more than that of Reference suspension II, express `ìt is minished by the color of the sample and eliminates the in-
not more than Reference suspension II''. In the same way, fluence of the color. When the measurement is performed by
when the turbidity is more than that of Reference suspension using an integrating sphere, it is particularly called the in-
II but not more than that of Reference suspension III, ex- tegrating sphere method, which measures the total transmit-
press `ìt is not more than Reference suspension III'', and ted light value as well as the scattered light value occurred
when the turbidity is more than that of Reference suspension with the suspended particles, and the turbidity can be deter-
III but not more than that of Reference suspension IV, ex- mined from the ratio of them.
press `ìt is not more than Reference suspension IV''. When 2.4. Application of photoelectric photometry for mono-
the turbidity is more than that of Reference suspension IV, graph requirements
express `ìt is more than Reference suspension IV''. The turbidity of the test solution, determined by the pho-
1.4. Reagent solutions toelectric photometry, can be used as an indicating standard
Formazin opalescence standard solution: To exactly 3 mL for the conformity to the clarity requirements by converting
of formazin stock suspension add water to make exactly 200 into NTU by using turbidity known reference solutions such
mL. Use within 24 hours after preparation. Shake thor- as Reference suspensions I – IV, if needed, and water or the
oughly before use. Degrees of opalescence of this standard solvent used. In an automatically compensable apparatus
solution is equivalent to 60 NTU. being calibrated with turbidity known reference solutions,
the measuring result is given in NTU and it can be compared
2. Photoelectric photometry
directly with required specified value.
The turbidity can also be estimated by instrumental meas-
NTU is often used as the unit in the turbidity determina-
urement of the light absorbed or scattered on account of
tions. It is the unit used in the case when the turbidity is esti-
submicroscopic optical density inhomogeneities of opales-
mated by the instrument which measures the 90 ± 309scat-
cent solutions and suspensions. The photoelectric photomet-
tered light against the incident light intensity, using tungsten
ry is able to provide more objective determination than the
lamp, and in the case the estimation is performed by the
visual method. Though they can determine the turbidity by
instrument which measures the 90 ± 2.59 scattered light
measuring the scattered or transmitted light, the measuring
against the incident light intensity using 860 nm infrared
system and light source must be specified in individual test
light, FNU is used as the unit. FNU is equivalent to NTU at
method, and for the comparison of observed data, the same
a range of smaller measurements (less than 40 NTU). For the
measuring system and light source should be used.
unit of formazin concentration, FTU is also used, which is
In each case, the linear relationship between turbidity and
defined as a suspension of 1 mg formazin in 1L of purified
concentration must be demonstrated by constructing a
water is 1 FTU.
calibration curve using at least 4 concentrations. For colored
samples, the turbidity value is liable to be estimated lower
because of attenuating both incident and scattered lights due
to the absorption by the color, and the transmission-disper- 2.62 Mass Spectrometry
sion method is principally used.
2.1. Turbidimetry Mass spectrometry (MS) is a method to separate and de-
When a light passes through a turbid liquid the transmit- tect the ions generated from the ionization of molecules ac-
ted light is decreased by scattering with the particles dis- cording to their m/z values, and it is used for the identifica-
persed in the liquid. A linear relationship is observed be- tion and purity test of the substances. The m/z value is the
tween turbidity and concentration when the particles with a dimensionless parameter obtained from dividing the relative
constant size are uniformly dispersed, the size is small and mass (m) of the ion (the ratio of the mass of the ion to the
the suspension is not higher concentration. The turbidity can unified atomic mass unit) by the charge number (z) of the
be measured by Ultraviolet-visual Spectrophotometry <2.24> ion. The unified atomic mass unit is defined as one-twelfth
using spectrophotometer or photoelectric photometer. The of the mass of a 12C atom in its ground state, and it is used to
turbidity of the sample in higher concentration can also be express masses of atoms, molecules and ions. The result of
measured, however, it is susceptible to the color of the sam- measurement is shown as the mass spectrum in which the

m/z values of the ions and the relative intensities of the sig- 1.2.1. Electron Ionization (EI) Method
nals corresponding to the ions are shown on the x-axis and In the EI method, the vaporized sample molecule (M) is
the y-axis, respectively. ionized by receiving the energy of thermal electrons (usually,
The precise mass of a molecule or an ion consisting of 70 eV), and the molecular ion (M＋・) and fragment ions with
only a single isotope (usually, the isotope with the greatest the structural information of the sample molecule are gener-
natural abundance) of each element comprising a sample ated. This method is suitable for ionizing nonpolar molec-
molecule is referred to as the ``monoisotopic mass.'' Usually ules such as volatile or gaseous samples with low molecular
not only a monoisotopic ion but also its isotopic ions are mass up to approximately 1000. It is used for the identifica-
seen in the mass spectrum. The molecular mass of the sample tion of substances using a data library or other source, be-
substance can be determined from the m/z value of the mo- cause mass spectra with reproducible fragmentation patterns
lecular ion. When the fragment ions are observed, the mo- can be obtained by this method.
lecular structure of the sample substance can be estimated 1.2.2. Chemical Ionization (CI) Method
and confirmed based on the masses of the fragment ions and In the CI method, the vaporized sample molecules are
the mass differences among the molecular ion and the frag- ionized through ion/molecule reactant with reaction ions
ment ions. In tandem mass spectrometry (MS/MS), the generated from reagent gases such as methane, isobutane
product ions, generated by the dissociation of the selected and ammonia. When a reagent gas is introduced into the
precursor ion with m/z value, are used for the mass spectro- ionization chamber, protonated ions of the molecules [M ＋
metry. The structural estimation and confirmation of the H]＋, deprotonated ions of the molecules [M － H]－ or
precursor ion can be performed using the m/z value of the reactant ion adducts of the molecules are generated. Since
product ion observed in the measurement. The schematic di- the ions generated by the CI method have internal energy
agram of the MS and the MS/MS is shown in Fig. 2.62-1. values that are much lower than those obtained by the EI
method, the fragmentation of sample molecules hardly
1. Mass Spectrometer
occurs.
A mass spectrometer usually consists of a sample in-
1.2.3. Electrospray Ionization (ESI) Method
troduction unit, an ionization unit (ion source), a mass
When the sample solution is sprayed through a capillary
analyzer, a detector and data processor, and an exhaust
with a tip to which high voltage is applied, atomized charged
system to maintain the mass analyzer under high vacuum
droplets are produced. Subsequently, the sample molecules
(Fig. 2.62-1).
will be ionized when the charge density of the droplets
1.1. Sample Introduction
increases, accompanied by the evaporation of the solvent;
For the introduction of the sample into the ion source, the
[M ＋ H]＋, [M － H]－, or alkali metal ion adduct of the
following methods are used; Direct infusion method, in
molecules is thus generated. This method is used for the ioni-
which solution samples are injected into the ion source by
zation of sample substances from those with low molecular
using a syringe pump or capillary tip, for example; Direct in-
mass and relatively high polarity to those with high molecu-
let method, in which a liquid or solid sample is placed in a
lar mass. The ESI method can also be applied for the meas-
glass tube or other appropriate vessel and introduced into
urement of biopolymers such as peptides, proteins and poly-
the vicinity of the electron beams or reactant ion atmosphere
saccharides, because the method makes it easy to generate
of the ion source. In addition, the method, in which each
multiply-charged ions such as [M ＋ nH]n＋ and [M －
component separated by the chromatographic technique
nH]n－.
such as gas chromatography or liquid chromatography and
1.2.4. Atmospheric Pressure Chemical Ionization (APCI)
capillary electrophoresis is introduced into the ion source
Method
successively, is also used.
In the APCI method, the sample solution is sprayed and
1.2. Ion Source
vaporized by passing through a heated capillary using nitro-
When the sample substances are introduced into the mass
gen as the carrier, and the corona discharge is induced at the
spectrometer, ions with a positive or negative charge are
time with a high-voltage needle electrode, and the solvent
generated from the substance in the ion source. There are
molecules are thus ionized. The sample molecules will be
various ionization methods in mass spectrometry, and it is
ionized through the ion/molecule reaction with the solvent
important to select the most suitable ionization method ac-
ions, and [M ＋ H]＋, [M － H]－, or alkali metal ion adduct
cording to the polarity and molecular mass of the sample
of the molecules will be generated. This method is suitable
substance to be measured and the purpose of the meas-
for ionizing nonpolar to highly polar compounds with a mo-
urement. Typical ionization methods are as follows.
lecular mass up to approximately 1500.
1.2.5. Matrix-assisted Laser Desorption/Ionization
(MALDI) Method
When a mixture of the sample and a matrix such as a-
cyano-4-hydroxycinnamic acid or sinapinic acid is irradiated
with a pulsed laser, the sample molecules will be vaporized
quickly and ionized, accompanied by the electronic excita-
tion of the matrix. At that time, the proton transfer occurs
between the matrix and the sample molecules, and [M ＋
H]＋, [M － H]－, or alkali metal ion adduct of the molecules
is generated. With the MALDI method, it is possible to
ionize the compounds from low molecular mass of several
hundreds to high molecular mass of several hundred thou-
sand by selecting the appropriate matrix. Since the amount
of the sample required for the measurement is very small,
this method is used for the ionization of samples of biologi-
Fig. 2.62-1 Schematic diagram of mass spectrometry (MS) cal origin such as peptides and proteins.
and tandem mass spectrometry (MS/MS)

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
88 2.62 Mass Spectrometry / General Tests JP XVII
1.2.6. Other Ionization Methods instrument with the sensitivity and dynamic range improved
Various other ionization methods have been developed, by using four electrodes with a hyperbolic surface is referred
including the field ionization (FI) method, the field desorp- to as a linear ion-trap (LIT) analyzer.
tion (FD) method, the fast atom bombardment (FAB) 1.3.2.2. Kingdon Trap
method, the secondary ion mass spectrometry (SIMS) In the Kingdon trap analyzer, ions are trapped while rotat-
method, the atmospheric pressure photoionization (APPI) ing around a spindle-shaped electrode. The image current in-
method, and an ionization method in which the volatile sub- duced by the ions oscillating according to their m/z values is
stances on the material surface can be directly ionized using measured. The mass spectrum is obtained by Fourier-tran-
the ionization by the collision reaction with helium in the ex- sforming the measured waveform data on the time axis to
cited state in the open space. those on the frequency axis. This analyzer is used for quali-
1.2.7. Sample Introduction Method and Ionization tative analyses such as structure analyses because it has ex-
Method tremely high mass resolving power and mass accuracy.
Each ionization technique is closely related to the sample 1.3.2.3. Penning Ion-trap
introduction methods. In the case of the gas chromatogra- The Penning ion-trap is used for Fourier transform-ion cy-
phy mass spectrometry (GC-MS), vaporized substances sepa- clotron resonance (FT-ICR). The ions that enter into the
rated by a capillary column are directly introduced into a strong magnetic field formed by a superconducting magnet
high-vacuum ion source and ionized by the EI method or CI show cyclotron movement due to the effect of the Lorenz
method, for example. In the case of liquid chromatography force. Here, the angular frequency (v) can be expressed by
mass spectrometry (LC-MS), the liquid phase containing the following equation.
sample substances separated by the LC column is sprayed
v ＝ qB/m
under atmospheric pressure, and the sample substances are
ionized by an ionization method described above at the inter- where m is the mass in atomic mass units of the ion, q is the
face to introduce the ions to the high-vacuum mass analyzer. electric charge of the ion, and B is the magnetic flux density.
At that time, it is necessary to ensure that the mobile phase When the high-frequency electric field with this frequency is
to be used has an appropriate composition for both the applied to the magnetic field, the ions move along the spiral
column separation and the ionization. In the case of capil- orbital. These rotating ion groups induce the electric current,
lary electrophoresis mass spectrometry, the flow rate is usu- which changes periodically according to its respective m/z
ally adjusted by adding an appropriate solution to the elec- value in the detecting electrode. The mass spectrum can be
trolyte at the end of the capillary, and the sample substance obtained by Fourier-transforming the signals measured
is ionized by the ESI method or other ionization method. above and further converting the frequencies to the m/z
1.3. Mass Analyzers values. The Penning ion-trap is used for precise structural
In a mass analyzer, the ions generated in the ion source are studies in combination with various dissociation techniques
separated according to their m/z values. As a result, the for precursor ions, since an FT-ICR analyzer has extremely
mass and the relative abundance of the ions derived from the high mass resolving power and mass accuracy.
samples to be analyzed can be measured. The following mass 1.3.3. Time-of-flight (TOF) Analyzer
analyzers are commonly used for MS. In the time-of-flight (TOF) analyzer, the ions are sepa-
1.3.1. Quadrupole (Q) Analyzer rated based on the difference of the flight times necessary for
The quadrupole (Q) analyzer has four rod electrodes set reaching the detector. For the ions with the mass m acceler-
parallel to each other, to which high-frequency alternating ated by the constant voltage V, the time t necessary for the
current voltage is applied and on which direct current volt- ions to fly a distance L and reach the detector can be ex-
age is superimposed. The ions that enter this space oscillate pressed by the following equation.
according to their m/z values, and only ions with a specific
L
m/z value have a stable trajectory and will be able to pass t ＝ m/z ×
through the space. The ions with different m/z values can 2eV
also become able to pass through the analyzer with a change The time of flight t is proportional to the square root of
in the applied voltage, and thereby the mass spectrum can be the m/z value, and consequently, the ions with smaller mass
obtained. The mass resolving power of a Q analyzer is gener- reach the detector faster. In the reflector mode in which the
ally low, but Q analyzers are widely used for the qualitative ions are reflected by the reflectron with the electrodes ar-
and quantitative analyses as general-purpose equipment, ranged side by side, high mass resolving power can be ob-
since they have a relatively wide dynamic range and simple tained by bringing the distribution of the kinetic energy of
composition that can be downsized. the ions into focus and doubling the flight distance of the
1.3.2. Ion-trap (IT) Analyzers ions. TOF analyzers are used for the analyses of high-
An ion-trap (IT) analyzer is made of an electric field or molecular-mass compounds such as proteins in combination
magnetic field or a combination of field, and is used to trap with the MALDI method and other techniques, since the
the ions in a space. The three most commonly used IT mass range measurable by this method does not have a mar-
analyzers are as follows. gin, theoretically. It is also frequently used for the qualita-
1.3.2.1. Paul Ion-trap tive analyses of low-molecular-mass substances, since it has
The Paul ion-trap is a synonym for quadrupole ion-trap high mass resolving power.
(QIT). Although it is similar to the quadrupole analyzer in 1.3.4. Magnetic Sector Analyzer
principle, it is able to trap ions stably by using ring elec- The ions that enter a magnetic sector analyzer are deflect-
trodes and end-capped electrodes instead of rod electrodes. ed by the Lorenz force of the magnetic field perpendicular to
The trapped ions are discharged into the detector according the ion current. At that time, ions with different m/z values
to their m/z values by a scan of the high-frequency voltage, (with the velocity v) fly into the magnetic field with different
and thereby the mass spectrum can be obtained. This radii of curvature r according to the following equation.
method is frequently used for qualitative analyses such as
mv
structure analysis because multiple-stage mass spectrometry r＝
(MSn) can be achieved by using only one analyzer. The qB

Only ions with a specific m/z value are able to pass ple-stage mass spectrometry (MSn) can be performed by
through the slit placed on the path of the ions. The mass repeating the selection and dissociation of the precursor ions
spectrum can be obtained by scanning the magnetic flux den- and the separation of the product ions multiple times.
sity B, and introducing the ions with different m/z values 2.1. Dissociation of Precursor Ions
passed through the slit into the detector in order. A magnetic 2.1.1. Collision-induced Dissociation (CID)
sector analyzer is usually used as a double-focusing-type In this dissociation method, a part or all of the collision
instrument in which the electric sector is combined with the energy is converted to the internal energy of the ions by the
magnetic sector, and the analyzer is used for both qualitative collision of the accelerated ions with the neutral collision
and quantitative analyses, since it has high mass resolving gases (He, Ar, N2, etc.), and subsequently the ions obtaining
power and is also highly quantitative. excess internal energy are excited and dissociated.
1.4. Detectors 2.1.2. Post-source Decay (PSD)
Ions that have passed through a mass analyzer are usually In the MALDI method, the ions generated at the ioniza-
transduced to the electric signal by releasing the electrons at tion source are dissociated during the interval between leav-
the detector. The following detectors are in current use. In ing the accelerating region and reaching the detector, due to
Fourier-transform-type instruments, the electric current in- the excess internal energy of the ions themselves or the colli-
duced by the movement of the ions at the detector is identi- sion with the residual gas. PSD is used for MS/MS using a
fied with a detection electrode. reflectron time-of-flight mass spectrometer.
1.4.1. Secondary Electron Multiplier (SEM) 2.1.3. Others
A secondary electron multiplier (SEM) has a multistage ar- Other dissociation methods are electron capture dissocia-
rangement of electrodes called dynodes. The secondary elec- tion, electron transfer dissociation, infrared multiphoton
trons emitted by the collision of the ions that enter the mul- dissociation, and surface-induced dissociation.
tiplier to the first dynode are sequentially multiplied, and 2.2. Constitutions of Principal Tandem Mass Spectrome-
finally transduced to the electric signal and recorded. This ters
multiplying effect of the secondary electrons enables the de- 2.2.1. Triple Quadrupole Mass Spectrometer (Q-q-Q)
tection of small amounts of ions. A triple quadrupole mass spectrometer (Q-q-Q) has a con-
1.4.2. Channel Electron Multiplier (CEM) figuration in which three quadrupoles are tandemly connect-
A channel electron multiplier (CEM) has a pipe-shaped ed so that the first quadrupole is used for the selection of the
channel configuration, and the secondary electrons are emit- precursor ions, the second quadrupole is used as the collision
ted by the collision of the ions that entered the multiplier to chamber for the dissociation of the precursor ions, and the
the inner wall of the channel. Multiple amplification is third quadrupole is used for the mass separation of the
achieved by repeating this process at every opposite side of product ions. Various scanning methods can be employed,
the inner wall. A CEM is simpler compared to SEMs, and and this type of spectrometer is frequently used for quantita-
with a CEM it is possible to downsize. tive analyses in particular.
1.4.3. Microchannel Plate (MCP) 2.2.2. Quadrupole Time-of-flight Mass Spectrometer (Q-
A microchannel plate (MCP) has a configuration in which TOF)
many very small CEMs are accumulated to form a detector. A quadrupole time-of-flight mass spectrometer (Q-TOF)
It is used for the detector of TOF-type instruments, since an has a configuration in which the third quadrupole in the Q-
MCP has a wide ion-receiving surface, and the time disper- q-Q is replaced with a TOF mass analyzer. The precursor
sion of the secondary electrons is small because of the very ions are selected at the first quadrupole, and the separation
thin structure of the MCP. of generated ions is conducted by the orthogonal-type TOF.
1.4.4. Faraday Cup (FC) Measurement with high sensitivity and high resolution is pos-
A Faraday cup (FC) is a simple detector that receives the sible.
charge of the ions that have entered an ion detector, and it 2.2.3. Time-of-flight Time-of-flight Mass Spectrometer
transduces the charge to the electric current. It has a cup- (TOF-TOF)
shaped configuration so that the secondary electrons emitted A time-of-flight time-of-flight mass spectrometer
from the ions can be captured. (TOF-TOF) consists of a TOF analyzer in which the precur-
sor ions are selected, the collision chamber, and a TOF
2. Tandem Mass Spectrometers
analyzer in which the mass separation of the product ions is
Tandem mass spectrometry (TMS) is a technique in which
performed. It is used for MALDI-TOF-TOF mass spectro-
precursor ions are selected from the fragment ions of the
metry.
sample substance at the first-stage mass analyzer, and the
2.2.4. Other Mass Spectrometers
product ions generated by dissociating the precursor ions are
The mass spectrometers other than those described above
separated and detected at the second-stage mass analyzer.
are the four-sector mass spectrometer with the configuration
TMS is used for (1) the structural estimation and confirma-
in which two double-focusing instruments are connected,
tion of fragment ions, and (2) specific and high sensitive ana-
and the LIT-Kingdon trap and QIT-TOF, in which an in-
lyses. There are two categories of TMS: TMA in space, and
time-type mass analyzer is used.
TMS in time.
With TMS in space, the selection of the precursor ions, the 3. Methods Used for Measurement
dissociation of the precursor ions, and the separation of the 3.1. Mass Spectrometry
product ions are conducted at the first-stage mass analyzer, The following measurement methods are used with mass
the intermediate region, and the second stage mass analyzer, spectrometry. An outline of the data obtained by each
respectively. With TMS in time, the selection/dissociation/ method is also described.
separation of the ions are conducted at the different time 3.1.1. Total Ion Monitoring (TIM)
zones in the same mass analyzer. TMS in space includes the Total ion monitoring (TIM) is also known as the full-scan
triple quadrupole-type, quadrupole/time-of-flight-type, and mode. It is the technique in which the mass spectrometer is
time-of-flight/time of flight-type mass analyzers. The latter operated so that all ions within the selected m/z range are
includes the ion-trap type mass analyzer, with which multi- detected and recorded, and the integrated value of the

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
90 2.62 Mass Spectrometry / General Tests JP XVII
amounts of ions observed in each scanning is called the total tensity and system sensitivity.
ion current (TIC). 4.1.2. Calibration
The chromatogram in which the total ion current obtained The mass calibration of a mass spectrometer is carried out
from the mass spectrum measured in LC-MS and GC-MS is based on the mass of standard material. The reproducibility
plotted against the retention time is called the total ion cur- of the measurement mass values is affected by the electrical
rent chromatogram (TICC), and the chromatogram in which variation of the instrument, the surface cleanliness of each
the relative intensity at the specific m/z value is expressed as component unit such as the ion source, and the room tem-
the function of time is called the extracted ion chromato- perature. There are the external and internal standard tech-
gram (EIC). niques for mass calibration. The number of points for the
3.1.2. Selected Ion Monitoring (SIM) calibration differs according to the type of mass spectrome-
In selected ion monitoring (SIM), the mass spectrometer is ter.
operated so that only the ions with a specific m/z value are 4.1.3. Mass Resolving Power
continuously detected and recorded instead of measuring the The ability to separate two adjacent ion peaks from each
mass spectrum. SIM is used for the assay and high-sensitivity other is referred to as the mass resolving power. Higher mass
detection of sample substances in LC-MS and GC-MS. resolving power capacitates to separate and detect the ion
3.2. Tandem Mass Spectrometry (TMS) peaks with a small mass difference. In magnetic-sector mass
The following methods are used for measurements using spectrometry, the mass resolving power R is calculated by
TMS. An outline of the data obtained by each method is also the following equation when two peaks with the mass of M
described. and M ＋ DM overlap each other to 10z of either peak
3.2.1. Product Ion Analysis height.
Product ion analysis is used to detect the product ions gen-
R ＝ M/D M
erated from the precursor ions with a selected m/z value,
and with this method the sample's qualitative information When an instrument other than a magnetic-sector mass
can be obtained. spectrometer is used, such as a quadrupole mass spectrome-
3.2.2. Precursor Ion Scan ter or a time-of-flight mass spectrometer, the mass resolving
Precursor ion scan is a method for scanning the precursor power can usually be calculated by the method using peak
ions from which the product ions with a specific m/z value width at half-height. When the width of the ion peak with
are generated by dissociation, and it is used for the specific the mass of m is Dm, the mass resolving power is calculated
detection of a substance with a specified substructure in the by R ＝ m/Dm, and is discriminated from that of the mag-
sample. netic-sector mass spectrometer.
3.2.3. Constant Neutral Loss Scan 4.2. Test for Identification
In constant neutral loss scan, the precursor ions that The identification of a test substance using mass spectro-
undergo the loss of specified mass (desorption of neutral metry is usually performed by the confirmation of the mass
species) due to dissociation are scanned. This method is used of the test substance molecule. The test should be performed
for the specific detection of substances with a specified sub- after confirming in advance that the measurement value is
structure in the sample. within the range specified in the monograph using the stand-
3.2.4. Selected Reaction Monitoring (SRM) ard solutions defined in the monograph, or the specified ion
Selected reaction monitoring (SRM) detects product ions can be detected. According to the mass resolving power of
with a specific m/z value generated by the dissociation of the the instrument and the mass of the test substance molecule,
precursor ions with a specified m/z value, and it is used for the mass of the test substance molecule obtained from mass
the quantitative detection of trace amounts of substances spectrometry can be adjusted to the monoisotopic mass or
present in a complex matrix. Although this method is similar the average mass.
to SIM, the specificity is improved by using the product ions In general, the mass of the molecule consisting of only
generated from the precursor ions for the detection. principle isotopes should be obtained from the monoisotopic
peak. However, when the monoisotopic peak cannot be
4. Application to Various Tests
In pharmaceutical analyses, mass spectrometry is used for identified because, for example, the molecular mass is high
or the resolution is not sufficient, the average mass should be
the identification and purity tests of molecules as a specific
calculated from the weighted average of the peak. When
detection method based on the mass and the structural infor-
samples with high molecular mass such as proteins are ana-
mation of the molecules.
lyzed by ESI/MS, the average mass should be calculated by
4.1. Optimization of Instruments
In mass spectrometry, in order to obtain a good shape, the deconvolution technique, because the ESI mass spectra
would show a series of multiply-charged ions with different
sensitivity, and mass accuracy of the ion peak it is necessary
charge states. It may be combined with the detection of the
to pre-optimize the measurement parameters of each compo-
nent unit of the instrument by using an appropriate standard fragment ions or the product ions generated from the test
substance molecule, which includes characteristic partial
material in accord with the ionization method and mass
range. structural information.
4.3. Purity test
4.1.1. Tuning
The purity test of a test substance using mass spectrometry
The shape, sensitivity, and relative intensity of the ion
peak detected are optimized by adjusting the parameters is usually performed in combination with a separation tech-
nique such as chromatography using a standard solution
such as the gas pressure, temperature, and voltage of the ion
source, mass analyzer, and detector. The various parameters with a concentration corresponding to the specified limit of
the impurity in the sample. The peak responses of the mo-
of the ion source affect the generation of ion species, the
lecular ions or the characteristic fragment ions and product
species transported to the mass analyzer, and the relative in-
tensity. The parameters related to the mass analyzer in- ions generated from the specified impurity in the sample so-
lution should be compared with those of the ions generated
fluence the peak width, mass accuracy, resolving power, and
sensitivity, and the detector parameters affect the signal in- from the substance in the standard solution. To obtain more

precise values, the method in which the stable isotope-la- by the ICP are separated into those with each m/z value and
beled compound of the analyte is added to the sample solu- the intensities of the separated ions are counted with a mass
tion as the internal standard is also important. When the test spectrometer. Compared to ICP-AES, ICP-MS is higher
is performed using mass spectrometry in combination with, sensitive, and with it an isotope analysis can be performed.
for example, chromatography, a system suitability test ICP-AES and ICP-MS are both excellent trace analysis
should also be required in accord with the chromatography. methods specific for the inorganic impurities or coexisting
elements in drug substances and drug products. Therefore,
using these methods, qualitative and quantitative analyses
can be performed not only for alkaline/alkaline-earth metals
2.63 Inductively Coupled and heavy metals, but also for many elements for which ade-
Plasma-Atomic Emission quate control is required to ensure the safety of pharmaceu-
tical products. It would be useful for the quality assurance of
Spectrometry and Inductively drug substances to apply these methods to the profile ana-
Coupled Plasma-Mass Spectrometry lyses of inorganic elements contained in the substances, be-
cause these methods enable the simultaneous analysis of
Inductively coupled plasma-atomic emission spectrometry many elements.
(ICP-AES) and inductively coupled plasma-mass spectro-
1. Instruments
metry (ICP-MS) are elemental analysis methods in which in-
1.1. Instrument Configuration of ICP-AES
ductively coupled plasma (ICP) is used as the excitation
An ICP-AES is composed of an excitation source, a sam-
source or the ion source.
ple introduction system, a light emission unit, a spectro-
ICP is an excitation source composed of high-temperature
scope, a photometer and a data processor.
argon plasma with intense thermal energy, which is formed
The excitation source consists of a high-frequency power
by the inductive coupling method. The atoms contained in
generator, a control circuit to supply and control the electric
the sample solution are excited when the solution is sprayed
energy to the light emission unit, and a gas source. The sam-
into the plasma. ICP-AES is the method used to measure the
ple introduction system, the main components of which are a
atomic emission spectrum of the light emitted from the plas-
nebulizer and a spray chamber, is used for introducing sam-
ma at the time and to identify and analyze the contents of
ple solutions to the light emission unit after nebulizing the
elements contained in the sample by determining the wave-
solutions.
length and intensity of its spectral lines. Since ICP is also a
The main components of the light emission unit, in which
good ionization source, the atoms in the sample solution are
the elements containing in the sample solution are atomized
ionized when the solution is sprayed into the plasma. ICP-
and excited to induce light emission, are a torch and a high-
MS is the method used to measure the mass spectrum of the
frequency induction coil. The torch has a triple tube struc-
element ions generated by ICP at the time by separating the
ture, and the sample solution is introduced through the
element ions into those with each m/z value and counting the
central tube. Argon gas is used to form the plasma and to
intensities of ion peaks using a mass spectrometer as the de-
transport the sample solution. For the observation method
tector.
of the light emitted from the light emission unit, there are
When intense energy is added to an atom from the out-
two viewing modes: the lateral viewing mode in which the
side, the peripheral electrons of the atom would transit to an
radial light of the plasma is observed, and the axial viewing
excited state by absorbing certain energy. The electron in the
mode in which the central light of the plasma is observed.
excited state would release the absorbed energy as the light
The spectroscope separates the light from the light emis-
when it returns to its ground state. The light released at the
sion unit to the spectral lines, and is composed of optical
time has a frequency n (or wavelength l) characteristic of
devices such as a light-converging system and diffraction
each element. When h is Planck's constant and c is the veloc-
grating. There are two types of spectrometers: wavelength-
ity of light, the energy DE of the released light is expressed
scanning spectrometers (monochromators) and simultane-
by the following equation.
ously measuring spectrometers (polychromators) of the
DE ＝ hn ＝ hc/l wavelength-fixed type. In addition, it is necessary to form a
vacuum or to substitute the air in the chamber of the pho-
Since there are many excited states with various energy
tometer with argon or nitrogen gas, when it is required to
levels to which peripheral electrons might transit, many
measure the spectral lines of the vacuum ultraviolet region
emission lines with various levels of energy can generate
(190 nm or shorter).
from one element, although some lines are strong and others The photometer, which consists of a detector and a signal
are weak. However, there is only a limited number of emis-
processing system, transduces the light energy of incident
sion lines observed in the ultraviolet/visible region and with
light to the electric signal proportional to the intensity of the
enough sensitivity for the qualitative and quantitative ana- light. For the detector, a photomultiplier or a semiconductor
lyses of each element. Since each element exhibits its own
detector is used.
spectral line with a characteristic frequency (or wavelength) The data processor is used to process the data obtained by
in the atomic emission spectrum, the elements contained in
the measurements, and it displays the calibration curves and
the sample solution can be identified by determining the
measurement results.
wavelengths of spectral lines in the spectrum. Quantitative 1.2. Instrument Configuration of ICP-MS
analyses of the elements in the sample solution can also be
An ICP-MS system is composed of an excitation source, a
performed by determining the intensity of the spectral line sample introduction system, an ionization port, an interface,
characteristic of each element. The elemental analysis
an ion lens, a mass analyzer, an ion detector and a data proc-
method using this principle is ICP-AES.
essor.
ICP-MS is the elemental analysis method alternative to The excitation source, sample introduction system and
optical analysis methods such as atomic absorption spectro-
ionization port have the same configuration as their counter-
metry and ICP-AES. In ICP-MS, the element ions generated parts in an ICP-AES system.

The interface is the boundary component for introducing up the instrument for 15 – 30 min. The operating parameters
the ions generated by the plasma under atmospheric pressure should usually be set as follows: high frequency power, 0.8 –
into a high-vacuum mass analyzer, and is composed of the 1.4 kW; argon gas flow rate, 10 – 18 L/min for the coolant
sampling cone and skimmer cone. gas (plasma gas), 0 – 2 L/min for the auxiliary gas, and 0.5 –
The ion lens brings the ions introduced via the interface 2 L/min for the carrier gas. In the lateral viewing mode, the
into focus and helps introduce the focused ions into the mass point for measuring the light emitted from the plasma
analyzer efficiently. should be set within the range of 10 – 25 mm from the top
For the mass analyzer, a common choice is a quadrupole edge of the induction coil, and the aspiration rate of the
mass analyzer. The interference caused by the polyatomic sample solution should be set at 0.5 – 2 mL/min. In the axial
ions described later can be suppressed by placing a col- viewing mode, the optical axis should be adjusted so that the
lision/reaction cell within the vacuum region before the mass maximum value can be obtained for the intensity of emission
analyzer, and introducing a gas such as hydrogen, helium, line measured. The integration time should be set within the
ammonia or methane into the cell. range of one to several tens of seconds, taking the stability
The ion detector transduces the energy of the ions that of the intensity of the emission line measured into account.
reached the detector to an electric signal which is amplified When a test using an ICP-AES system is defined in a JP
by the multiplier. The data processor is used to process the monograph, the operating conditions such as the analytical
data of the electric signal from the ion detector, and to dis- line (nm), high-frequency power (kW), and argon gas flow
play the calibration curves and measurement results, etc. rate (L/min) should be prescribed in the monograph.
However, it is necessary to optimize the operating conditions
2. Pretreatment of Sample
individually for each instrument and for each viewing mode
When the samples to be analyzed are organic compounds
used for the measurement.
such as pharmaceutical drug substances, they are usually
3.3. Interference and Its Suppression or Correction
digested and ashed by the dry ash method or the wet diges-
In the term ICP-AES, the word ``interference'' is used as a
tion method, and the sample solutions for ICP-AES or ICP-
general term that indicates the influence of the coexisting
MS are prepared by dissolving the residues in small quanti-
components or matrix on the measurement results. Various
ties of nitric acid or hydrochloric acid. When a sample is
interferences are roughly classified as either non-spectral in-
difficult to digest in the usual manner, the sample can be
terference (such as physical interference and ionization inter-
sealed in a closed, pressurized container and digested using
ference) or spectral interference. Their effects can be elimi-
microwave digestion equipment. Although liquid samples
nated or reduced by applying the appropriate suppression or
containing small amounts of organic solvents can be intro-
correction methods for the measurement.
duced directly into an ICP-AES or ICP-MS instrument with-
Physical interference means that the measurement results
out pretreatment, another alternative is introducing oxygen
are influenced by the difference between the spray efficien-
as the option gas to prevent the build-up of carbon generated
cies of the sample solution and the standard solution used
from the solvent onto the torch and the interface by con-
for its calibration in the light emission unit, when the physi-
tributing to the incineration of organic solvents.
cal properties (such as viscosity, density and surface tension)
3. Operation of ICP-AES of the solutions differ. The effective methods for eliminating
The operation of an ICP-AES system is as follows. The or reducing this type of physical influence are as follows.
argon plasma is formed by setting the argon gas flow at the The sample solution should be diluted to the level at which
specified rate and turning the high-frequency power source such interference will not occur; the properties between the
on. After confirmation that the state of the plasma is stable, sample solution and the standard solution used for its
a quantity of the sample solution or the standard solution calibration should be matched as much as possible (matrix-
prepared by the method prescribed in the monograph is in- matching method); and the internal standard method (inten-
troduced into the instrument via the sample injection port, sity ratio method) or the standard addition method should
and the emission intensity of the analytical line specified for be used.
the element is measured. When it is necessary to perform a Ionization interference indicates the influence due to the
test for the confirmation or identification of some elements, change in the ionization rate caused by the increase of elec-
the emission spectrum in the wavelength range in which ana- tron density in the plasma, which is induced by a large num-
lytical lines specified for the elements that appear is meas- ber of electrons generated from the elements coexisting in
ured. the sample solution at a high concentration. The suppression
3.1. Performance Evaluations of Spectrometers or correction method against the ionization interference is
Since each spectrometer requires its own calibration essentially the same as the method used in the case of physi-
method that accords with its properties, a wavelength cal interference. The measurement conditions with low ioni-
calibration must be performed according to the procedure zation interference can also be set by the selection and
indicated by the manufacturer. adjustment of the observation method of emitted light, the
For expressing the wavelength-resolving power of a spec- height for viewing, high-frequency power and carrier gas
trometer, the half height width of the analytical line in the flow rate, and so on.
emission spectrum of a specified element is usually defined Spectral interference is the phenomenon which influences
in the form of ``not more than xxx nm (a constant value).'' the analytical results of the sample by overlapping the vari-
The following emission lines, from the line with a low wave- ous emission lines and/or the light with a continuous spec-
length to that with a high wavelength, are usually selected trum with the analytical line of the analyte element. To avoid
for the above purpose: arsenic (As: 193.696 nm), manganese this type of interference, it is necessary to select another ana-
(Mn: 257.610 nm), copper (Cu: 324.754 nm) and barium lytical line which will not suffer from the spectral interfer-
(Ba: 455.403 nm). ence. However, when no suitable analytical lines can be
3.2. Optimization of Operating Conditions found, it is necessary to carry out the correction of the spec-
The operating conditions usually adopted are as follows. tral interference. In addition, when the pretreatment of the
The operating conditions of the instrument should be op- organic samples is not sufficient, the molecular band spectra
timized after stabilizing the state of the plasma by warming (NO, OH, NH, CH, etc.) derived from nitrogen, oxygen,

hydrogen and carbon remaining in the sample solution might analyte element, for example, the overlap of 40Ar with 40Ca
appear at the wavelength close to the analytical line of the and 204Hg with 204Pb. Since argon plasma is used as the
analyte element, and could interfere with the analysis. ionization source, the polyatomic ions such as 40Ar16O,
40Ar16O1H, 40Ar might be generated, and they would inter-
2
4. Operation of ICP-MS
fere with the measurements of 56Fe, 57Fe and 80Se, respec-
In the operation of an ICP-MS system, after the confirma-
tively. When an instrument equipped with a collision/reac-
tion that the state of the plasma is stable, the optimization of
tion cell is used, these polyatomic ions can be decreased in
the instrument is performed and the system's suitability is
the cell. Doubly charged ions are the ions exhibiting their ion
confirmed. A quantity of the sample solution or the stand-
peaks at 1/2 the m/z value of the corresponding monovalent
ard solution prepared by the method prescribed in the mono-
ions, and the interference might occur when the element with
graph is introduced, and the ion count numbers of the signal
an isotope with the mass number twice that of the analyte
at the m/z value specified for the analyte element are deter-
element might be present in the sample solution.
mined. When it is necessary to perform a test for the con-
Non-spectral interference includes not only the physical
firmation or identification of some elements, the mass
interference and the ionization interference as in the case of
spectrum in the m/z value range specified for the analyte
the ICP-AES, but also the matrix interference unique to
elements is measured.
ICP-MS. Matrix interference is the phenomenon in which
4.1. Performance Evaluation of Mass Spectrometer
the ion count numbers of every analyte element generally
The performance evaluation items for mass spectrometers
decrease when large amounts of other elements might co-
are the mass accuracy and the mass resolving power. The
exist in the sample solution. This tendency becomes more
mass accuracy should be adjusted by matching the m/z value
significant when the mass number of a co-existing element is
of the mass axis of the mass analyzer to that of the standard
larger and its concentration is higher, and when the mass
element in the standard solution for the optimization speci-
number of the analyte element is smaller. The extent of non-
fied in the operating conditions section of the monograph.
spectral interference can be estimated based on the recovery
With quadrupole mass spectrometers, it is preferable that the
rate obtained by adding a known amount of the analyte ele-
mass accuracy be within ±0.2. For the mass resolving
ment to the unknown sample. When it is found that the
power, it is preferable that the peak width at 10z of the
recovery rate is low and the reliability of the analysis is not
peak height in the observed ion peak is not more than 0.9.
assured, the correction should be carried out by using the in-
4.2. Optimization of Operating Conditions
ternal standard method or the standard addition method.
When a limit test or a quantitative test is performed, the
For ICP-MS in particular, the influence of non-spectral in-
sensitivity, background and generation ratio of oxide ions
terference can be reduced by using the isotope dilution
and doubly charged ions defined below should be optimized
method.
previously to assure that the performance of the instrument
is suitable. For the optimization of operating conditions, the 5. System Suitability
solutions of the elements which represent the low mass num- When a limit test or quantitative test is performed using
ber elements, intermediate mass number elements and high these methods, it is necessary to confirm that the per-
mass number elements and are unlikely to be contaminated formance of the instrument is suitable by carrying out a sys-
from the environment (e.g., 7Li, 9Be, 59Co, 89Y, 115In, 140Ce, tem suitability test as defined below in advance of the limit
205Tl and 209Bi) are usually used as the standard solutions test or quantitative test.
after adjusting to adequate concentrations. 5.1. Evaluation for Required Detectability and Linearity
The sensitivity is evaluated by the ion count numbers per In an evaluation of an ICP-MS system for the required
second of integration time (cps). When a limit test or quan- detectability and linearity, a solution is prepared in which
titative test is performed, it is preferable to have the sensi- the analyte element is not contained and the standard solu-
tivity of several tens of thousands cps per 1 mg/L (ppb) for tion with the concentration of the specification limit of the
each element with a low mass number, intermediate mass analyte element, and these solutions are used as the blank so-
number or high mass number. lution and the solution for the system suitability test, respec-
For the background, it is preferable to be not more than tively. The spectra obtained with these solutions are meas-
10 cps, when the measurement is performed at the m/z value ured according to the test conditions optimized individually
at which no elements exist naturally (e.g., m/z value of 4, 8 for each instrument, and it must be confirmed whether the
or 220). emission line (or ion peak) of the analyte element is clearly
For the generation ratio of oxide ions and doubly charged observed at the specified wavelength (or m/z value) in the so-
ions, the count numbers of oxide ions (e.g., 140Ce: 140Ce16O＋, lution for system suitability test when compared with the
m/z 156), doubly charged ions (140Ce2＋, m/z 70) and blank solution. In this regard, the limit of the analyte ele-
monovalent ions (140Ce＋, m/z 140) should be measured, and ment should be specified at the concentration of more than
the generation ratios are calculated by dividing the ion count the quantitation limit (10s). The test for required detectabil-
number of the oxide ions and doubly charged ions by that of ity is not required in the assay.
the monovalent ions. It is preferable that the generation For the evaluation of linearity, it should be confirmed that
ratio of oxide ions (i.e., 140Ce16O＋/140Ce＋) is not more than the correlation coefficient of the calibration curve prepared
0.03 and that of doubly charged ions (i.e., 140Ce2＋/140Ce＋) is by the procedure described in the section below, ``6.2. Quan-
not more than 0.05. titative Analysis'' is not less than 0.99. The confirmation of
4.3. Interferences and their Suppression or Correction linearity is not required in quantitative analyses in the sec-
In measurements using ICP-MS, attention must be paid to tion 6.1. or when isotope dilution in the section 6.2. is per-
spectral interference and non-spectral interference. formed.
Spectral interference includes isobaric interference and 5.2. Evaluation for System Repeatability
the interference caused by overlapping the mass spectrum of Unless otherwise specified, when the test is repeated six
the analyte element with those of polyatomic ions or doubly times using the solution with the lowest concentration among
charged ions. Isobaric interference is the interference by the those used for plotting the calibration curve according to the
isobaric element with the atomic mass adjacent to that of the test conditions optimized individually for each instrument, it

should be confirmed that the relative standard deviation of in ICP-AES, the following requirements are to be met for
the observed values for the emission intensity (or ion count the internal standard element: the changes in the emission in-
number) of the analyte element is not more than the speci- tensity due to the measurement conditions and properties of
fied value (e.g., not more than 3z for an assay, and not the solution should be similar to those of the analyte ele-
more than 5z for a purity test). ment, and the emission line which does not cause spectral in-
terference to the analytical line of the analyte element should
6. Qualitative and Quantitative Analyses
be selected for the analysis. In contrast, in ICP-MS, it is
6.1. Qualitative Analyses
preferable to select an internal standard element which does
In ICP-AES, when the wavelengths and relative emission
not cause spectral interference to the analyte element and has
intensities of multiple emission lines from the sample solu-
the ionization efficiency and mass number equivalent to the
tion conform to those of the emission lines from the ele-
analyte element.
ments contained in the standard solution, the presence of the
(iii) Standard addition method: Take 4 portions or more
elements can be confirmed. In addition, the library of ICP-
of the sample solution with the same volume, and prepare
emission spectra attached to each instrument or the wave-
the following solutions; the solution in which the analyte ele-
length table of the spectra can also be used instead of the
ment is not added; the standard solutions for plotting
standard solution. Since the mass number region covering all
calibration curve in which the analyte element is added at
the elements can be scanned in a short time in ICP-MS, the
different concentrations (3 or more). Measure the emission
elements contained in the sample solution can be analyzed
intensities at the specified analytical line or the ion count
qualitatively based on the m/z value of the ion peak in the
numbers at the specified m/z value for these solutions. Plot
mass spectrum obtained from the sample solution.
the obtained data against the concentrations calculated from
It would be feasible to list the metal catalysts and inorgan-
the added amount of the analyte element. Calibrate the con-
ic elements that might be contained in the sample as impuri-
centration of the analyte element in the sample solution from
ties, and for some elements (such as arsenic and lead) it
the absolute value of the horizontal axis (concentration)-in-
might be necessary to monitor them in a routine manner
tercept of the regression line.
from the point of view of safety, and to carry out the profile
In ICP-AES, this method is useful for the correction of
of these inorganic impurities as a part of the manufacturing
non-spectral interference caused by coexisting substances in
controls for a drug substance. In addition, the standard solu-
the sample solution, and it is applicable only to the cases in
tion of each element should be prepared at an appropriate
which spectral interference does not exist, or the background
concentration considering the acceptance limit of each ele-
and the spectral interference are exactly corrected and the
ment to be specified separately.
relationship between the emission intensity and the concen-
6.2. Quantitative Analyses
tration shows good linearity. In ICP-MS, this method is use-
The quantitative analysis of an inorganic element in the
ful for the correction of non-spectral interference caused by
sample solution is usually performed by one of the following
coexisting substances in the sample solution, and it is applic-
methods based on the emission intensity or ion count num-
able only to the cases in which the spectral interference is ex-
bers obtained by the integration of measurement data in a
actly corrected and the relationship between the ion count
specified time.
number and the concentration shows good linearity down to
(i) Calibration curve method: Prepare standard solutions
the low concentration region.
for plotting a calibration curve with different concentrations
(iv) Isotope dilution method: Isotope dilution method is
(four or more) of the analyte element. Using these standard
applicable only to the ICP-MS. The concentration of the
solutions, the emission intensities at the analytical line speci-
analyte element is determined from the change of the isotope
fied for the analyte element by ICP-AES or the ion count
composition ratio of the element by adding a substance con-
numbers at the m/z value specified for the analysis of the
taining a concentrated isotope with a known isotope compo-
element by ICP-MS are measured. The data obtained are
sition that is different from the natural composition to the
then plotted against the concentrations, and this plot is used
sample solution. It is applicable only to the element which
as the calibration curve. The concentration of the analyte
has two or more stable isotopes naturally and is able to per-
element in the sample solution is determined by using this
form the isotope analysis. It is the feature of this method
calibration curve.
that the analytical precision is high and is not influenced by
(ii) Internal standard method: Prepare standard solu-
non-spectral interference, because the quantitation can be
tions for plotting a calibration curve with a fixed concentra-
performed only by adding an adequate amount of a sub-
tion of the internal standard element and different concen-
stance containing a concentrated isotope and measuring the
trations (four or more) of the analyte element. Using these
isotope composition ratio of the sample solution.
standard solutions, the ratios of the emission intensities (or
ion count numbers) of the analyte element to those of the in- 7. Note
ternal standard element are determined. The data obtained Water and reagents and the standard solutions used in this
are plotted against the concentrations, and this plot is used test are as follows.
as the calibration curve. The internal standard element is (i) For water, water for an ICP analysis should be used.
also added to the sample solution, so that the concentration It should be verified prior to the test that the impurities con-
of internal standard element in the solution becomes the tained in the water do not interfere with the analysis of the
same as that in the standard solution. The concentration of analyte element. Here, the water for an ICP analysis has the
the analyte element in the sample solution is determined by electric conductivity of 1 mS･cm－1 or less (259C).
using the calibration curve plotted above. (ii) Reagents that are suitable for ICP analyses and are
Before this method is applied, it is necessary to verify that of high quality should be used.
the internal standard element to be added is not contained in (iii) For argon gas, either liquefied argon or compressed
the sample solution. If the internal standard element to be argon gas with the purity of 99.99 volz or higher should be
added is present in the sample solution, it is necessary to used.
verify that the contaminated amount of standard element is (iv) For the standard solutions, they should be prepared
negligible compared to the amount to be added. In addition, by diluting the Standard Solution (e.g., the Standard Solu-

tion defined in the JP, or a standard solution with a concen- 1.1. Release of monosaccharides
tration certified by a public institution or scientific organiza- 1.1.1. Neutral and amino sugars
tion) to the specified concentration using the water for ICP Neutral and amino sugars are generally released by acid
analysis. However, in cases in which interference with the hydrolysis. The hydrolysis rate is dependent on the identity
analysis might occur, it is preferable to match the properties of monosaccharide and linkage, and the degradation rate of
of the standard solution to those of the sample solution. released monosaccharide is different between the individual
(v) When a standard solution containing multiple ele- monosaccharides. Thus, the conditions of acid hydrolysis
ments is prepared, a combination of the test solutions and should be optimized in order to release and recover the
elements should be selected so that precipitation and/or monosaccharides with high efficiency. Standard materials of
mutual interference does not occur. neutral and amino sugars should be treated as tested sam-
ples.
1.1.2. Sialic acids
Sialic acids are released by mild acid hydrolysis or siali-
2.64 Glycosylation Analysis of dase digestion, because they are labile. In general, sialidases
Glycoprotein with broad substrate specificity, such as those from Ar-
throbacter ureafaciens or Clostridium perfringens may be
Glycosylation analysis is a method to confirm the con- used.
sistency of the oligosaccharides attached to glycoprotein 1.2. Quantitative analysis of monosaccharides
drug. The main types of oligosaccharides observed in Released monosaccharides are quantitatively analyzed by
glycoprotein drug are N-linked oligosaccharides, which are several methods, including high-pH anion-exchange chroma-
attached to asparagine residue, and O-linked oligosaccha- tography/pulsed amperometric detection as underivatized
rides, which are attached to serine or threonine residues. monosaccharides, and liquid chromatography with fluoro-
Oligosaccharides have a diverse variety in structure, and metoric or UV detection after derivatization. Content of
attached oligosaccharides may be heterogeneous even in each monosaccharide is determined by internal standard
same glycoprotein and at same attachment site. Glycoprotein method or absolute calibration method. For derivatization
generally consists of a mixture of heterogeneous molecules of neutral and amino sugar, 2-aminobenzoic acid, 2-
(glycoforms), which differ only in glyosylation. Some aminopyridine, ethyl 4-aminobenzoate, 3-methyl-1-phenyl-
oligosaccharides of glycoprotein may involve in stabilization 5-pyrazolone may be used. For derivatization of sialic acid,
of glycoprotein structure, prevention of enzymatic degrada- 1,2-diamino-4,5-methylenedioxybenzene, or 1,2 phenyl-
tion, modulation of the biological activities, clearance from enediamine can be used. Analysis of derivatized monosac-
the bloodstream, intake into the cell, and immunogenicity. charides can be performed by reversed-phase chromatogra-
Since the oligosaccharide structures and their heterogeneity phy or anion-exchange chromatography with formation of
in the recombinant glycoproteins may change depending on borate complexes, etc. The analytical results are typically
the cell lines, culture conditions, etc., it is important to en- expressed as molar ratio of individual monosaccharide to
sure consistency of the glycosylation for ensurement of the glycoprotein, and then confirmed to be within the specified
efficacy and safety of glycoprotein drug. Methods to evalu- criteria.
ate the glycosylation of glycoprotein are classified into 1)
2. Oligosaccharide analysis/Oligosaccharide profiling
analysis of released monosaccharides obtained after degra-
Oligosaccharide analysis is a method to confirm consisten-
dation of glycoprotein (monosaccharide analysis), 2) analysis
cy of the oligosaccharide identities, structures and distribu-
of released oligosaccharides obtained from the glycoprotein
tion. Oligosaccharides attached to glycoprotein are released
(oligosaccharide analysis/oligosaccharide profiling), 3)
by enzymatic or chemical treatments, and then the released
analysis of glycopeptides obtained after proteolytic treat-
oligosaccharides were analyzed using liquid chromatography
ment of the glycoprotein (glycopeptide analysis), and 4)
<2.01>, capillary electrophoresis, mass spectrometry <2.62>
analysis of intact glycoprotein (glycoform analysis). In the
and in combination of them as underivatized or derivatized
setting specification for glycosylation analysis, methods
for the purpose of improvement in sensitivity and separa-
should be properly selected and used alone or in combina-
tion. The analytical results were obtained as chromatogram,
tion, in consideration of the structural characteristics in the
electropherogram or mass spectrum, respectively, and they
oligosaccharides, which affect efficacy and safety of the sub-
are referred to as oligosaccharide profile which provides
stance being tested.
oligosaccharide identities and distribution. If oligosaccha-
1. Monosaccharide analysis ride profiling is performed after decreasing heterogeneity by
Monosaccharide analysis is a method to provide the infor- exoglycosidase digestions for insufficient resolutions due to
mation of the identity and contents of monosaccharides, high oligosaccharide heterogeneity, the relationship of
which constitute the oligosaccharides attached to the oligosaccharide structures to the efficacy and safety of the
glycoprotein drugs, etc. The kind of the monosaccharides therapeutic glycoprotein are considered, and enzymes should
that constitute oligosaccharide is limited. Typically, amino be selected so that structures to be evaluated are not lost.
sugars, such as N-acetyl glucosamine and N-acetyl galactosa- 2.1. Release and purification of oligosaccharides
mine, neutral sugars, such as galactose, mannose, glucose Enzymatic or chemical treatment are used for releasing
and fucose, and sialic acids, such as N-acetyl neuraminic oligosaccharides from the glycoprotein. N-linked oligosac-
acid and N-glycolyl neuraminic acid, may be analyzed. charides are released by peptide N-glycosidase digestion or
Monosaccharide analysis consist of releasing monosaccha- hydrazinolysis, and O-linked oligosaccharides are released
rides from the glycoprotein and quantitative analysis of by alkaline b-elimination, hydrazine degradation, and endo
released monosaccharides. Monosaccharide analysis is gen- O-glycanase. Because releasing efficacy may be affected by
erally performed after isolation and purification of the identity of protein, glycosylation sites and oligosaccharide
glycoprotein using appropriate methods, because excipients structure, releasing conditions should be optimized for each
and salts can interfere the analysis. glycoprotein. Careful attention should be taken to the pos-
sibility of structural changes in oligosaccharides, such as loss

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
96 2.65 Methods for Color Matching / General Tests JP XVII
of sialic acids, anomerization at reducing end residues, and lyzed by mass spectrometer, where they are ionized by soft
successive degradation from reducing end (peeling reaction). ionization techniques, such as electrospray ionization or
There are several methods to recover the oligosaccharides matrix-assisted laser desorption/ionization, then separated
from the reaction mixtures after releasing, including deple- based on m/z values, and detected. Both of positive and
tion of protein by ice-cold ethanol precipitation and then ex- negative ion mode are available. Because ionization efficien-
traction from the supernatant, and solid phase extraction cy of oligosaccharides depends on their structures, ionization
using a media, which oligosaccharides have adsoption to, polarity should be selected according to the characteristics of
etc. Reproducibility of oligosaccharide recovery should be the oligosaccharides. Mass spectrometry coupled with liquid
evaluated, and it should be confirmed that recovery does not chromatography or capillary electrophoresis provides not
differ between oligosaccharides. only elution time or migration time but also information of
2.2. Analysis of released oligosaccharides molecular mass, and allow us more specific oligosaccharide
Derivatization of released oligosaccharides is usually car- profiling. It is noted that the reproducibility of oligosaccha-
ried out by reacting aldehyde group at reducing end with ride profile obtained by mass spectrometry is lower than liq-
derivatizing agent. Given that oligosaccharide react with uid chromatography and capillary electrophoresis, and that
derivatizing agent stoichiometrically, relative peak responses sialic acid residues in the sialo-oligosaccharides tend to be
may suggest molar ratios of the oligosaccharides attached to lost in positive ion mode. Mass spectrometry should be used
the glycoprotein. It should be confirmed that derivatizing with consideration of the characteristics of oligosaccharides
method has sufficient reaction yield and reproducibility, and responsible for efficacy and safety.
that structural changes during derivatization, such as loss of
3. Glycopeptide analysis
sialic acid residues, are at minimum. If needed, excessive
Glycopeptide analysis is a method to provide the informa-
reagents are removed or derivatized oligosaccharides are
tion about site-specific glycosylation properties, such as the
purified not to affect the test results. It should be confirmed
degree of occupancy, oligosaccharide structures and heter-
that oligosaccharide profile does not change due to different
ogeneity. If particular oligosaccharides at specific sites affect
recovery rates. Test methods should be appropriately select-
biological activity or pharmacokinetics, glycopeptide analy-
ed considering structures and distribution of oligosaccha-
sis should be performed. Glycoprotein is digested by specific
rides which affect efficacy and safety.
protease, and resultant mixture of glycopeptides and pep-
Oligosaccharide profile obtained from the product under
tides are subjected to liquid chromatography coupled with
test is compared with that of reference material, treated in
mass spectrometer, and mass spectra of the glycopeptides are
the same conditions, and then it is confirmed that peaks of
obtained. Glycopeptide ion is assigned based on masses of
oligosaccharides that is considered important for efficacy
peptides and information of product ions obtained by tan-
and safety are comparable from the view point of peak posi-
dem mass spectrometry or multiple-stage mass spectrometry.
tion and peak response by visual inspection. Otherwise, rela-
Glycopptides may be fractionated by liquid chromatogra-
tive abundance of each oligosaccharide is calculated as a per-
phy, and then glycopeptides are subjected to mass spectro-
centage of the total peak response or as a relative peak
metry by offline coupling, or oligosaccharides released from
response, and then it is confirmed to be within the specified
glycopeptides were subjected to oligosaccharide analysis/
range.
oligosaccharide profiling using liquid chromatography or
2.2.1. Liquid chromatography <2.01>
capillary electrophoresis.
Oligosaccharides derivatized with a tag, such as 2-
aminobenzamide, 2-aminobenzoic acid, 2-aminopyridine, or 4. Glycoform analysis of glycoprotein
others, may be separated by chromatography based on Glycoform analysis is a method to confirm overall
hydrophilic interaction, reversed-phase, ion-exchange, or glycosylation characteristics and their consistency as
mix mode of them, and then detected using fluorometric glycoprotein. It is desirable to obtain the glycoform profiles
detector. Underivatized oligosaccharides may be analyzed to reflect oligosaccharide structures that play a role in
using high-pH anion-exchange chromatography/pulsed am- efficacy and safety of the glycoprotein. If the degree of
perometric detection. Analytical methods should be selected sialylation significantly contributes to the efficacy of the
and optimized according to the characteristics of the glycoprotein, isoelectric focusing, capillary isoelectric focus-
oligosaccharides to be tested. ing, capillary zone electrophoresis, or ion-exchange chroma-
2.2.2. Capillary electrophoresis tography, etc, may be performed to provide the charge-
Derivatizing agents with multiple negative charges, such as based glycoform profiles. Mass spectrometry provides the
8-aminopyrene-1,3,6-trisulfonic acid, are often used for glycoform profiles based on molecular mass. Size-exclusion
analysis of lower sialylated oligosaccharides, to reduce the chromatography, capillary gel electrophoresis and SDS-
time required for analysis. Derivatizing agents with low PAGE may be useful for confirmation of the glycosylation
negative charge, such as 2-aminobenzoic acid may be used status of a protein. Glycoform profile of the product under
for analysis of highly sialylated oligosaccharides in order to test is confirmed that peak position and response are com-
achieve the separation based on the number of sialylation. parable with that of similarly treated standard material, or
Electrolyte buffer containing borate may be used for adding that the distribution of glycorom is within the specified
negative charge and increasing resolution by forming ranges. If glycoprotein has high molecular mass and/or
oligosaccharide-borate complex. Derivatized oligosaccha- many glycosylation sites, it may be difficult to separate each
rides are separated by capillary zone electrophoresis using glycoform peak adequately. Separation and reproducibility
appropriate electrolyte buffer, and then detected using laser- of glycoform profile should be evaluated during method de-
induced fluorometric detector, etc. In general, capillary is velopment.
used with the inner wall surface modified using neutral poly-
mers covalently or dynamically in order to prevent electroos-
motic flow. pH and compositions of the electrolyte are
selected so that good separation is achieved. 2.65 Methods for Color Matching
2.2.3. Mass spectrometry <2.62>
Methods for Color Matching are applied to the purity test
Derivatized or underivatized oligosaccharides may be ana-

where the color of a test solution is examined by comparing Table 2.65-1 Composition of matching fluids for color A
with a matching fluid for color. to T
1. Matching fluids for color Matching Cobalt (II) Iron (III) Copper (II)
Matching fluids for color A to T are prepared by measur- Water
fluid for Chloride Chloride Sulfate
ing exactly the volume of three colorimetric stock solutions (mL)
color CS (mL) CS (mL) CS (mL)
and water as directed in Table 2.65-1 with a buret or a pipet
graduated to less than 0.1 mL, and mixing. Store the solu- A 0.1 0.4 0.1 4.4
tions in glass-stoppered bottles. B 0.3 0.9 0.3 3.5
For each of the matching fluids of B-series (B1 to B9), BY- C 0.1 0.6 0.1 4.2
series (BY1 to BY7), Y-series (Y1 to Y7), GY-series (GY1 to D 0.3 0.6 0.4 3.7
GY7) and R-series (R1 to R7), the primary matching solu- E 0.4 1.2 0.3 3.1
tions for individual color are prepared first by mixing the F 0.3 1.2 — 3.5
three colorimetric stock solutions as directed in Table 2.65-2, G 0.5 1.2 0.2 3.1
then mix the primary matching solution for corresponding H 0.2 1.5 — 3.3
color as directed in Table 2.65-3 to prepare desired matching I 0.4 2.2 0.1 2.3
fluid for color. J 0.4 3.5 0.1 1.0
K 0.5 4.5 — —
2. Procedure L 0.8 3.8 0.1 0.3
Compare a test solution with a matching fluid for color M 0.1 2.0 0.1 2.8
specified in monograph according to the following manners, N — 4.9 0.1 —
and confirm that the test solution has no more color than the O 0.1 4.8 0.1 —
specified matching fluid for color. P 0.2 0.4 0.1 4.3
When the matching fluids for color A to T are used, unless Q 0.2 0.3 0.1 4.4
otherwise specified, place the test solution and the matching R 0.3 0.4 0.2 4.1
fluid for color in Nessler tubes, and view transversely against S 0.2 0.1 — 4.7
a white background. T 0.5 0.5 0.4 3.6
When the matching fluids for color of B-series, BY-series,
Y-series, GY-series or R-series are used, compare the color
by the following two methods, and state the used method
Table 2.65-2 Primary matching solutions for color used for
number in the monograph. A solution is colourless if it has
preparation of a series of matching fluids for color (B-
the appearance of water or the solvent or is not more inten-
series, BY-series, Y-series, GY-series, R-series)
sely coloured than matching solution B9.
Method 1: Place separately 2.0 mL each of a test solution Primary Volumes to mix (mL)
and a reference liquid such as water, solvent or the matching matching
fluid for color specified in the monograph in clear and color- solutions for Iron (III) Cobalt (II) Copper (II) Diluted dilute
less glass test tubes, 12 mm in outside diameter, and compare individual Chloride Chloride Sulfate hydrochloric
color CS CS CS acid 1 in 10
the color by viewing transversely against a white background
under scattering light. Brown primary
Method 2: Place separately a test solution and a reference matching solu- 3.0 3.0 2.4 1.6
liquid such as water, solvent or the matching fluid for color tion
specified in the monograph in clear and colorless flat-bottom Brownish yel-
test tubes, 15 – 25 mm in internal diameter, so that the depth low primary
of the layer is 40 mm, and compare the color by viewing ver- 2.4 1.0 0.4 6.2
matching solu-
tically against a white background under scattering light. tion
3. Colorimetric stock solutions Yellow prima-
Cobalt (II) Chloride CS: Dissolve 65 g of cobalt (II) chloride ry matching 2.4 0.6 0.0 7.0
hexahydrate in 25 mL of hydrochloric acid and water to solution
make 1000 mL. Pipet 10 mL of this solution, add water to Greenish yel-
make exactly 250 mL. Pipet 25 mL of the solution, add 75 low primary
mL of water and 50 mg of murexide-sodium chloride indica- 9.6 0.2 0.2 0.0
matching solu-
tor, and add dropwise diluted ammonia solution (28) (1 in tion
10) until the color of the solution changes from red-purple to
Red primary
orange-yellow. Titrate <2.50> with 0.01 mol/L disodium di-
matching solu- 1.0 2.0 0.0 7.0
hydrogen ethylenediamine tetraacetate VS until the color of tion
the solution changes from yellow to red-purple, after the ad-
dition of 0.2 mL of diluted ammonia solution (28) (1 in 10)
near the end-point.
Each mL of 0.01 mol/L disodium dihydrogen Copper (II) Sulfate CS: Dissolve 65 g of copper (II) sulfate
ethylenediamine tetraacetate VS pentahydrate in 25 mL of hydrochloric acid and water to
＝ 2.379 mg of CoCl2.6H2O make 1000 mL. Pipet 10 mL of this solution, and add water
to make exactly 250 mL. Pipet 25 mL of this solution, add
According to the titrated value, add diluted hydrochloric
75 mL of water, 10 mL of a solution of ammonium chloride
acid (1 in 40) to make a solution containing 59.5 mg of
(3 in 50), 2 mL of diluted ammonia solution (28) (1 in 10)
cobalt (II) chloride hexahydrate (CoCl2.6H2O: 237.93) in
and 50 mg of murexide-sodium chloride indicator. Titrate
each mL, and use. Store the solution in a glass-stoppered
<2.50> with 0.01 mol/L disodium dihydrogen ethylene-
bottle.
diamine tetraacetate VS until the color of the solution

1.1. Method 1: Measurement in a graduated cylinder cup and determine the mass (m) of the powder to the nearest
1.1.1. Procedure 0.1 per cent. Calculate the bulk density in g per mL by the
Pass a quantity of powder sufficient to complete the test formula m/V0 in which V0 is the volume of the cup and
through a sieve with apertures greater than or equal to 1.0 record the average of 3 determinations using 3 different
mm, if necessary, to break up agglomerates that may have powder samples.
formed during storage; this must be done gently to avoid 1.3. Method 3: Measurement in a vessel
changing the nature of the material. Into a dry graduated 1.3.1. Apparatus
cylinder of 250 mL (readable to 2 mL), gently introduce, The apparatus consists of a 100 mL cylindrical vessel of
without compacting, approximately 100 g of the test sample stainless steel with dimensions as specified in Fig. 3.01-2.
(m) weighed with 0.1 per cent accuracy. Carefully level the 1.3.2. Procedure
powder without compacting, if necessary, and read the Pass a quantity of powder sufficient to complete the test
unsettled apparent volume (V0) to the nearest graduated through a 1.0 mm sieve, if necessary, to break up agglomer-
unit. Calculate the bulk density in g per mL by the formula ates that may have formed during storage and allow the ob-
m/V0. Generally, replicate determinations are desirable for tained sample to flow freely into the measuring vessel until it
the determination of this property. overflows. Carefully scrape the excess powder from the top
If the powder density is too low or too high, such that the of the vessel as described for Method 2. Determine the mass
test sample has an untapped apparent volume of either more (m0) of the powder to the nearest 0.1 per cent by subtraction
than 250 mL or less than 150 mL, it is not possible to use of the previously determined mass of the empty measuring
100 g of powder sample. Therefore, a different amount of vessel. Calculate the bulk density (g/mL) by the formula
powder has to be selected as test sample, such that its m0/100 and record the average of 3 determinations using 3
untapped apparent volume is 150 mL to 250 mL (apparent different powder samples.
volume greater than or equal to 60 per cent of the total
2. Tapped density
volume of the cylinder); the mass of the test sample is speci-
The tapped density is an increased bulk density attained
fied in the expression of results.
after mechanically tapping a container containing the pow-
For test samples having an apparent volume between 50
der sample.
mL and 100 mL, a 100 mL cylinder readable to 1 mL can be
The tapped density is obtained by mechanically tapping a
used; the volume of the cylinder is specified in the expression
graduated measuring cylinder or vessel containing the pow-
of results.
der sample. After observing the initial powder volume or
1.2. Method 2: Measurement in a volumeter
mass, the measuring cylinder or vessel is mechanically
1.2.1. Apparatus
tapped, and volume or mass readings are taken until little
The apparatus (Fig. 3.01-1) consists of a top funnel fitted
further volume or mass change is observed. The mechanical
with a 1.0 mm sieve. The funnel is mounted over a baffle
tapping is achieved by raising the cylinder or vessel and
box containing four glass baffle plates over which the pow-
allowing it to drop, under its own mass, a specified distance
der slides and bounces as it passes. At the bottom of the baf-
by either of 3 methods as described below. Devices that
fle box is a funnel that collects the powder and allows it to
rotate the cylinder or vessel during tapping may be preferred
pour into a cup mounted directly below it. The cup may be
to minimize any possible separation of the mass during tap-
cylindrical (25.00 ± 0.05 mL volume with an inside diameter
ping down.
of 30.00 ± 2.00 mm) or cubical (16.39 ± 0.20 mL volume
2.1. Method 1
with inside dimensions of 25.400 ± 0.076 mm).
2.1.1. Apparatus
1.2.2. Procedure
The apparatus (Fig. 3.01-3) consists of the following:
Allow an excess of powder to flow through the apparatus
(i) a 250 mL graduated cylinder (readable to 2 mL) with
into the sample receiving cup until it overflows, using a mini-
a mass of 220 ± 44 g,
mum of 25 cm3 of powder with the cubical cup and 35 cm3 of
(ii) a settling apparatus capable of producing, in 1 min,
powder with the cylindrical cup. Carefully, scrape excess
either nominally 250 ± 15 taps from a height of 3 ± 0.2
powder from the top of the cup by smoothly moving the
mm, or nominally 300 ± 15 taps from a height of 14 ± 2
edge of the blade of a spatula perpendicular to and in con-
mm. The support for the graduated cylinder, with its holder,
tact with the top surface of the cup, taking care to keep the
has a mass of 450 ± 10 g.
spatula perpendicular to prevent packing or removal of pow-
2.1.2. Procedure
der from the cup. Remove any material from the side of the
Proceed as described above for the determination of the
bulk volume (V0).
Secure the cylinder in the holder. Carry out 10, 500 and
1250 taps on the same powder sample and read the corre-

Fig. 3.01-1 Volumeter Fig. 3.01-2 Measuring vessel (left) and cap (right)

2.2. Method 2 method to determine specific surface area (the total surface
2.2.1. Procedure area of powder per unit mass) of a pharmaceutical powder
Proceed as directed under Method 1 except that the sample by using gas adsorption method. The specific sur-
mechanical tester provides a fixed drop of 3 ± 0.2 mm at a face area of a powder is determined by physical adsorption
nominal rate of 250 taps per minute. of a gas on the surface of the solid and by calculating the
2.3. Method 3 amount of adsorbate gas corresponding to a monomolecular
2.3.1. Procedure layer on the surface. Physical adsorption results from rela-
Proceed as described in the method for measuring the bulk tively weak forces (van der Waals forces) between the adsor-
density using the measuring vessel equipped with the cap bate gas molecules and the adsorbent surface of the test
shown in Fig. 3.01-2. The measuring vessel with the cap is powder. The determination is usually carried out at the tem-
lifted 50-60 times per minute by the use of a suitable tapped perature of liquid nitrogen. The amount of gas adsorbed can
density tester. Carry out 200 taps, remove the cap and care- be measured by a volumetric or continuous flow procedure.
fully scrape excess powder from the top of the measuring
1. Measurements
vessel as described in Method 3 for measuring the bulk den-
1.1. Multi-point measurement
sity. Repeat the procedure using 400 taps. If the difference
When the gas is physically adsorbed by the powder sam-
between the 2 masses obtained after 200 and 400 taps exceeds
ple, the following relationship (Brunauer, Emmett and Teller
2 per cent, carry out a test using 200 additional taps until the
(BET) adsorption isotherm) holds when the relative pressure
difference between succeeding measurements is less than 2
(P/P0) is in the range of 0.05 to 0.30 for pressure P of the
per cent. Calculate the tapped density (g/mL) using the for-
adsorbate gas in equilibrium for the volume of gas adsorbed,
mula mf/100 where mf is the mass of powder in the measur-
Va.
ing vessel. Record the average of 3 determinations using 3

1/[Va{(P0/P) － 1}] proaches zero. The single-point method may be employed in-
＝ {(C － 1)/VmC} × (P/P0) ＋ (1/VmC ) (1) directly for a series of very similar powder samples of a given
material for which the material constant C is not infinite but
P: Partial vapour pressure of adsorbate gas in equilibrium
may be assumed to be invariant. Under these circumstances,
with the surface at －195.89C (b.p. of liquid nitrogen),
the error associated with the single-point method can be
in pascals
reduced or eliminated by using the multiple-point method to
P0: Saturated pressure of adsorbate gas, in pascals
evaluate C for one of the samples of the series from the BET
Va: Volume of gas adsorbed at standard temperature and
plot, from which C is calculated as (1 ＋ slope/intercept).
pressure (STP) [09C and atmospheric pressure (1.013
Then Vm is calculated from the single value of Va measured
× 105 Pa)], in milliliters
at a single value of P/P0 by the equation:
Vm: Volume of gas adsorbed at STP to produce an appar-
ent monolayer on the sample surface, in milliliters Vm ＝ Va{(P0/P) － 1}[(1/C ) ＋ {(C － 1)/C} × (P/P0)] (4)
C: Dimensionless constant that is related to the enthalpy
2. Sample preparation
of adsorption of adsorbate gas on the powder sample
Before the specific surface area of the sample can be deter-
A value of Va is measured at each of not less than 3 values mined, it is necessary to remove gases and vapors that may
of P/P0. Then the BET value, 1/[Va{(P0/P) － 1}], is plotted have become physically adsorbed onto the surface during
against P/P0 according to equation (1). This plot should storage and handling. If outgassing is not achieved, the spe-
yield a straight line usually in the approximate relative pres- cific surface area may be reduced or may be variable because
sure range 0.05 to 0.3. The data are considered acceptable if some parts of surface area are covered with molecules of the
the correlation coefficient, r, of the linear regression is not previously adsorbed gases or vapors. The outgassing condi-
less than 0.9975; that is, r 2 is not less than 0.995. From the tions are critical for obtaining the required precision and ac-
resulting linear plot, the slope, which is equal to (C － 1)/ curacy of specific surface area measurements on pharmaceu-
VmC, and the intercept, which is equal to 1/(VmC ), are eval- ticals because of the sensitivity of the surface of the mate-
uated by linear regression analysis. From these values, Vm is rials. The outgassing conditions must be demonstrated to
calculated as 1/(slope ＋ intercept ), while C is calculated as yield reproducible BET plots, a constant weight of test pow-
(slope/intercept ) ＋ 1. From the value of Vm so determined, der, and no detectable physical or chemical changes in the
the specific surface area, S, in m2g－1, is calculated by the test powder.
equation: The outgassing conditions defined by the temperature,
pressure and time should be so chosen that the original sur-
S ＝ (Vm Na)/(m × 22,400) (2)
face of the solid is reproduced as closely as possible.
N: Avogadro constant (6.022 × 1023 mol－1), Outgassing of many substances is often achieved by apply-
a: Effective cross-sectional area of one adsorbate molec- ing a vacuum, by purging the sample in a flowing stream of a
ule, in square meters (0.162 × 10－18 m2 for nitrogen non-reactive, dry gas, or by applying a desorption-adsorp-
and 0.195 × 10－18 m2 for krypton) tion cycling method. In either case, elevated temperatures
m: Mass of test powder, in grams are sometimes applied to increase the rate at which the
22,400: Volume, in milliliters, occupied by one mole of contaminants leave the surface. Caution should be exercised
the adsorbate gas at STP allowing for minor when outgassing powder samples using elevated tempera-
departures from the ideal tures to avoid affecting the nature of the surface and the in-
tegrity of the sample.
A minimum of 3 data points is required. Additional meas-
If heating is employed, the recommended temperature
urements may be carried out, especially when non-linearity is
and time of outgassing are as low as possible to achieve
obtained at a P/P0 value close to 0.3. Because non-linearity
reproducible measurement of specific surface area in an ac-
is often obtained at a P/P0 value below 0.05, values in this
ceptable time. For outgassing sensitive samples, other out-
region are not recommended. The test for linearity, the treat-
gassing methods such as the desorption-adsorption cycling
ment of the data, and the calculation of the specific surface
method may be employed.
area of the sample are described above.
The standard technique is the adsorption of nitrogen at
1.2. Single-point measurement
liquid nitrogen temperature.
Normally, at least 3 measurements of Va each at different
For powders of low specific surface area (＜0.2 m2g－1) the
values of P/P0 are required for the determination of specific
proportion adsorbed is low. In such cases the use of krypton
surface area by the dynamic flow gas adsorption technique
at liquid nitrogen temperature is preferred because the low
(Method I ) or by volumetric gas adsorption (Method II ).
vapor pressure exerted by this gas greatly reduces error. All
However, under certain circumstances described below, it
gases used must be free from moisture.
may be acceptable to determine the specific surface area of a
Accurately weigh a quantity of the test powder such that
powder from a single value of Va measured at a single value
the total surface of the sample is at least 1 m2 when the ad-
of P/P0 such as 0.300 (corresponding to 0.300 mole of nitro-
sorbate is nitrogen and 0.5 m2 when the adsorbate is kryp-
gen or 0.001038 mole fraction of krypton), using the follow-
ton. Lower quantities of sample may be used after appropri-
ing equation for calculating Vm:
ate validation.
Vm ＝ Va{1 － (P/P0)} (3) Because the amount of gas adsorbed under a given pres-
sure tends to increase on decreasing the temperature, adsorp-
The single-point method may be employed directly for a
tion measurements are usually made at a low temperature.
series of powder samples of a given material for which the
Measurement is performed at －195.89C, the boiling point
material constant C is much greater than unity. These cir-
of liquid nitrogen.
cumstances may be verified by comparing values of specific
Adsorption of gas should be measured either by Method I
surface area determined by the single-point method with that
or Method II.
determined by the multiple-point method for the series of
powder samples. Close similarity between the single-point
values and multiple-point values suggests that 1/C ap-

Fig. 3.02-1 Schematic diagram of the dynamic flow method Fig. 3.02-2 Schematic diagram of the volumetric method
apparatus apparatus

calibration ball of known volume for particle density meas-

3.03 Powder Particle Density urement. The final pressures (Pf) are determined for the ini-
tial empty test cell followed by the test cell placed with the
Determination calibration ball for particle density measurement in accor-
dance with the procedures, and Vc and Vr are calculated
This test is harmonized with the European Pharmacopoeia
using the equation described in the section of Procedure.
and the U.S. Pharmacopoeia. The parts of the test that are
Calculation can be made taking into account that the sample
not harmonized are marked with symbols ( ).
volume (Vs) is zero in the first run.
Powder Particle Density Determination is a method to
3. Procedure
determine particle density of powdered pharmaceutical drugs
The gas pycnometric density measurement is performed at
or raw materials of drugs, and generally performed using a
a temperature between 159 C and 309C and must not vary by
gas displacement pycnometer. The gas pycnometric density
more than 29 C during the course of measurement.
is determined by measuring the volume occupied by a known
Volatile contaminants in the powder are removed by
mass of powder which is equivalent to the volume of gas dis-
degassing the powder under a constant purge of helium prior
placed by the powder using a gas displacement pycnometer.
to the measurement. Occasionally, powders may have to be
In gas pycnometric density measurements, the volume deter-
degassed under vacuum. Because volatiles may be evolved
mined excludes the volume occupied by open pores;
during the measurement, weighing of the sample is done
however, it includes the volume occupied by sealed pores or
after the pycnometric measurement of volume.
pores inaccessible to the gas.
Weigh the mass of the test cell and record it. After weigh-
Usually, helium is used as a test gas due to its high diffu-
ing out the amount of the sample as described in the individ-
sivity into small open pores. If gases other than helium are
ual monograph and placing it in the test cell, seal the cell in
used, different values would be obtained, since the penetra-
the pycnometer.
tion of the gas is dependent on the size of the pore as well as
Open the valve (A) which connects the expansion cell with
the cross-sectional area of the gas.
the test cell, confirm with the manometer (M) that the pres-
The measured density is a volume weighted average of the
sure inside the system is stable, and then read the system ref-
densities of individual powder particles. It is called the parti-
erence pressure (Pr). Secondly, close the valve that connects
cle density, distinct from the true density of solid or the bulk
to the two cells, and introduce the measurement gas into the
density of powder. The density of solids are expressed in
test cell to achieve positive pressure. Confirm with the
grams per cubic centimeter (g/cm3), although the interna-
manometer that the pressure inside the system is stable, and
tional unit is the kilogram per cubic meter (1 g/cm3 ＝ 1000
then read the initial pressure (Pi). Open the valve to connect
kg/m3).
the test cell with the expansion cell. After confirming that
1. Apparatus the indicator of the manometer is stable, read the final pres-
The schematic diagram of particle density apparatus for sure (Pf), and calculate the sample volume (Vs) with the fol-
gas displacement pycnometric measurement is shown in Fig. lowing equation.
3.03-1. The apparatus consists of a test cell in which the sam-
Vr
ple is placed, an expansion cell and a manometer (M). The Vs ＝ Vc －
Pi － Pr
test cell, with an empty cell volume (Vc), is connected －1
through a valve (A) to an expansion cell, with a volume (Vr). Pf － Pr
Generally, helium is used as the measurement gas. The Vr: Expansion volume (cm3)
apparatus has to be equipped with a system capable of pres- Vc: Cell volume (cm3)
suring the test cell to the defined pressure (P) through the Vs: Sample volume (cm3)
manometer. Pi: Initial pressure (kPa)
Pf: Final pressure (kPa)
2. Calibration of apparatus
Pr: System reference pressure (kPa)
The volumes of the test cell (Vc) and the expansion cell (Vr)
must be accurately determined to the nearest 0.001 cm3, and Repeat the measurement sequence for the same powder
to assure accuracy of the results of volume obtained, calibra- sample until consecutive measurements of the sample
tion of the apparatus is carried out as follows using a volume agree to within 0.2z, and calculate the mean of
sample volumes (Vs). Finally, unload the test cell, weigh the
mass of the test cell, and calculate the final sample mass (m)
by deducting the empty cell mass from the test cell mass. The
powder particle density r is calculated by the following equa-
tion:
r ＝ m/Vs
r: Powder particle density in g/cm3,
m: Final sample mass in g,
Vs: Sample volume in cm3
If the pycnometer differs in operation or construction
from the one shown in Fig. 3.03-1, follow the instructions of
the manufacturer of the pycnometer. The sample condi-
tioning is indicated with the results. For example, indicate
whether the sample was tested as is or dried under specific
conditions such as those described for loss on drying.

Fig. 3.03-1 Schematic diagram of a gas pycnometer

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
104 3.04 Particle Size Determination / General Tests JP XVII
1.1.1.2. Visual Characterization
3.04 Particle Size Determination The magnification and numerical aperture should be
sufficiently high to allow adequate resolution of the images
This test is harmonized with the European Pharmacopoeia of the particles to be characterized. Determine the actual
and the U. S. Pharmacopeia. The parts of the text that are magnification using a calibrated stage micrometer to
not harmonized are marked with symbols ( ). calibrate an ocular micrometer. Errors can be minimized if
Particle the magnification is sufficient that the image of the particle
Size Determination is a method to determine di-
is at least 10 ocular divisions. Each objective must be cali-
rectly or indirectly morphological appearance, shape, size
brated separately. To calibrate the ocular scale, the stage
and its distribution of powdered pharmaceutical drugs and
micrometer scale and the ocular scale should be aligned. In
excipients to examine their micromeritic properties. Optical
this way, a precise determination of the distance between
microscopy and analytical sieving method may be used de-
ocular stage divisions can be made.
pending on the measuring purpose and the properties of test When the particle size is measured, an ocular micrometer
specimen.
is inserted at the position of the ocular diaphragm, and a
1. Method 1. Optical Microscopy calibrated stage micrometer is placed at the center of the
Theoptical microscopy is used to observe the morpho- microscope stage and fixed in place. The ocular is attached
logical appearance and shape of individual particle either di- to the lens barrel and adjusted to the focus point of the stage
rectly with the naked eye or by using a microscopic photo- micrometer scale. Then, the distance between the scales of
graph, in order to measure the particle size. The particle size the two micrometers is determined, and the sample size
distribution can also be determined by this method. It is also equivalent 1 division of the ocular scale is calculated using
possible with this method to measure the size of the individ- the following formula:
ual particle even when different kinds of particles mingle if
The particle size equivalent 1 division on the ocular scale
they are optically distinguishable. Data processing tech-
( mm) ＝ Length on the stage micrometer (mm)/Number of
niques, such as image analysis, can be useful for determining
scale divisions on the ocular micrometer
the particle size distribution.
This method for particle characterization can generally be The stage micrometer is removed and the test specimen is
applied to particles 1 mm and greater. The lower limit is im- placed on the microscope stage. After adjusting the focus,
posed by the resolving power of the microscope. The upper the particle sizes are determined from the number of scale
limit is less definite and is determined by the increased divisions read through the ocular.
difficulty associated with the characterization of larger parti- Several different magnifications may be necessary to
cles. Various alternative techniques are available for particle characterize materials having a wide particle size distribu-
characterization outside the applicable range of optical tion.
microscopy. Optical microscopy is particularly useful for 1.1.1.3. Photographic Characterization
characterizing particles that are not spherical. This method If particle size is to be determined by photographic
may also serve as a base for the calibration of faster and methods, take care to ensure that the object is sharply
more routine methods that may be developed. focused at the plane of the photographic emulsion. Deter-
1.1. Apparatus mine the actual magnification by photographing a calibrated
Use a microscope that is stable and protected from vibra- stage micrometer, using photographic film of sufficient
tion. The microscope magnification (product of the objec- speed, resolving power, and contrast. Exposure and process-
tive magnification, ocular magnification, and additional ing should be identical for photographs of both the test
magnifying components) must be sufficient to allow ade- specimen and the determination of magnification. The ap-
quate characterization of the smallest particles to be classi- parent size of a photographic image is influenced by the ex-
fied in the test specimen. The greatest numerical aperture of posure, development, and printing processes as well as by
the objective should be sought for each magnification range. the resolving power of the microscope.
Polarizing filters may be used in conjunction with suitable 1.2. Preparation of the Mount
analyzers and retardation plates. Color filters of relatively The mounting medium will vary according to the physical
narrow spectral transmission should be used with achromatic properties of the test specimen. Sufficient, but not excessive,
objectives and are preferable with apochromats and are re- contrast between the specimen and the mounting medium is
quired for appropriate color rendition in photomicrography. required to ensure adequate detail of the specimen edge. The
Condensers corrected for at least spherical aberration should particles should rest in one plane and be adequately dis-
be used in the microscope substage and with the lamp. The persed to distinguish individual particles of interest. Further-
numerical aperture of the substage condenser should match more, the particles must be representative of the distribution
that of the objective under the condition of use; this is af- of sizes in the material and must not be altered during prepa-
fected by the actual aperture of the condenser diaphragm ration of the mount. Care should be taken to ensure that this
and the presence of immersion oils. important requirement is met. Selection of the mounting me-
1.1.1. Adjustment dium must include a consideration of the analyte solubility.
The precise alignment of all elements of the optical system 1.3. Characterization
and proper focusing are essential. The focusing of the ele- 1.3.1. Crystallinity Characterization
ments should be done in accordance with the recommenda- The crystallinity of a material may be characterized to de-
tions of the microscope manufacturer. Critical axial align- termine compliance with the crystallinity requirement where
ment is recommended. stated in the individual monograph of a drug substance. Un-
1.1.1.1. Illumination less otherwise specified in the individual monograph, mount
A requirement for good illumination is a uniform and a few particles of the specimen in mineral oil on a clean glass
adjustable intensity of light over the entire field of view; slide. Examine the mixture using a polarizing microscope:
Kohler illumination is preferred. With colored particles, the particles show birefringence (interference colors) and ex-
choose the color of the filters used so as to control the con- tinction positions when the microscope stage is revolved.
trast and detail of the image.

1.3.2. Limit Test of Particle Size by Microscopy that has the same projected are as the particle.
Weigh a suitable quantity of the powder to be examined (iv) Length: The longest dimension from edge to edge of
(for example, 10 to 100 mg), and suspend it in 10 mL of a a particle oriented parallel to the ocular scale.
suitable medium in which the powder does not dissolve, add- (v) Width: The longest dimension of the particle meas-
ing, if necessary, a wetting agent. A homogeneous suspen- ured at right angles to the length.
sion of particles can be maintained by suspending the parti- 1.3.4. Particle Shape Characterization
cles in a medium of similar or matching density and by For irregularly shaped particles, characterization of parti-
providing adequate agitation. Introduce a portion of the ho- cle size must also include information on particle shape. The
mogeneous suspension into a suitable counting cell, and scan homogeneity of the powder should be checked using ap-
under a microscope an area corresponding to not less than 10 propriate magnification. The following defines some com-
mg of the powder to be examined. Count all the particles monly used descriptors of particle shape (see Fig. 3.04-2).
having a maximum dimension greater than the prescribed (i) Acicular: Slender, needle-like particle of similar width
size limit. The size limit and the permitted number of parti- and thickness.
cles exceeding the limit are defined for each substance. (ii) Columnar: Long, thin particle with a width and
1.3.3. Particle Size Characterization thickness that are greater than those of an acicular particle.
The measurement of particle size varies in complexity de- (iii) Flake: Thin, flat particle of similar length and
pending on the shape of the particle and the number of parti- width.
cles characterized must be sufficient to insure an acceptable (iv) Plate: Flat particles of similar length and width but
level of uncertainty in the measured parameters1). For spheri- with greater thickness than flakes.
cal particles, size is defined by the diameter. For irregular (v) Lath: Long, thin, and blade-like particle.
particles, a variety of definitions of particle size exist. In gen- (vi) Equant: Particles of similar length, width, and thick-
eral, for irregularly shaped particles, characterization of par- ness; both cubical and spherical particles are included.
ticle size must also include information on the type of diame- 1.3.5. General Observations
ter measured as well as information on particle shape. Sever- A particle is generally considered to be the smallest dis-
al commonly used measurements of particle size are defined crete unit. A particle may be a liquid or semisolid droplet; a
below (see Fig. 3.04-1). single crystal or polycrystalline; amorphous or an agglomer-
(i) Feret's Diameter: The distance between imaginary ate. Particles may be associated.
parallel lines tangent to a randomly oriented particle and This degree of association may be described by the follow-
perpendicular to the ocular scale. ing terms.
(ii) Martin's Diameter: The diameter of the particle at (i) Lamellar: Stacked plates.
the point that divides a randomly oriented particle into two (ii) Aggregate: Mass of adhered particles.
equal projected areas. (iii) Agglomerate: Fused or cemented particles.
(iii) Projected area Diameter: The diameter of a circle (iv) Conglomerate: Mixture of two or more types of par-
ticles.
(v) Spherulite: Radial cluster.
(vi) Drusy: Particle covered with tiny particles.
Particle condition may be described by the following
terms.
(i) Edges: Angular, rounded, smooth, sharp, fractured.
(ii) Optical: Color (using proper color balancing filters),
transparent, translucent, opaque.
(iii) Defects: Occlusions, inclusions.
Surface characteristics may be described by the following
terms.
(i) Cracked: Partial split, break, or fissure.
(ii) Smooth: Free of irregularities, roughness, or projec-
tions.
(iii) Porous: Having openings or passageways.
(iv) Rough: Bumpy, uneven, not smooth.
(v) Pitted: Small indentations.
2. Method 2. Analytical Sieving Method
Fig. 3.04-1 Commonly used measurements of particle size Theanalytical sieving method is a method to estimate the
particle size distribution of powdered pharmaceutical drugs
by sieving. The particle size determined by this method is
shown as the size of a minimum sieve opening through which
the particle passes. ``Powder'' here means a gathering of
numerous solid particles.
Sieving is one of the oldest methods of classifying powders
and granules by particle size distribution. When using a
woven sieve cloth, the sieving will essentially sort the parti-
cles by their intermediate size dimension (i.e., breadth or
width). Mechanical sieving is most suitable where the
majority of the particles are larger than about 75 mm. For
smaller particles, the light weight provides insufficient force
during sieving to overcome the surface forces of cohesion
and adhesion that cause the particles to stick to each other
Fig. 3.04-2 Commonly used descriptions of particle shape

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
106 3.04 Particle Size Determination / General Tests JP XVII
and to the sieve, and thus cause particles that would be ISO sieves listed in the Table 3.04-1 as recommended in the
expected to pass through the sieve to be retained. For such particular region.
materials other means of agitation such as air-jet sieving or Sieves are selected to cover the entire range of particle
sonic sifting may be more appropriate. Nevertheless, sieving sizes present in the test specimen. A nest of sieves having
can sometimes be used for some powders or granules having a 2 progression of the area of the sieve openings is recom-
median particle sizes smaller than 75 mm where the method mended. The nest of sieves is assembled with the coarsest
can be validated. In pharmaceutical terms, sieving is usually screen at the top and the finest at the bottom. Use microme-
the method of choice for classification of the coarser grades ters or millimeters in denoting test sieve openings.
of single powders or granules. It is a particularly attractive [Note—Mesh numbers are provided in the table for conver-
method in that powders and granules are classified only on sion purposes only.] Test sieves are made from stainless steel
the basis of particle size, and in most cases the analysis can or, less preferably, from brass or other suitable non-reactive
be carried out in the dry state. wire.
Among the limitations of sieving method are the need for 2.1.1.1. Calibration of test sieves
an appreciable amount of sample (normally at least 25 g, Calibration and recalibration of test sieves is in accor-
depending on the density of the powder or granule, and the dance with the most current edition of ISO 3310-12). Sieves
diameter of test sieves) and difficulty in sieving oily or other should be carefully examined for gross distortions and
cohesive powders or granules that tend to clog the sieve fractures, especially at their screen frame joints, before use.
openings. The method is essentially a two-dimensional esti- Sieves may be calibrated optically to estimate the average
mate of size because passage through the sieve aperture is opening size, and opening variability, of the sieve mesh.
frequently more dependent on maximum width and thick- Alternatively, for the evaluation of the effective opening of
ness than on length. test sieves in the size range of 212 to 850 mm, Standard Glass
This method is intended for estimation of the total particle Spheres are available. Unless otherwise specified in the
size distribution of a single material. It is not intended for individual monograph, perform the sieve analysis at con-
determination of the proportion of particles passing or trolled room temperature and at ambient relative humidity.
retained on one or two sieves. 2.1.1.2. Cleaning Test Sieves
Estimate the particle size distribution as described under Ideally, test sieves should be cleaned using only an air jet
Dry Sieving Method, unless otherwise specified in the in- or a liquid stream. If some apertures remain blocked by test
dividual monograph. Where difficulty is experienced in particles, careful gentle brushing may be used as a last
reaching the endpoint (i.e., material does not readily pass resort.
through the sieves) or when it is necessary to use the finer 2.1.2. Test Specimen
end of the sieving range (below 75 mm), serious consideration If the test specimen weight is not given in the monograph
should be given to the use of an alternative particle-sizing for a particular material, use a test specimen having a weight
method. between 25 and 100 g, depending on the bulk density of the
Sieving should be carried out under conditions that do not material, and test sieves having a 200 mm diameter. For 76
cause the test sample to gain or lose moisture. The relative mm sieves the amount of material that can be accommodat-
humidity of the environment in which the sieving is carried ed is approximately 1/7th that which can be accommodated
out should be controlled to prevent moisture uptake or loss on a 200 mm sieve. Determine the most appropriate weight
by the sample. In the absence of evidence to the contrary, for a given material by test sieving accurately weighed speci-
analytical test sieving is normally carried at ambient hu- mens of different weights, such as 25, 50, and 100 g, for the
midity. Any special conditions that apply to a particular ma- same time period on a mechanical shaker. [Note—If the test
terial should be detailed in the individual monograph. results are similar for the 25-g and 50-g specimens, but the
Principles of Analytical Sieving: Analytical test sieves are 100-g specimen shows a lower percentage through the finest
constructed from a woven-wire mesh, which is of simple sieve, the 100-g specimen size is too large.] Where only a
weave that is assumed to give nearly square apertures and is specimen of 10 to 25 g is available, smaller diameter test
sealed into the base of an open cylindrical container. The sieves conforming to the same mesh specifications may be
basic analytical method involves stacking the sieves on top of substituted, but the endpoint must be re-determined. The use
one another in ascending degrees of coarseness, and then of test samples having a smaller mass (e.g. down to 5 g) may
placing the test powder on the top sieve. be needed. For materials with low apparent particle density,
The nest of sieves is subjected to a standardized period of or for materials mainly comprising particles with a highly
agitation, and then the weight of material retained on each iso-diametrical shape, specimen weights below 5 g for a 200
sieve is accurately determined. The test gives the weight per- mm screen may be necessary to avoid excessive blocking of
centage of powder in each sieve size range. the sieve. During validation of a particular sieve analysis
This sieving process for estimating the particle size distri- method, it is expected that the problem of sieve blocking will
bution of a single pharmaceutical powder is generally in- have been addressed.
tended for use where at least 80z of the particles are larger If the test material is prone to picking up or losing sig-
than 75 mm. The size parameter involved in determining par- nificant amounts of water with varying humidity, the test
ticle size distribution by analytical sieving is the length of the must be carried out in an appropriately controlled environ-
size of the minimum square aperture through which the par- ment. Similarly, if the test material is known to develop an
ticle will pass. electrostatic charge, careful observation must be made to
2.1. Procedure ensure that such charging is not influencing the analysis. An
2.1.1. Test Sieves antistatic agent, such as colloidal silicon dioxide and/or alu-
Test sieves suitable for pharmacopoeial tests conform to minum oxide, may be added at a 0.5 percent (m/m) level to
the most current edition of International Organisation for minimize this effect. If both of the above effects cannot
Standardization (ISO) Specification ISO 3310-1; Test eliminated, an alternative particle-sizing technique must be
sieves—Technical requirements and testing (see Table selected.
3.04-1). 2.1.3. Agitation Methods
Unless otherwise specified in the monograph, use those Several different sieve and powder agitation devices are

Table 3.04-1. Size of standard sieve series in range of interest

ISO Nominal Aperture

Recommended
Principal sizes Supplementary sizes US Sieve USP Sieves European Japan
No. Sieve No. Sieve No.
(microns)
R 20/3 R 20 R 40/3

11.20 mm 11.20 mm 11.20 mm 11200

10.00 mm
9.50 mm
9.00 mm
8.00 mm 8.00 mm 8.00 mm
7.10 mm
6.70 mm
6.30 mm
5.60 mm 5.60 mm 5.60 mm 5600 3.5
5.00 mm
4.75 mm 4
4.50 mm
4.00 mm 4.00 mm 4.00 mm 5 4000 4000 4.7
3.55 mm
3.35 mm 6 5.5
3.15 mm
2.80 mm 2.80 mm 2.80 mm 7 2800 2800 6.5
2.50 mm
2.36 mm 8 7.5
2.24 mm
2.00 mm 2.00 mm 2.00 mm 10 2000 2000 8.6
1.80 mm
1.70 mm 12 10
1.60 mm
1.40 mm 1.40 mm 1.40 mm 14 1400 1400 12
1.25 mm
1.18 mm 16 14
1.12 mm
1.00 mm 1.00 mm 1.00 mm 18 1000 1000 16
900 mm
850 mm 20 18
800 mm
710 mm 710 mm 710 mm 25 710 710 22
630 mm
600 mm 30 26
560 mm
500 mm 500 mm 500 mm 35 500 500 30
450 mm
425 mm 40 36
400 mm
355 mm 355 mm 355 mm 45 355 355 42
315 mm
300 mm 50 50
280 mm
250 mm 250 mm 250 mm 60 250 250 60
224 mm
212 mm 70 70
200 mm
180 mm 180 mm 180 mm 80 180 180 83
160 mm
150 mm 100 100
140 mm
125 mm 125 mm 125 mm 120 125 125 119
112 mm
106 mm 140 140
100 mm
90 mm 90 mm 90 mm 170 90 90 166
80 mm
75 mm 200 200
71 mm
63 mm 63 mm 63 mm 230 63 63 235
56 mm
53 mm 270 282
50 mm
45 mm 45 mm 45 mm 325 45 45 330
40 mm
38 mm 38 391

commercially available, all of which may be used to perform often includes the use of finer test sieves than used in ordina-
sieve analyses. However, the different methods of agitation ry dry sieving. This technique is more suitable where only
may give different results for sieve analyses and endpoint de- oversize or undersize fractions are needed.
terminations because of the different types and magnitude of In the sonic sifting method, a nest of sieves is used, and
the forces acting on the individual particles under test. the test specimen is carried in a vertically oscillating column
Methods using mechanical agitation or electromagnetic agi- of air that lifts the specimen and then carries it back against
tation, and that can induce either a vertical oscillation or a the mesh openings at a given number of pulses per minute. It
horizontal circular motion, or tapping or a combination of may be necessary to lower the sample amount to 5 g, when
both tapping and horizontal circular motion are available. sonic shifting is employed.
Entrainment of the particles in an air stream may also be The air jet sieving and sonic sieving methods may be use-
used. The results must indicate which agitation method was ful for powders or granules when mechanical sieving tech-
used and the agitation parameters used (if they can be va- niques are incapable of giving a meaningful analysis.
ried), since changes in the agitation conditions will give These methods are highly dependent upon proper disper-
different results for the sieve analysis and endpoint determi- sion of the powder in the air current. This requirement may
nations, and may be sufficiently different to give a failing be hard to achieve if the method is used at the lower end of
result under some circumstances. the sieving range (i.e., below 75 mm), when the particles tend
2.1.4. Endpoint Determination to be more cohesive, and especially if there is any tendency
The test sieving analysis is complete when the weight on for the material to develop an electrostatic charge. For the
any of the test sieves does not change by more than 5z or above reasons endpoint determination is particularly critical,
0.1 g (10z in the case of 76 mm sieves) of the previous and it is very important to confirm that the oversize material
weight on that sieve. If less than 5z of the total specimen comprises single particles and is not composed of aggregates.
weight is present on a given sieve, the endpoint for that sieve 2.3. Interpretation
is increased to a weight change of not more than 20z of the The raw data must include the weight of test specimen, the
previous weight on that sieve. total sieving time, and the precise sieving methodology and
If more than 50z of the total specimen weight is found on the set values for any variable parameters, in addition to the
any one sieve, unless this is indicated in the monograph, the weights retained on the individual sieves and in the pan. It
test should be repeated, but with the addition to the sieve may be convenient to convert the raw data into a cumulative
nest of a more coarse sieve intermediate between that weight distribution, and if it is desired to express the distri-
carrying the excessive weight and the next coarsest sieve in bution in terms of a cumulative weight undersize, the range
the original nest, i.e., addition of the ISO series sieve omit- of sieves used should include a sieve through which all the
ted from the nest of sieves. material passes. If there is evidence on any of the test sieves
2.2. Sieving Methods that the material remaining on it is composed of aggregates
2.2.1. Mechanical Agitation (Dry Sieving Method) formed during the sieving process, the analysis is invalid.
Tare each test sieve to the nearest 0.1 g. Place an accu-
rately weighed quantity of test specimen on the top (coarsest) 1)Additional information on particle size measurement, sample size,

sieve, and place the lid. Agitate the nest of sieves for 5 and data analysis is available, for example, in ISO 9276.
minutes. Then carefully remove each from the nest without 2)International Organization for Standardization (ISO) Specification

loss of material. Reweigh each sieve, and determine the ISO 3310-1; Test sieves-Technical requirements and testing
weight of material on each sieve. Determine the weight of
material in the collecting pan in a similar manner. Reassem-
ble the nest of sieves, and agitate for 5 minutes. Remove and
weigh each sieve as previously described. Repeat these steps 3.05 Water-Solid Interactions:
until the endpoint criteria are met (see Endpoint Determina- Determination of Sorption-
tion under Test Sieves). Upon completion of the analysis,
reconcile the weights of material. Total losses must not Desorption Isotherms and of
exceed 5z of the weight of the original test specimen. Water Activity
Repeat the analysis with a fresh specimen, but using a
single sieving time equal to that of the combined times used This test is harmonized with the European Pharmacopoeia
above. Confirm that this sieving time conforms to the re- and the U. S. Pharmacopeia. The parts of the text that are
quirements for endpoint determination. When this endpoint not harmonized are marked with symbols ( ).
has been validated for a specific material, then a single fixed The powder of drug as drug substances or preparations
time of sieving may be used for future analyses, providing
often contacts with water during the production process or
the particle size distribution falls within normal variation.
storage. For the assessment of the water-solid interactions,
If there is evidence that the particles retained on any sieve
determinations of sorption-desorption isotherms and water
are aggregates rather than single particles, the use of
activity are used. Water is interacted physically with solid in
mechanical dry sieving is unlikely to give good reproducibili-
two ways, by an adsorption onto the surface of solid or an
ty, a different particle size analysis method should be used.
absorption permeating into the solid. In the case where both
2.2.2. Air Entrainment Methods (Air Jet and Sonic Shifter
the adsorption and absorption are occurred, the term ``sorp-
Sieving)
tion'' is usually used.
Different types of commercial equipment that use a mov-
ing air current are available for sieving. A system that uses a 1. Determination of Sorption-Desorption Isotherms
single sieve at a time is referred to as air jet sieving. It uses 1.1 Principle
the same general sieving methodology as that described The tendency to take up water vapour is best assessed by
under the Dry Sieving Method, but with a standardized air measuring sorption or desorption as a function of relative
jet replacing the normal agitation mechanism. It requires se- humidity, at constant temperature, and under conditions
quential analyses on individual sieves starting with the finest where sorption or desorption is essentially occurring in-
sieve to obtain a particle size distribution. Air jet sieving dependently of time, i.e. equilibrium. Relative humidity,

RH, is defined by the following equation: a good baseline stability as well as accurate control of the
relative humidity generation. The required relative humidi-
RH = (Pc/P0) × 100
ties can be generated, e.g. by accurately mixing dry and
Pc: pressure of water vapour in the system; saturated vapour gas with flow controllers. The electrostatic
P0: saturation pressure of water vapour under the same behaviour of the powder must also be considered. The verifi-
conditions. cation of the temperature and the relative humidity (con-
trolled with, for example, a certified hygrometer, certified
The ratio Pc/P0 is referred to as the relative pressure.
salt solutions or deliquescence points of certified salts over
Sorption or water uptake is best assessed by starting with
an adequate range), must be consistent with the instrument
dried samples and subjecting them to a known relative
specification. The balance must provide a sufficient mass
humidity. Desorption is studied by beginning with a system
resolution and long term stability.
already containing sorbed water and reducing the relative
It is also possible to measure amounts of water uptake not
humidity. As the name indicates, the sorption-desorption
detectable gravimetrically using volumetric techniques. In
isotherm is valid only for the reference temperature, hence a
the case of adsorption, to improve sensitivity, one can in-
special isotherm exists for each temperature. Ordinarily, at
crease the specific surface area of the sample by reducing
equilibrium, moisture content at a particular relative hu-
particle size or by using larger samples to increase the total
midity must be the same, whether determined from sorption
area. It is important, however, that such comminution of the
or desorption measurements. However, it is common to see
solid does not alter the surface structure of the solid or ren-
sorption-desorption hysteresis.
der it more amorphous or otherwise less ordered in crystal-
1.2 Methods
linity. For absorption, where water uptake is independent of
Samples may be stored in chambers at various relative
specific surface area, only increasing sample size will help.
humidities. The mass gained or lost for each sample is then
Increasing sample size, however, will increase the time to
measured. The major advantage of this method is con-
establish some type of equilibrium. To establish accurate
venience, while the major disadvantages are the slow rate of
values, it is important to get desolvation of the sample as
reaching constant mass, particularly at high relative humidi-
thoroughly as possible. Higher temperatures and lower pres-
ties, and the error introduced in opening and closing the
sures (vacuum) facilitate this process; however, one must be
chamber for weighing. Dynamic gravimetric water sorption
aware of any adverse effects this might have on the solid
systems allow the on-line weighing of a sample in a con-
such as dehydration, chemical degradation or sublimation.
trolled system to assess the interaction of the material with
Using higher temperatures to induce desorption, as in a ther-
moisture at various programmable levels of relative humidity
mogravimetric apparatus, likewise must be carefully carried
at a constant temperature. The major benefit of a controlled
out because of these possible pitfalls.
system is that isothermal conditions can be more reliably
1.3. Report and interpretation of the data
established and that the dynamic response of the sample to
Sorption data are usually reported as a graph of the appar-
changing conditions can be monitored. Data points for the
ent mass change in per cent of the mass of the dry sample as
determination of the sorption isotherm (e.g. from 0z to
a function of relative humidity or time. Sorption isotherms
approximately 95z RH, non condensing) are only taken
are reported both in tabular form and as a graph. The meas-
after a sufficiently constant signal indicates that the sample
urement method must be traceable with the data.
has reached equilibrium at a given level of humidity. In some
Adsorption-desorption hysteresis can be interpreted, for
cases (e.g. deliquescence), the maximum time may be res-
example, in terms of the porosity of the sample, its state of
tricted although the equilibrium level is not reached. The
agglomeration (capillary condensation), the formation of
apparatus must adequately control the temperature to ensure
hydrates, polymorphic change, or liquefying of the sample.
Certain types of systems, particularly those with micro-
porous solids and amorphous solids, are capable of sorbing
large amounts of water vapour. Here, the amount of water
associated with the solid as relative humidity is decreased, is
greater than the amount that originally sorbed as the relative
humidity was increased. For microporous solids, vapour
adsorption-desorption hysteresis is an equilibrium phenome-
non associated with the process of capillary condensation.
This takes place because of the high degree of irregular cur-
vature of the micropores and the fact that they ``fill''
(adsorption) and ``empty'' (desorption) under different
equilibrium conditions. For non-porous solids capable of
absorbing water, hysteresis occurs because of a change in the
degree of vapour-solid interaction due to a change in the
equilibrium state of the solid, e.g. conformation of polymer
A. Humidity controller
chains, or because the time scale for structural equilibrium is
B. Temperature controlled chamber longer than the time scale for water desorption. In measur-
C. Balance module
ing sorption-desorption isotherms, it is therefore important
D. Humidity regulated module
to establish that something close to an equilibrium state has
E. Reference
been reached. Particularly with hydrophilic polymers at high
F. Sample
relative humidities, the establishment of water sorption or
G. Vapour humidifier desorption values independent of time is quite difficult, since
H. Flow control module
one is usually dealing with a polymer plasticised into its
I. Dry gas
``fluid'' state, where the solid is undergoing significant
Fig. 3.05-1 Example of an apparatus for the determination change.
of the water sorption (other designs are possible) In the case of crystal hydrate formation, the plot of water

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
110 4.01 Bacterial Endotoxins Test / General Tests JP XVII
uptake versus pressure or relative humidity will in these cases equilibrium with the test sample, is directed at the mirror
exhibit a sharp increase in uptake at a particular pressure which cools until condensation occurs on the mirror. The
and the amount of water taken up will usually exhibit a temperature at which this condensation begins is the dew
stoichiometric mole: mole ratio of water to solid. In some point from which the ERH is determined. Commercially
cases, however, crystal hydrates will not appear to undergo a available instruments using the dew point/chilled mirror
phase change or the anhydrous form will appear amorphous. method or other technologies need to be evaluated for
Consequently, water sorption or desorption may appear suitability, validated, and calibrated when used to make
more like that seen with adsorption processes. X-ray crystal- water activity determinations.
lographic analysis and thermal analysis are particularly use- These instruments are typically calibrated over an ade-
ful for the study of such systems. quate range, for example, using some saturated salt solutions
For situations where water vapour adsorption occurs at 259C such as those listed in Table 3.05-1.
predominantly, it is very helpful to measure the specific sur-
face area of the solid by an independent method and to ex-
press adsorption as mass of water sorbed per unit area of Table 3.05-1 Standard saturated salt solutions
solid surface. This can be very useful in assessing the possi-
ble importance of water sorption in affecting solid proper- Saturated salt solutions at 259C ERH (z) Aw
ties. For example, 0.5z m/m uptake of water could hardly Potassium sulphate (K2SO4) 97.3 0.973
cover the bare surface of 100 m2/g, while for 1.0 m2/g this
Barium chloride (BaCl2) 90.2 0.902
amounts to 100 times more surface coverage. In the case of Sodium chloride (NaCl) 75.3 0.753
pharmaceutical solids which have a specific surface area in
Magnesium nitrate (Mg(NO3)2) 52.9 0.529
the range of 0.01 m2/g to 10 m2/g, what appears to be low
Magnesium chloride (MgCl2) 32.8 0.328
water content could represent a significant amount of water Lithium chloride (LiCl) 11.2 0.112
for the available surface. Since the ``dry surface area'' is not
a factor in absorption, sorption of water with amorphous or
1) AOAC International Official Method 978.18.
partially amorphous solids can be expressed on the basis of
unit mass corrected for crystallinity, when the crystal form
does not sorb significant amounts of water relative to the
amorphous regions.
2. Determination of the Water Activity 4. Biological Tests/Biochemical
2.1. Principle
Water activity, Aw, is the ratio of vapour pressure of water Tests/Microbial Tests
in the product (P ) to saturation pressure of water vapour
(P0) at the same temperature. It is numerically equal to 1/100
of the relative humidity (RH) generated by the product in a 4.01 Bacterial Endotoxins Test
closed system. RH can be calculated from direct measure-
ments of partial vapour pressure or dew point, or from in- This test is harmonized with the European Pharmacopoeia
direct measurement by sensors whose physical or electric and the U.S. Pharmacopeia.
characteristics are altered by the RH to which they are Bacterial Endotoxins Test is a test to detect or quantify
exposed. Ignoring activity coefficients, the relationship bacterial endotoxins of gram-negative bacterial origin using
between Aw and equilibrium relative humidity (ERH) are an amoebocyte lysate prepared from blood corpuscle ex-
represented by the following equations: tracts of horseshoe crab (Limulus polyphemus or Tachypleus
Aw = P/P0 tridentatus). There are two types of techniques for this test:
ERH (z) = Aw × 100 the gel-clot techniques, which are based on gel formation by
the reaction of the lysate TS with endotoxins, and the photo-
2.2. Method metric techniques, which are based on endotoxin-induced
The water activity is determined by placing the sample in a optical changes of the lysate TS. The latter include turbidi-
small airtight cup inside which the equilibrium between the metric techniques, which are based on the change in lysate
water in the solid and the headspace can be established. The TS turbidity during gel formation, and chromogenic tech-
volume of the headspace must be small in relation to the niques, which are based on the development of color after
sample volume in order not to change the sorption state of cleavage of a synthetic peptide-chromogen complex.
sample during the test. The equilibration as a thermody- Proceed by any one of these techniques for the test. In the
namic process takes time but may be accelerated by forced event of doubt or dispute, the final decision is made based
circulation within the cell. The acquired water activity value on the limit test of the gel-clot techniques, unless otherwise
is only valid for the simultaneously determined temperature. indicated.
This requires a precise temperature-measuring device as part The test is carried out in a manner that avoids endotoxin
of the equipment. Furthermore, the probe must be thermally contamination.
insulated to guarantee a constant temperature during the
test. The sensor measuring the humidity of the headspace air 1. Apparatus
above the sample is a key component. Theoretically, all Depyrogenate all glassware and other heat-stable materials
types of hygrometers can be used, but for analytical pur- in a hot-air oven using a validated process. Commonly used
poses miniaturization and robustness are a precondition. minimum time and temperature settings are 30 minutes at
The Aw measurement may be conducted using the dew 2509C. If employing plastic apparatus, such as multi-well
point/chilled mirror method1). A polished, chilled mirror is plates and tips for micropipettes, use only that which has
used as a condensing surface. The cooling system is electro- been shown to be free of detectable endotoxin and which
nically linked to a photoelectric cell into which light is does not interfere with the test.
reflected from the condensing mirror. An air stream, in

2. Preparation of Solutions tivity (4.1.1.) and for interfering factors (4.1.2.) as described
2.1. Standard Endotoxin Stock Solution under Preparatory testing (4.1.).
Prepare Standard Endotoxin Stock Solution by dissolving 4.1. Preparatory testing
Japanese Pharmacopoeia Reference Standard Endotoxin 4.1.1. Test for confirmation of labeled lysate reagent sensi-
that has been calibrated to the current WHO International tivity
Standard for Endotoxin, using water for bacterial endo- The labeled sensitivity of lysate is defined as the lowest
toxins test (BET). Endotoxin is expressed in Endotoxin Units concentration of endotoxin that is needed to cause the lysate
(EU). One EU is equal to one International Unit (IU) of TS to clot under the conditions specified for the lysate to be
endotoxin. used.
2.2. Standard Endotoxin Solution The test for confirmation of the labeled lysate sensitivity is
After mixing Standard Endotoxin Stock Solution thor- to be carried out when each new lot of lysate is used or when
oughly, prepare appropriate serial dilutions of Standard there is any change in the experimental conditions which
Endotoxin Solution, using water for BET. Use dilutions as may affect the outcome of the test.
soon as possible to avoid loss of activity by adsorption. Prepare standard solutions having four concentrations
2.3. Sample Solutions equivalent to 2 l, l, 0.5 l and 0.25 l by diluting the Stand-
Unless otherwise specified, prepare sample solutions by ard Endotoxin Stock Solution with water for BET. Mix a
dissolving or diluting drugs, using water for BET. By the volume of the lysate TS with an equal volume of one of the
sample, an aqueous solution other than water for BET may standard solutions (usually, 0.1 mL aliquots) in each test
be used to dissolve or dilute. If necessary, adjust the pH of tube. When single test vials or ampoules containing lyophi-
the sample solution so that the pH of the mixture of the lized lysate are used, add solutions directly to the vial or am-
lysate TS and sample solution falls within the specified pH poule.
range for the lysate to be used. The pH of the sample solu- Keep the tubes (or containers such as vials or ampoules)
tion may be in the range of 6.0 to 8.0. For adjustment of containing the reaction mixture usually at 37 ± 19C for 60
pH, acid, base or a suitable buffer solution may be used. ± 2 minutes, avoiding vibration. To test the integrity of the
The acid and base are prepared from their concentrated solu- gel after incubation, invert each tube or container through
tions or solids using water for BET, and then stored in con- approximately 1809in one smooth motion. If a firm gel has
tainers free of detectable endotoxin. The buffer solutions formed that remains in place upon inversion, record the
must be validated to be free of detectable endotoxin and result as positive. A result is negative if either a firm gel is
interfering factors. not formed, or if a fragile gel has formed but flows out upon
inversion.
3. Determination of Maximum Valid Dilution
Making the standard solutions of four concentrations one
The Maximum Valid Dilution (MVD) is the maximum
set, test four replicates of the set.
allowable dilution of a sample solution at which the endo-
The test is valid when 0.25 l of the standard solution
toxin limit can be determined.
shows a negative result in each set of tests. If the test is not
Determine the MVD from the following equation:
valid, repeat the test after verifying the test conditions.
MVD The endpoint is the last positive test in the series of
＝ (Endotoxin limit × Concentration of sample solu- decreasing concentrations of endotoxin. Calculate the geo-
tion)/l metric mean endpoint concentration of the four replicate
series using the following formula:
Endotoxin limit:
The endotoxin limit for injections, defined on the basis Geometric Mean Endpoint Concentration ＝ antilog (Se/f )
of dose, equals K/M, where K is a threshold pyrogenic
Se: The sum of the log endpoint concentrations of the
dose of endotoxin per kg body mass (EU/kg), and M is
dilution series used
equal to the maximum bolus dose of product per kg
f: The number of replicates
body mass. When the product is to be injected at
frequent intervals or infused continuously, M is the If the geometric mean endpoint concentration is not less
maximum total dose administered in a single hour than 0.5 l and not more than 2.0 l, the labeled sensitivity is
period. confirmed, and is used in tests performed with this lysate.
Concentration of sample solution: 4.1.2. Test for interfering factors
mg/mL in the case of endotoxin limit specified by mass This test is performed to check for the presence of enhanc-
(EU/mg) ing or inhibiting factors for the reaction in sample solutions.
mEq/mL in the case of endotoxin limit specified by Prepare the solutions A, B, C and D according to Table
equivalent (EU/mEq) 4.01-1, and test solutions A and B and solutions C and D in
Units/mL in the case of endotoxin limit specified by quadruplicate and in duplicate, respectively. Concerning the
biological unit (EU/Unit) incubation temperature, incubation time, and procedure for
mL/mL in the case of endotoxin limit specified by the confirmation of gel formation, follow the procedure
volume (EU/mL) described in 4.1.1.
l: the labeled lysate reagent sensitivity in the gel-clot tech- The geometric mean endpoint concentrations of B and C
niques (EU/mL) or the lowest point used (EU/mL) in solutions are determined by using the formula described in
the standard regression curve of the turbidimetric or 4.1.1.
chromogenic techniques This test must be repeated when there is any change in the
experimental conditions which may affect the outcome of
4. Gel-clot techniques
the test.
The gel-clot techniques detect or quantify endotoxins
The test is valid if solutions A and D show no reaction and
based on clotting of the lysate TS in the presence of endo-
the result for solution C confirms the labeled sensitivity.
toxin.
If the geometric mean endpoint concentration of solution
To ensure both the precision and validity of the test, per-
B is not less than 0.5 l and not greater than 2.0 l, the sample
form the tests for confirming the labeled lysate reagent sensi-

1 2l 1 2l
Water for 2 1l Water for 2 1l
C*3 2l/Water for BET 2 C*3 2l/Water for BET 2
BET 4 0.5l BET 4 0.5l
8 0.25l 8 0.25l
D*4 0/Water for BET — — — 2 D*4 0/Water for BET — — — 2
*1 Negative control. Sample solution only. *1 Sample solutions for the Quantitative test. The dilution range of the dilu-
*2 Sample solutions added with standard endotoxin (for testing interfering tion series may be changed as appropriate, but not exceeding the MVD.
factors). *2 Positive control. Sample solution at the same dilution as the solution A
*3 Standard endotoxin solutions for confirmation of the labeled lysate rea- diluted at the lowest dilution factor, containing standard endotoxin at a
gent sensitivity. concentration of 2l.
*4 Negative control. Water for BET only. *3 Standard endotoxin solutions for confirmation of the labeled lysate
sensitivity.
*4 Negative control. Water for BET only.
Table 4.01-2
Endotoxin concentration/Solution Number of
Solution
to which endotoxin is added replicates
ple solutions complying with 4.1.2.
A*1 0/Sample solution 2 Concerning the test conditions including the incubation
B*2 2l/Sample solution 2 temperature, incubation time, and procedure for the confir-
mation of gel formation, follow the procedure under 4.1.1.
C*3 2l/Water for BET 2 4.2.2. Interpretation
D*4 0/Water for BET 2 The test is valid when both replicates of solutions B and C
are positive and those of solution D are negative.
*1 Sample solution for the limit test. The solution may be diluted not to
exceed the MVD.
When a negative result is found for both replicates of solu-
*2 Positive control. Sample solution at the same dilution as solution A, tion A, the sample complies with the Bacterial Endotoxins
containing standard endotoxin at a concentration of 2l. Test.
*3 Positive control. Standard endotoxin solution containing standard en- When a positive result is found for both replicates of solu-
dotoxin concentration of 2l.
tion A, the sample does not comply with the test.
*4 Negative control. Water for BET only.
When a positive result is found for one replicate of solu-
tion A and a negative result is found for the other, repeat the
test. In the repeat test, the sample complies with the test if a
negative result is found for both replicates of solution A.
solution being examined does not contain interfering factors The sample does not comply with the test if a positive result
and complies with the test for interfering factors. Otherwise is found for one or both replicates of solution A.
the sample solution interferes with the test. However, if the sample does not comply with the test at a
If the sample under test does not comply with the test at a dilution less than the MVD, the test may be repeated using a
dilution less than the MVD, repeat the test using a greater di- grater dilution, not exceeding the MVD.
lution, not exceeding the MVD. The use of a more sensitive 4.3. Quantitative Test
lysate permits a grater dilution of the sample to be examined. This method measures endotoxin concentrations of sam-
Furthermore, interference of the sample solution or diluted ples by determining an endpoint of gel formation.
sample solution may be eliminated by suitable treatment, 4.3.1. Procedure
such as filtration, neutralization, dialysis or heat treatment. Prepare solutions A, B, C and D according to Table
To establish that the treatment chosen effectively eliminates 4.01-3. Making these four solutions one set, test two repli-
interference without loss of endotoxins, perform the assay cates of the set. When preparing solutions A and B, use sam-
described above using the preparation to be examined to ple solutions complying with 4.1.2. Concerning the test con-
which Standard Endotoxin has been added and which has ditions, follow the procedure described in 4.1.1.
then been submitted to the chosen treatment. 4.3.2. Calculation and interpretation
4.2. Limit test The test is valid when the following three conditions are
This method tests whether or not a sample contains met: (a) both replicates of the negative control solution D are
endotoxins grater than the endotoxin limit specified in the negative, (b) both replicates of the positive product control
individual monograph based on the gel formation in the solution B are positive and (c) the geometric mean endpoint
presence of endotoxins at a concentration of more than the concentration of solution C is in the range of 0.5 l to 2 l.
labeled lysate sensitivity. The endpoint is defined as the maximum dilution showing
4.2.1. Procedure the last positive test in the dilution series of solution A, and
Prepare solutions A, B, C and D according to Table the endotoxin concentration of the sample solution is calcu-
4.01-2. Making these four solutions one set, test two repli- lated by multiplying the endpoint dilution factor by l.
cates of the set. In preparing solutions A and B, use the sam- If none of the dilutions of solution A is positive, report

the endotoxin concentration of the sample solution as less Table 4.01-4

than l × the lowest dilution factor of the sample solution.
Solution to which Number of test
If all dilutions are positive, the endotoxin concentration of Solution Endotoxin concentration
endotoxin is added tubes or wells
the sample solution is reported as equal to or greater than
the greatest dilution factor of solution A multiplied by l. A*1 0 Sample solution Not less than 2
Calculate the endotoxin concentration (in EU/mL, EU/ Middle concentration of the
mg, EU/mEq or EU/Unit) of the sample based on the endo- B*2 Sample solution Not less than 2
standard curve
toxin concentration of the sample solution. The sample com-
plies with the Bacterial Endotoxins Test if the endotoxin C*3 At least 3 concentrations Water for BET Each not less than 2
concentration of the sample in both replicates meets the D*4 0 Water for BET Not less than 2
requirement for the endotoxin limit (in EU/mL, EU/mg,
*1 Sample solution only (for assaying endotoxin concentration in the sample
EU/mEq or EU/Unit) specified in the individual mono- solution). The sample solution may be diluted not to exceed the MVD.
graph. *2 Sample solution at the same dilution as solution A, containing added
standard endotoxin at a concentration equal to or near the middle of the
5. Photometric quantitative techniques standard curve.
5.1. Turbidimetric technique *3 Standard endotoxin solutions at the concentrations used in 5.3.1. (for the
This technique measures the endotoxin concentrations of standard curve).
samples based on the measurement of turbidity change ac- *4 Negative control. Water for BET only.
companying gel formation of the lysate TS. This technique is
classified as either endpoint-turbidimetric or kinetic-turbidi-
metric.
standards should be included to bracket each log increase in
The endpoint-turbidimetric technique is based on the
the range of the standard curve.
quantitative relationship between the concentration of endo-
If the absolute value of the correlation coefficient, |r|, is
toxins and the turbidity of the reaction mixture at a specified
greater than or equal to 0.980 for the range of endotoxin
reaction time.
concentrations set up, the criteria for the standard curve are
The kinetic-turbidimetric technique is based on the quan-
valid and the curve complies with the test.
titative relationship between the concentration of endotoxins
If the standard curve does not comply with the test, repeat
and either the time needed to reach a predetermined tur-
the test after verifying the test conditions.
bidity of the reaction mixture or the rate of turbidity de-
5.3.2. Test for interfering factors
velopment.
Prepare solutions A, B, C and D according to Table
The test is usually carried out at 37 ± 19 C, and turbidity
4.01-4. Perform the test on these solutions following the
is expressed in terms of either absorbance or transmission.
optimal conditions for the lysate reagent used (with regard to
5.2. Chromogenic technique
volume of sample solution and lysate TS, volume ratio of
This technique measures the endotoxin concentrations of
sample solution to lysate TS, incubation time, etc.).
sample solutions based on the measurement of chromophore
The test for interfering factors must be repeated when any
released from a synthetic chromogenic substrate by the
condition changes, which is likely to influence the result of
reaction of endotoxins with the lysate TS. This technique is
the test.
classified as either endpoint-chromogenic or kinetic-chromo-
The test is valid when the following conditions are met.
genic.
1: The absolute value of the correlation coefficient of the
The endpoint-chromogenic technique is based on the
standard curve generated using solution C is greater
quantitative relationship between the concentration of endo-
than or equal to 0.980.
toxins and the release of chromophore at the end of an incu-
2: The result with solution D does not exceed the limit of
bation period.
the blank value required in the description of the
The kinetic-chromogenic technique is based on the quan-
lysate employed, or it is less than the endotoxin detec-
titative relationship between the concentration of endotoxins
tion limit of the lysate employed.
and either the time needed to reach a predetermined absor-
Calculate the recovery of the endotoxin added to solution
bance (or transmittance) of the reaction mixture or the rate
B from the concentration found in solution B after subtract-
of color development.
ing the endotoxin concentration found in solution A. When
The test is usually carried out at 37 ± 19C.
the recovery of the endotoxin added to solution B is within
5.3. Preparatory testing
50z to 200z, the sample solution under test is considered to
To assure the precision and validity of the turbidimetric or
be free of interfering factors and the solution complies with
chromogenic techniques, perform both Test for assurance of
the test.
criteria for the standard curve (5.3.1.) and Test for interfer-
When the endotoxin recovery is out of the specified range,
ing factors (5.3.2.), as indicated below.
the sample solution under test is considered to contain inter-
5.3.1. Test for assurance of criteria for the standard curve
fering factors. If the sample under test does not comply with
The test is to be carried out when each new lot of lysate
the test, repeat the test using a greater dilution, not
reagent is used or when there is any change in the experimen-
exceeding the MVD. Furthermore, interference of the sam-
tal conditions which may affect the outcome of the test.
ple solution or diluted sample solution not to exceed the
Using the Standard Endotoxin Solution, prepare at least
MVD may be eliminated by suitable treatment, such as
three endotoxin concentrations to generate the standard
filtration, neutralization, dialysis or heat treatment. To es-
curve within the range of endotoxin concentrations indicated
tablish that the treatment chosen effectively eliminates inter-
by the instructions for the lysate reagent used. Perform the
ference without loss of endotoxins, perform the assay
test using at least three replicates of each standard endotoxin
described above using the preparation to be examined to
concentration according to the optimal conditions for the
which Standard Endotoxin has been added and which has
lysate reagent used (with regard to volume ratios, incubation
then been submitted to the chosen treatment.
time, temperature, pH, etc.).
If the desired range is greater than two logs, additional

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
114 4.02 Microbial Assay for Antibiotics / General Tests JP XVII
5.4. Quantitative test TS, potassium hydroxide TS or 1 mol/L hydrochloric acid
5.4.1. Procedure TS. A different medium to the one specified for each test or-
Prepare solutions A, B, C and D according to Table ganism may be used if it has both a similar composition and
4.01-4, and follow the procedure described in 5.3.2. an equal or better growth efficiency of the test organism in
5.4.2. Calculation of endotoxin concentration comparison with the specified medium. Unless otherwise
Calculate the mean endotoxin concentration of solution A specified, sterilize the media to be used in an autoclave.
using the standard curve generated with solution C. The test (1) Agar media for seed and base layer
is valid when all the following requirements are met. 1) Media for test organism Bacillus subtilis ATCC 6633
1: The absolute value of the correlation coefficient of the i. Peptone 5.0 g
standard curve generated using solution C is greater Meat extract 3.0 g
than or equal to 0.980. Agar 15.0 g
2: The endotoxin recovery, calculated from the concen- Water 1000 mL
tration found in solution B after subtracting the con- Mix all the ingredients, and sterilize. Adjust the pH of the
centration of endotoxin found in solution A, is within solution so that it will be 7.8 to 8.0 after sterilization.
the range of 50z to 200z. ii. Peptone 5.0 g
3: The result with solution D does not exceed the limit of Meat extract 3.0 g
the blank value required in the description of the Trisodium citrate dihydrate 10.0 g
lysate employed, or it is less than the endotoxin detec- Agar 15.0 g
tion limit of the lysate employed. Water 1000 mL
5.4.3. Interpretation Mix all the ingredients, and sterilize. Adjust the pH of the
The sample complies with the Bacterial Endotoxins Test if solution so that it will be 6.5 to 6.6 after sterilization.
the endotoxin concentration of the sample calculated from 2) Medium for test organism Saccharomyces cerevisiae
the mean endotoxin concentration of solution A meets the ATCC 9763
requirement of the endotoxin limit (in EU/mL, EU/mg, Glucose 10.0 g
EU/mEq or EU/Unit) specified in the individual mono- Peptone 9.4 g
graph. Meat extract 2.4 g
Yeast extract 4.7 g
Sodium chloride 10.0 g
Agar 15.0 g
4.02 Microbial Assay for Water 1000 mL
Antibiotics Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.0 to 6.2 after sterilization.
Microbial Assay for Antibiotics is a method to determine 3) Media for other organisms
the antimicrobial potency of antibiotics based on their anti- i. Glucose 1.0 g
microbial activities. There are three methods for this test: the Peptone 6.0 g
cylinder-plate, perforated plate, and turbidimetric methods. Meat extract 1.5 g
The former two are based on the measurement of the size of Yeast extract 3.0 g
the zones of microbial growth inhibition in a nutrient agar Agar 15.0 g
medium, and the turbidimetric method is based on the meas- Water 1000 mL
urement of the inhibition of turbidity development in a fluid Mix all the ingredients, and sterilize. Adjust the pH of the
medium with microbial growth. Unless otherwise specified in solution so that it will be 6.5 to 6.6 after sterilization.
the individual monograph, tests specified to be carried out ii. Glucose 1.0 g
by the cylinder-plate method may be conducted under the Meat peptone 6.0 g
same test conditions using the perforated plate method in- Casein peptone 4.0 g
stead. If necessary, first sterilize water, isotonic sodium chlo- Meat extract 1.5 g
ride solution, buffer solutions, reagents, test solutions and Yeast extract 3.0 g
essential parts of measuring instruments and appliances to be Agar 15.0 g
used for the test. In performing the test, precautions must be Water 1000 mL
taken to prevent biohazard. Mix all the ingredients, and sterilize. Adjust the pH of the
solution so that it will be 6.5 to 6.6 after sterilization.
1. Cylinder-plate method
iii. Peptone 10.0 g
The cylinder-plate method is a method to determine the
Meat extract 5.0 g
antimicrobial potency of the antibiotic to be tested, and is
Sodium chloride 2.5 g
based on the measurement of the size of the zone of growth
Agar 15.0 g
inhibition of a test organism by the use of cylinder-agar
Water 1000 mL
plates.
Mix all the ingredients, and sterilize. Adjust the pH of the
1.1. Test organisms
solution so that it will be 6.5 to 6.6 after sterilization.
Use the test organism specified in the individual mono-
(2) Agar media for transferring test organisms
graph.
1) Medium for test organism Saccharomyces cerevisiae
1.2. Culture media
ATCC 9763
Unless otherwise specified, use media with the following
Glucose 15.0 g
compositions. When `peptone' is indicated as an ingredient
Peptone 5.0 g
of a medium, either meat peptone or casein peptone is ap-
Yeast extract 2.0 g
plicable. Use sodium hydroxide TS or 1 mol/L hydrochloric
Magnesium sulfate heptahydrate 0.5 g
acid TS to adjust the pH of the medium to obtain the speci-
Potassium dihydrogen phosphate 1.0 g
fied value after sterilization. In the case of the medium for
Agar 15.0 g
Bacillus subtilis ATCC 6633, adjust the pH using ammonia
Water 1000 mL

Mix all the ingredients, and sterilize. Adjust the pH of the occasion demands. Store the stock suspensions of the test or-
solution so that it will be 6.0 to 6.2 after sterilization. ganisms at a temperature not exceeding 59C, and use within
2) Media for other organisms 30 days.
i. Glucose 1.0 g (iii) Preparation of a stock suspension of other test or-
Meat peptone 6.0 g ganisms
Casein peptone 4.0 g Inoculate the test organism onto the slant or the plate of
Meat extract 1.5 g the agar medium which has been prepared for transferring
Yeast extract 3.0 g the test organisms specified in 1.2. (2) 2) i. Incubate the in-
Agar 15.0 g oculated slant at 32 to 379 C for 16 to 24 hours. The subcul-
Water 1000 mL ture should be performed at least three times. Inoculate the
Mix all the ingredients, and sterilize. Adjust the pH of the subcultured test organism onto another slant or plate agar
solution so that it will be 6.5 to 6.6 after sterilization. medium (described above), and incubate the slant at 32 to 37
ii. Peptone 10.0 g 9C for 16 to 24 hours. Scrape away and suspend the resulting
Meat extract 5.0 g growth from the agar surface in isotonic sodium chloride so-
Sodium chloride 2.5 g lution, and use this as a stock suspension of the test organ-
Agar 15.0 g ism. The concentration of the test organism is confirmed
Water 1000 mL with the turbidity or absorbance, as occasion demands. Store
Mix all the ingredients, and sterilize. Adjust the pH of the the stock suspensions of the test organisms at a temperature
solution so that it will be 6.5 to 6.6 after sterilization. not exceeding 59C, and use within 5 days.
1.3. Preparation of agar slant or plate media 1.5. Preparation of agar base layer plates
Unless otherwise specified, dispense approximately 9 mL Unless otherwise specified, dispense 20 mL of the melted
of melted agar medium in each test tube (approximately 16 agar medium for the base layer into each Petri dish, and in
mm in inside diameter), and make them as slant media, or the case of a large dish, dispense a quantity of the agar me-
dispense approximately 20 mL of melted agar medium in dium to form a uniform layer 2 to 3 mm thick. Distribute the
each Petri dish (approximately 90 mm in inside diameter), agar evenly in each dish on a flat, level surface, and allow it
and make them as plate media. to harden.
1.4. Preparation of stock suspensions of test spores or or- 1.6. Preparation of seeded agar layers
ganisms Unless otherwise specified, determine the volume of the
Unless otherwise specified, prepare stock suspensions of stock suspension of the spore or the test organism with
test spore or organism cultures as follows. Check the aspects which the employed standard solution shows a clear and
of the test spores or organisms as occasion demands. definite zone of growth inhibition. Prepare the seeded agar
(i) Preparation of a stock spore suspension of test organ- layer by mixing thoroughly the previously determined
ism Bacillus subtilis ATCC 6633 volume of stock suspension of spore or test organism with
Inoculate the test organism onto the slant or plate of the agar medium for the seed layer kept at 48 to 519C. Usually,
agar medium which was prepared for transferring the test or- the rate of a stock spore suspension and a stock suspension
ganisms specified in 1.2. (2) 2) i. Incubate at 32 to 379C for of the test organism to add to the agar medium for the seed
16 to 24 hours. Inoculate the subcultured test organism onto layer are 0.1 to 1.0 volz and 0.5 to 2.0 volz, respectively.
a suitable volume of slant or plate of the agar medium 1.7. Preparation of cylinder-agar plates
(described above), which was prepared for transferring the Dispense 4 to 6 mL of the seeded agar layer, which is spe-
test organisms specified in 1.2. (2) 2) ii. Then incubate at 32 cified in the individual monograph, on an agar base layer
to 379 C for not less than 1 week to prepare spores. Suspend plate in a Petri dish. In the case of large dishes, dispense a
the spores in isotonic sodium chloride solution, heat at 659C quantity of the agar medium to form a uniform layer 1.5 to
for 30 minutes, and then centrifuge. Wash the spore sedi- 2.5 mm thick, and spread evenly over the surface before
ment three times with isotonic sodium chloride solution by hardening. After coagulating the agar, allow the plate to
means of centrifugation. Re-suspend the spore sediment in stand under a clean atmosphere to exhale moisture vapor of
water or isotonic sodium chloride solution, and heat again at the inside of Petri or large dishes and water on the agar sur-
659 C for 30 minutes to prepare the stock spore suspension. face. Place 4 cylinders on an agar plate in a Petri dish so that
The concentration of the test organism is confirmed with the the individual cylinders are equidistant from the center of
turbidity or absorbance, as occasion demands. Store the the plate and equally spaced from one another (the cylinders
stock spore suspension at a temperature not exceeding 59C, are set on the circumference of a circle of 25 to 28 mm
and use within 6 months. If the stock spore suspension radius). When large dish plates are used, place cylinders on
shows a clear and definite zone of growth inhibition in an each plate according to the method of preparation for Petri
antibiotics potency test using adequate antibiotics, it may be dish agar plates. A set of 4 cylinders on each large dish plate
used for further 6 months. is considered to be equivalent to one Petri dish plate. Use
(ii) Preparation of a stock suspension of the test organ- stainless steel cylinders with the following dimensions: out-
ism Saccharomyces cerevisiae ATCC 9763 side diameter 7.9 to 8.1 mm; inside diameter 5.9 to 6.1 mm;
Inoculate test organism onto the slant or plate agar me- length 9.9 to 10.1 mm. The cylinders should not interfere
dium which has been prepared for transferring test organism with the test. Prepare the cylinder-agar plates before use.
specified in 1.2. (2) 1). Incubate at 25 to 269C for 40 to 48 1.8. Standard solutions
hours. The subculture should be performed at least three Use both a standard solution of high concentration and
times. Inoculate the subcultured test organism onto another one of low concentration, as specified in the individual
slant or plate of the agar medium (described above), and in- monograph. Unless otherwise specified, prepare the stand-
cubate at 25 to 269C for 40 to 48 hours. Scrape away and ard solutions before use.
suspend the resulting growth from the agar surface in iso- 1.9. Sample solutions
tonic sodium chloride solution, and use this as a stock sus- Use both a sample solution of high concentration and one
pension of the test organism. The concentration of the test of low concentration, as specified in the individual mono-
organism is confirmed with the turbidity or absorbance, as graph. Unless otherwise specified, prepare the sample solu-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
116 4.02 Microbial Assay for Antibiotics / General Tests JP XVII
tions before use. suitable tool, prepare 4 circular cavities having a diameter of
1.10. Procedure 7.9 to 8.1 mm on a Petri dish agar plate so that the individ-
Unless otherwise specified, use 5 cylinder-agar plates as ual cavities are equidistant from the center of the plate. The
one assay set when Petri dishes are employed. When large cavities spaced equally from one another on the circumfer-
dishes are employed, the number of cylinders for one assay ence of a circle with radius 25 to 28 mm, and are deep
set should be equal to that defined when using Petri dishes. enough to reach the bottom of dish. When large dish plates
Apply the standard solution of high concentration and that are used, prepare the circular cavities on each plate accord-
of low concentration to a pair of cylinders set opposite each ing to the method of preparation for Petri dish agar plates.
other on each plate. Apply the high and low concentration A set of 4 cavities on each large dish plate is considered to be
sample solutions to the remaining 2 cylinders. The same equivalent to one Petri dish plate. Prepare the perforated
volume of these solutions must be added to each cylinder. agar plates before use.
Incubate the plates at 32 to 379C for 16 to 20 hours. Using a 2.2. Procedure
suitable measuring tool, measure the diameters of circular Unless otherwise specified, use 5 perforated agar plates as
inhibition zones with a precision that can discriminate differ- one assay set when Petri dishes are employed. When large
ences of at least 0.25 mm. Each procedure should be per- dishes are employed, the number of cavities for one assay set
formed quickly under clean laboratory conditions. should be equal to that defined when using Petri dishes.
1.11. Estimation of potency Apply the high and low concentration standard solutions to
The following correlation between the potency (P) of solu- a pair of cavities prepared opposite each other on each plate,
tion in a cylinder and the diameter (d ) of zone of inhibition and apply the high and low concentration sample solutions
is established. to the remaining 2 cavities. The same volume of these solu-
tions must be added to each cavity. Incubate the plates at 32
d ＝ a log P ＋ b
to 379 C for 16 to 20 hours. Using a suitable measuring tool,
where, a and b are constants. measure the diameters of the circular inhibition zones with a
If necessary, ascertain the values in the above equation. precision that can discriminate differences of at least 0.25
Based on this equation, estimate the potency of the sample mm. Each procedure should be performed quickly under
solutions by application of the following equation: clean laboratory conditions.
Amount (potency) of sample 3. Turbidimetric method
＝ A × Potency of SH per mL × Dilution factor of UH The turbidimetric method is a method to determine the an-
timicrobial potency of an antibiotic, based on the meas-
where:
urement of the inhibition of growth of a microbial culture in
IV a fluid medium. The inhibition of growth of a test organism
logA ＝
W is photometrically measured as changes in turbidity of the
microbial culture.
I ＝ log (potency of SH/potency of SL)
3.1. Test organisms
V ＝ S UH ＋ S UL － SSH － S SL
Use the test organism specified in the individual mono-
W ＝ S UH ＋ SSH － S UL － S SL
graph.
The sum of the diameter (mm) of the inhibitory zone
3.2. Culture media
measured in each plate is designated as follows:
Unless otherwise specified, use media with the following
for standard solution of high concentration (SH) ＝ SSH
compositions. When peptone is indicated as an ingredient of
for standard solution of low concentration (SL) ＝ SSL
a medium, either meat peptone or casein peptone is applica-
for sample solution of high concentration (UH) ＝ SUH
ble. Use sodium hydroxide TS or 1 mol/L hydrochloric acid
for sample solution of low concentration (UL) ＝ SUL
TS to adjust the pH of the medium to obtain the specified
2. Perforated plate method value after sterilization. A different medium to the one spe-
The perforated plate method is a method to determine the cified for each test organism may be used if it has both a
antimicrobial potency of an antibiotic, based on the meas- similar composition and an equal or better growth efficiency
urement of the size of the zone of growth inhibition of a test of the test organism in comparison with the specified me-
organism by the use of perforated agar plates. dium. Unless otherwise specified, sterilize the media to be
This method is carried out by the use of perforated agar used in an autoclave.
plates in lieu of cylinder-agar plates used in Cylinder-plate (1) Agar media for transferring test organisms
method. Glucose 1.0 g
Proceed as directed below, but comply with the require- Peptone 6.0 g
ments of Cylinder-plate method, such as test organisms, me- Meat extract 1.5 g
dia, preparation of agar slant or plate media, preparation of Yeast extract 3.0 g
stock suspensions of spores or test organisms, preparation of Sodium chloride 2.5 g
agar base layer plates, preparation of seeded agar layers, Agar 15.0 g
standard solutions, sample solutions, and estimation of po- Water 1000 mL
tency. Mix all the ingredients, and sterilize. Adjust the pH of the
2.1. Preparation of perforated agar plates solution so that it will be 6.5 to 6.6 after sterilization.
Dispense 4 to 6 mL of the seeded agar layer specified in (2) Liquid media for suspending test organisms
the individual monograph on each agar base layer plate of Glucose 1.0 g
the Petri dish. In the case of large dishes, dispense a quantity Peptone 5.0 g
of the agar medium to form a uniform layer 1.5 to 2.5 mm Meat extract 1.5 g
thick, and spread evenly over the surface before hardening. Yeast extract 1.5 g
After coagulating the agar, allow the plate to stand under a Sodium chloride 3.5 g
clean atmosphere to exhale moisture vapor of the inside of Potassium dihydrogen phosphate 1.32 g
Petri or large dishes and water on the agar surface. Using a Disodium hydrogen phosphate* 3.0 g

Water 1000 mL a, b, c, d, e: Average transmittance or absorbance values

Mix all the ingredients, and sterilize. Adjust the pH of the for each standard dilution, where a is the value from
solution so that it will be 7.0 to 7.1 after sterilization. the lowest concentration standard solution, b, c and d
*Dipotassium hydrogen phosphate (3.68 g) may be used in are the values from each geometrically increased con-
lieu of disodium hydrogen phosphate (3.0 g). centration standard solution, respectively, and e is the
3.3. Preparation of agar slant or plate media value from the highest concentration standard solu-
Unless otherwise specified, proceed as directed in Prepara- tion.
tion of agar slant or plate media under Cylinder-plate
method.
3.4. Preparation of stock suspensions of test organisms
Unless otherwise specified, inoculate the test organism 4.03 Digestion Test
onto the slant or plate of the agar medium which was pre-
Digestion Test is a test to measure the activity of digestive
pared for transferring the specified test organism. Incubate
enzymes, as crude materials or preparations, on starch, pro-
the inoculated medium at 32 to 379C for 16 to 24 hours. The
tein and fat.
subculture should be performed at least three times. Check
the aspects of the test spores or organisms as occasion de- 1. Assay for Starch Digestive Activity
mands. Inoculate the subcultured test organism onto another The assay for starch digestive activity is performed
slant or plate of the agar medium (described above), and in- through the measurement of starch saccharifying activity,
cubate the slant at 32 to 379 C for 16 to 24 hours. After incu- dextrinizing activity, and liquefying activity.
bation, suspend the test organism in the liquid medium for 1.1. Measurement of starch saccharifying activity
suspending the test organism, and use as the suspension of The starch saccharifying activity can be obtained by meas-
the test organism. The concentration of the test organism is uring an increase of reducing activity owing to the hydrolysis
confirmed with the turbidity or absorbance, as occasion de- of the glucoside linkages when amylase acts on the starch.
mands. Under the conditions described in Procedure, one starch sac-
3.5. Standard solutions charifying activity unit is the amount of enzyme that cata-
Use the standard solutions specified in the individual lyzes the increase of reducing activity equivalent to 1 mg of
monograph. Unless otherwise specified, prepare the stand- glucose per minute.
ard solutions before use. 1.1.1. Preparation of Sample Solution
3.6. Sample solutions Dissolve the sample in an appropriate amount of water, or
Use the sample solutions specified in the individual mono- a buffer or salts solution specified in the monograph so that
graph. Unless otherwise specified, prepare the sample solu- the reducing activity increases in proportion to the concen-
tions before use. tration of the sample solution, when measuring under the
3.7. Procedure conditions described in Procedure. The concentration is nor-
Unless otherwise specified, proceed as follows: mally 0.4 to 0.8 starch saccharifying activity unit/mL. Filter
Distribute 1.0 mL of each concentration of the standard if necessary.
solution, the sample solution, and water used as a control, 1.1.2. Preparation of Substrate Solution
into each set composed of 3 test tubes (about 14 mm in inside Use potato starch TS for measuring the starch digestive
diameter and about 13 cm in length). Add 9.0 mL of the sus- activity. If necessary, add 10 mL of buffer or salts solution
pension of the test organism to each tube, and then incubate specified in the monograph, instead of 10 mL of 1 mol/L
in a water bath maintained at 35 to 379C for 3 to 4 hours. acetic acid-sodium acetate buffer solution (pH 5.0).
After incubation, add 0.5 mL of dilute formaldehyde (1 in 3) 1.1.3. Procedure
to each tube, and read each transmittance or absorbance at a Pipet 10 mL of the substrate solution, stand at 37 ±
wavelength of 530 nm. 0.59C for 10 minutes, add exactly 1 mL of the sample solu-
3.8. Estimation of potency tion, and shake immediately. Allow this solution to stand at
Average the transmittance or absorbance values of each 37 ± 0.59C for exactly 10 minutes, add exactly 2 mL of
concentration of the standard solution, the sample solution alkaline tartrate solution of the Fehling's TS for amylolytic
and water used as a control, respectively. Generate the activity test, and shake immediately. Then, add exactly 2 mL
standard curve based on the average values of transmittance of copper solution of the Fehling's TS for amylolytic activity
or absorbance of each concentration of the standard solu- test, shake gently, heat the solution in a water bath for ex-
tion, and estimate the potency of the sample solution from actly 15 minutes, and then immediately cool to below 259C.
its average value of transmittance or absorbance using the Then, add exactly 2 mL of concentrated potassium iodide TS
obtained standard curve. and 2 mL of diluted sulfuric acid (1 in 6), and titrate <2.50>
If the standard dilutions of five concentrations in geomet- the released iodine with 0.05 mol/L sodium thiosulfate VS to
ric progression are used, calculate the L and H values from the disappearance of the blue color produced by addition of
the following equations. Plot point L and point H on graph 1 to 2 drops of soluble starch TS (a mL). Separately, pipet 10
paper and construct a straight line for the standard curve. mL of water instead of the substrate solution and titrate
<2.50> in the same manner (b mL).
3a ＋ 2b ＋ c － e
L＝
5 Starch saccharifying activity (unit/g)
3e ＋ 2d ＋ c － a 1 1
H＝＝ amount (mg) of glucose × ×
5 10 M
where: Amount (mg) of glucose ＝ (b － a) × 1.6
L: Calculated value of transmittance or absorbance for
M: Amount (g) of sample in 1 mL of sample solution
the lowest concentration of the standard curve.
H: Calculated value of transmittance or absorbance for 1.2. Measurement of starch dextrinizing activity
the highest concentration of the standard curve. The starch dextrinizing activity can be obtained by meas-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
118 4.03 Digestion Test / General Tests JP XVII
uring a decrease in starch coloration by iodine resulting from 1.3.4. Procedure
hydrolysis of the straight chain component (amylose) in Put 50 mL of the 50z standard sucrose solution in a
starch when amylase acts on the starch. Under the conditions 100-mL conical flask, and allow it to stand in a thermostat at
described in Procedure, one starch dextrinizing activity unit 37 ± 0.59 C for 15 minutes. Fix a viscometer shown in Fig.
is the amount of enzyme required to reduce the coloration of 4.03-1 so that its lower end almost touches the bottom of the
potato starch by iodine by 10z per minute. flask and that the water in the thermostat circulates around
1.2.1. Preparation of Sample Solution the outer cylinder of the viscometer. After slowly pulling up
Dissolve the sample in an appropriate amount of water or the 50z standard sucrose solution by suction to the middle
a buffer or salts solution specified in the monograph so that of the upper bulb of the viscometer, let it flow down by grav-
the coloration of starch by iodine decreases in proportion to ity, measuring the time taken for the solution to fall from
the concentration of the sample solution, when measuring the upper to the lower indicators (t1 seconds). Take exactly
under the conditions described in Procedure. The concentra- 50 g of the substrate solution in another 100-mL conical
tion is normally 0.2 to 0.5 starch dextrinizing activity flask, and stand it in another thermostat at 37 ± 0.59 C for
unit/mL. Filter if necessary. 20 minutes. Add exactly 1 mL of the sample solution to it,
1.2.2. Preparation of Substrate Solution and shake the flask immediately. Fix a viscometer vertically
Prepare the substrate solution in the same manner as the so that its lower end almost touches the bottom of the flask
substrate solution in the measurement of starch sac- and that the water in the thermostat circulates around the
charifying activity. outer cylinder of the viscometer. Occasionally pull the reac-
1.2.3. Procedure tion solution up by suction to the middle of the upper bulb
Pipet 10 mL of the substrate solution, stand at 37 ± slowly, then let it flow down by gravity, measuring the time
0.59 C for 10 minutes, add exactly 1 mL of the sample solu- taken for the solution to fall from the upper to the lower in-
tion, and shake immediately. Allow this solution to stand at dicators (t seconds). Repeat this operation until t becomes
37 ± 0.59C for exactly 10 minutes. Pipet 1 mL of this solu- shorter than t1. At each measurement, record the time (T?
tion, add it to 10 mL of 0.1 mol/L hydrochloric acid TS, and seconds) from the moment that the sample solution is added
shake immediately. Pipet 0.5 mL of this solution, add ex- to the moment that the solution surface in the flask passes
actly 10 mL of 0.0002 mol/L iodine TS, and shake. Deter- the upper indicator. (T? ＋ t/2) is the reaction time (T ) cor-
mine the absorbance AT of this solution at the wavelength of responding to t. Draw a curve for both t and T. Obtain T1
660 nm as directed under Ultraviolet-visible Spectropho- and T2 that correspond to t1 and (2 × t1) by interpolation.
tometry <2.24>. Separately, using 1 mL of water instead of
60 1.5
the sample solution, determine the absorbance AB in the Starch liquefying activity (unit/g) ＝ ×
(T1 － T2) M
same manner.
M: Amount (g) of sample in 1 mL of sample solution
Starch dextrinizing activity (unit/g)
2. Assay for Protein Digestive Activity
(AB － AT) 1
＝ × The protein digestive activity can be obtained by the
AB M
colorimetric measurement, making use of Folin's reaction,
M: Amount (g) of sample in 1 mL of sample solution of the amount of acid-soluble low-molecular products,
which is increased owing to the hydrolysis of the peptide
1.3. Measurement of starch liquefying activity
linkages when protease acts on casein. One protein digestive
The starch liquefying activity can be obtained by measur-
activity unit is the amount of enzymes that produces Folin's
ing a decrease in the viscosity of starch solution resulting
TS-colorable substance equivalent to 1 mg of tyrosine per
from the hydrolysis of molecules when amylase acts on the
minute under the conditions described in Procedure.
starch. Under the conditions described in Procedure, one
2.1. Preparation of Sample Solution
starch liquefying activity unit is the amount of enzyme re-
Dissolve the sample in an appropriate amount of water, or
quired to reduce the viscosity of the substrate solution
a buffer or salts solution specified in the monograph so that
equivalent to 1 g of potato starch from 200z to 100z of
the amount of non-protein, Folin's TS-colorable substances
that of the 50z sucrose standard solution.
increase in proportion to the concentration of the sample so-
1.3.1. Preparation of Sample Solution
lution, when measuring under the conditions described in
Dissolve the sample in an appropriate amount of water, or
Procedure. The concentration is normally 15 to 30 protein
a buffer or salts solution specified in the monograph so that
digestive activity unit/mL.
the viscosity decreases in proportion to the concentration of
2.2. Tyrosine Calibration Curve
the sample solution, when measuring under the conditions
Weigh exactly 50 mg of Tyrosine Reference Standard, pre-
described in Procedure. The concentration is normally 0.15
viously dried at 1059 C for 3 hours, and dissolve in 0.2 mol/L
to 0.25 starch liquefying activity unit/mL. Filter if neces-
hydrochloric acid TS to make exactly 50 mL. Pipet 1 mL, 2
sary.
mL, 3 mL and 4 mL of this solution separately, and add 0.2
1.3.2. Preparation of Substrate Solution
mol/L hydrochloric acid TS to each solution to make them
Weigh accurately about 1 g of potato starch, and measure
exactly 100 mL. Pipet 2 mL of each solution, and add ex-
the loss on drying at 1059C for 2 hours. Weigh exactly pota-
actly 5 mL of 0.55 mol/L sodium carbonate TS and 1 mL of
to starch equivalent to 15.00 g calculated on the dried basis,
diluted Folin's TS (1 in 3) to each solution, shake immedi-
add 300 mL of water, then add gradually 25 mL of 2 mol/L
ately, then stand them at 37 ± 0.59 C for 30 minutes. Deter-
sodium hydroxide TS under thorough shaking, until the mix-
mine the absorbances, A1, A2, A3 and A4, of these solutions
ture forms a paste. Heat the mixture in a water bath for 10
at 660 nm as directed under Ultraviolet-visible Spectropho-
minutes, shaking it occasionally. After cooling, neutralize
tometry <2.24>, using a solution prepared with exactly 2 mL
the mixture with 2 mol/L hydrochloric acid TS, and add 50
of 0.2 mol/L hydrochloric acid TS in the same manner as the
mL of the buffer solution specified in the monograph and
blank. Then, draw a calibration curve with the absorbances,
water to make exactly 500 g. Prepare before use.
A1, A2, A3 and A4 as the ordinate, and with the amount ( mg)
1.3.3. Preparation of 50z Standard Sucrose Solution
of tyrosine in 2 mL of each solution as the abscissa. Obtain
Dissolve 50.0 g of sucrose in 50.0 mL of water.

trate, exactly measure the subsequent 2 mL of the filtrate,

add exactly 5 mL of 0.55 mol/L sodium carbonate TS and 1
mL of diluted Folin's TS (1 in 3) to the solution, shake well,
and stand it at 37 ± 0.59C for 30 minutes. Determine the
absorbance AT of this solution at 660 nm as directed under
Ultraviolet-visible Spectrophotometry <2.24>, using water as
the blank. Separately, pipet 1 mL of the sample solution,
add exactly 5 mL of trichloroacetic acid TS A or B to the
solution as specified in the monograph, and shake. To this
solution add exactly 5 mL of the substrate solution specified
in the monograph, shake immediately, and stand it at 37 ±
0.59C for 30 minutes. Follow the same procedure for the
sample solution, and determine the absorbance AB at 660
nm.
Protein digestive activity (unit/g)
11 1 1
＝ (AT － AB) × F × × ×
2 10 M
M: Amount (g) of sample in 1 mL of sample solution
F: Amount ( mg) of tyrosine for absorbance 1 determined
from Tyrosine Calibration Curve
3. Assay for Fat Digestive Activity
The fat digestive activity can be obtained by back titration
of the amount of fatty acid produced from the hydrolysis of
the ester linkage, when lipase acts on olive oil. One fat diges-
tive activity unit is the amount of enzymes that produces 1
mmole of fatty acid per minute under the conditions
described in Procedure.
Fig. 4.03-1 3.1. Preparation of Sample Solution
Dissolve or suspend the sample in an appropriate amount
of cold water, or a buffer or salts solution specified in the
monograph so that the amount of fatty acid increases in pro-
the amount ( mg) of tyrosine for the absorbance 1. portion to the concentration of the sample solution, when
2.3. Preparation of Substrate Solution measuring under the conditions described in Procedure. The
(i) Substrate solution 1: Weigh accurately about 1 g of concentration is normally 1 to 5 fat digestive activity
milk casein, and measure the loss on drying at 1059C for 2 unit/mL.
hours. Weigh exactly an amount of milk casein equivalent to 3.2. Preparation of Substrate Solution
1.20 g calculated on the dried basis, add 12 mL of lactic acid Take 200 to 300 mL of a mixture of emulsifier and olive
TS and 150 mL of water, and warm to dissolve in a water oil (3:1) in a blender (see Fig. 4.03-2), and emulsify it at
bath. After cooling in running water, adjust to the pH speci- 12,000 to 16,000 revolutions per minute for 10 minutes,
fied in the monograph with 1 mol/L hydrochloric acid TS or while cooling the solution to a temperature below 109C.
sodium hydroxide TS, and add water to make exactly 200 Stand this solution in a cool place for 1 hour, and make sure
mL. Prepare before use. before use that the oil does not separate.
(ii) Substrate solution 2: Weigh accurately about 1 g of 3.3. Preparation of Emulsifier
milk casein, and measure the loss on drying at 1059C for 2 Dissolve 20 g of polyvinyl alcohol specified in the mono-
hours. Weigh exactly an amount of milk casein equivalent to graph in 800 mL of water by heating between 759C and 809 C
1.20 g calculated on the dried basis, add 160 mL of 0.05 for 1 hour while stirring. After cooling, filter the solution if
mol/L disodium hydrogenphosphate TS, and warm to dis- necessary, and add water to make exactly 1000 mL.
solve in a water bath. After cooling in running water, adjust 3.4. Procedure
to the pH specified in the monograph with the 1 mol/L hy- Pipet 5 mL of the substrate solution and 4 mL of the
drochloric acid TS or sodium hydroxide TS, and add water buffer solution specified in the monograph, transfer them to
to make exactly 200 mL. Prepare before use. a conical flask, and shake. After standing the mixture at 37
2.4. Preparation of Precipitation Reagent ± 0.59C for 10 minutes, add exactly 1 mL of the sample so-
(i) Trichloroacetic acid TS A: Dissolve 7.20 g of lution, and shake immediately. Stand this solution at 37 ±
trichloroacetic acid in water to make 100 mL. 0.59C for exactly 20 minutes, add 10 mL of a mixture of
(ii) Trichloroacetic acid TS B: Dissolve 1.80 g of ethanol (95) and acetone (1:1), and shake. Then add exactly
trichloroacetic acid, 1.80 g of anhydrous sodium acetate and 10 mL of 0.05 mol/L sodium hydroxide VS, add 10 mL of a
5.5 mL of 6 mol/L acetic acid TS in water to make 100 mL. mixture of ethanol (95) and acetone (1:1), and shake. Titrate
2.5. Procedure <2.50> the excess sodium hydroxide with 0.05 mol/L hydro-
Pipet 5 mL of the substrate solution specified in the mono- chloric acid VS (b mL) (indicator: 2 to 3 drops of phenol-
graph, stand at 37 ± 0.59 C for 10 minutes, add exactly 1 mL phthalein TS). Separately, pipet 5 mL of the substrate solu-
of the sample solution, and shake immediately. After stand- tion and 4 mL of buffer solution specified in the mono-
ing this solution at 37 ± 0.59C for exactly 10 minutes, add graph, transfer them to a conical flask, and shake. After
exactly 5 mL of trichloroacetic acid TS A or B as specified in standing it at 37 ± 0.59 C for 10 minutes, add 10 mL of a
the monograph, shake, stand it at 37 ± 0.59 C for 30 mixture of ethanol (95) and acetone (1:1), then add exactly 1
minutes, and then filter. Discard the first 3 mL of the fil- mL of the sample solution, and shake. Add exactly 10 mL of

organisms used for inoculation are not more than 5 passages

4.05 Microbiological Examination removed from the original master seed-lot. Grow each of the
bacterial and fungal test strains separately as described in
of Non-sterile Products Table 4.05-I-1.
Use buffered sodium chloride-peptone solution pH 7.0 or
This chapter includes microbial enumeration tests and
phosphate buffer solution pH 7.2 to make test suspensions;
tests for specified micro-organisms. For the test, use a mix-
to suspend Aspergillus brasiliensis spores, 0.05 per cent of
ture of several portions selected at random from the bulk or
polysorbate 80 may be added to the buffer. Use the suspen-
from the contents of a sufficient number of containers. If
sions within 2 hours or within 24 hours if stored at 2 – 89 C.
test specimens are diluted with fluid medium, the test should
As an alternative to preparing and then diluting a fresh
be performed quickly. In performing the test, precautions
suspension of vegetative cells of Aspergillus brasiliensis or
must be taken to prevent biohazard.
Bacillus subtilis, a stable spore suspension is prepared and
I. Microbiological Examination of Non-sterile Products: then an appropriate volume of the spore suspension is used
Microbial Enumeration Tests for test inoculation. The stable spore suspension may be
These tests are harmonized with the European Pharmaco- maintained at 2 – 89C for a validated period of time.
poeia and the U.S. Pharmacopeia. 3.2. Negative control
To verify testing conditions, a negative control is per-
The tests described hereafter will allow quantitative
formed using the chosen diluent in place of the test prepara-
enumeration of mesophilic bacteria and fungi which may
tion. There must be no growth of micro-organisms. A nega-
grow under aerobic conditions.
tive control is also performed when testing the products as
The tests are designed primarily to determine whether a
described under 4. Testing of Products. A failed negative
substance or preparation complies with an established spe-
control requires an investigation.
cification for microbiological quality. When used for such
3.3. Growth promotion of the media
purposes follow the instructions given below, including the
Test each batch of ready-prepared medium and each batch
number of samples to be taken and interpret the results as
of medium, prepared either from dehydrated medium or
stated below.
from the ingredients described.
The methods are not applicable to products containing
Inoculate portions/plates of casein soya bean digest broth
viable micro-organisms as active ingredients.
and casein soya bean digest agar with a small number (not
Alternative microbiological procedures, including auto-
more than 100 CFU) of the micro-organisms indicated in
mated methods, may be used, provided that their equiva-
Table 4.05-I-1, using a separate portion/plate of medium for
lence to the Pharmacopoeial method has been demonstrated.
each. Inoculate plates of Sabouraud-dextrose agar with a
1. General Procedures small number (not more than 100 CFU) of the micro-organ-
Carry out the determination under conditions designed to isms indicated in Table 4.05-I-1, using a separate plate of
avoid extrinsic microbial contamination of the product to be medium for each. Incubate in the conditions described in
examined. The precautions taken to avoid contamination Table 4.05-I-1.
must be such that they do not affect any micro-organisms For solid media, growth obtained must not differ by a fac-
which are to be revealed in the test. tor greater than 2 from the calculated value for a stand-
If the product to be examined has antimicrobial activity, ardized inoculum. For a freshly prepared inoculum, growth
this is insofar as possible removed or neutralized. If inactiva- of the micro-organisms comparable to that previously ob-
tors are used for this purpose their efficacy and their absence tained with a previously tested and approved batch of me-
of toxicity for micro-organisms must be demonstrated. dium occurs. Liquid media are suitable if clearly visible
If surface-active substances are used for sample prepara- growth of the micro-organisms comparable to that previ-
tion, their absence of toxicity for micro-organisms and their ously obtained with a previously tested and approved batch
compatibility with inactivators used must be demonstrated. of medium occurs.
3.4. Suitability of the counting method in the presence of
2. Enumeration Methods
product
Use the membrane filtration method, or the plate-count
3.4.1. Preparation of the sample
methods, as prescribed. The most probable number (MPN)
The method for sample preparation depends on the physi-
method is generally the least accurate method for microbial
cal characteristics of the product to be tested. If none of the
counts, however, for certain product groups with very low
procedures described below can be demonstrated to be satis-
bioburden, it may be the most appropriate method.
factory, an alternative procedure must be developed.
The choice of a method is based on factors such as the na-
(i) Water-soluble products: Dissolve or dilute (usually a
ture of the product and the required limit of micro-organ-
1 in 10 dilution is prepared) the product to be examined in
isms. The method chosen must allow testing of a sufficient
buffered sodium chloride-peptone solution pH 7.0, phos-
sample size to judge compliance with the specification. The
phate buffer solution pH 7.2 or casein soya bean digest
suitability of the chosen method must be established.
broth. If necessary adjust to pH 6 – 8. Further dilutions,
3. Growth Promotion Test, Suitability of the Counting where necessary, are prepared with the same diluent.
Method and Negative Controls (ii) Non-fatty products insoluble in water: Suspend the
The ability of the test to detect micro-organisms in the product to be examined (usually a 1 in 10 dilution is pre-
presence of product to be tested must be established. pared) in buffered sodium chloride-peptone solution pH 7.0,
Suitability must be confirmed if a change in testing per- phosphate buffer solution pH 7.2 or casein soya bean digest
formance, or the product, which may affect the outcome of broth. A surface-active agent such as 1 g/L of polysorbate
the test is introduced. 80 may be added to assist the suspension of poorly wettable
3.1. Preparation of test strains substances. If necessary adjust to pH 6 – 8. Further dilu-
Use standardised stable suspensions of test strains or pre- tions, where necessary, are prepared with the same diluent.
pare as stated below. Seed lot culture maintenance tech- (iii) Fatty products: Dissolve in isopropyl myristate,
niques (seed-lot systems) are used so that the viable micro- sterilised by filtration or mix the product to be examined

Growth promotion Suitability of counting method in

the presence of the product
Micro-organism Preparation of
test strain
Total aerobic Total yeasts and Total aerobic Total yeasts and
microbial count moulds count microbial count moulds count

Staphylococcus Casein soya bean Casein soya bean Casein soya bean
aureus digest agar or digest agar and digest agar/MPN
casein soya bean casein soya bean casein soya bean
such as ATCC digest broth digest broth digest broth
6538, NCIMB 30 – 359C ≦100 CFU ≦100 CFU
9518, CIP 4.83 or 18 – 24 hours 30 – 359C 30 – 359C
NBRC 13276 ≦3 days ≦3 days

Pseudomonas Casein soya bean Casein soya bean Casein soya bean
aeruginosa digest agar or digest agar and digest agar/MPN
casein soya bean casein soya bean casein soya bean
such as ATCC digest broth digest broth digest broth
9027, NCIMB 30 – 359C ≦100 CFU ≦100 CFU
8626, CIP 82.118 18 – 24 hours 30 – 359C 30 – 359C
or NBRC 13275 ≦3 days ≦3 days

Bacillus subtilis Casein soya bean Casein soya bean Casein soya bean
digest agar or digest agar and digest agar/MPN
such as ATCC casein soya bean casein soya bean casein soya bean
6633, NCIMB digest broth digest broth digest broth
8054, CIP 52.62 or 30 – 359C ≦100 CFU ≦100 CFU
NBRC 3134 18 – 24 hours 30 – 359C 30 – 359C
≦3 days ≦3 days

Candida albicans Sabouraud-dextrose Casein soya bean Sabouraud-dex- Casein soya bean Sabouraud-dex-
agar or Sabouraud- digest agar trose agar digest agar trose agar
such as ATCC dextrose broth ≦100 CFU ≦100 CFU ≦100 CFU ≦100 CFU
10231, NCPF 20 – 259C 30 – 359C 20 – 259C 30 – 359C 20 – 259C
3179, IP 48.72 or 2 – 3 days ≦5 days ≦5 days ≦5 days ≦5 days
NBRC 1594 MPN: not applicable

Aspergillus Sabouraud-dextrose Casein soya bean Sabouraud-dex- Casein soya bean Sabouraud-dex-
brasiliensis agar or potato-dex- digest agar trose agar digest agar trose agar
trose agar ≦100 CFU ≦100 CFU ≦100 CFU ≦100 CFU
such as ATCC 20 – 259C 30 – 359C 20 – 259C 30 – 359C 20 – 259C
16404, IMI 5 – 7 days, or ≦5 days ≦5 days ≦5 days ≦5 days
149007, IP until good sporula- MPN: not applicable
1431.83 or NBRC tion is achieved
9455

with the minimum necessary quantity of sterile polysorbate them, adhesive side upwards, on sterile glass or plastic trays.
80 or another non-inhibitory sterile surface-active reagent, Cover the adhesive surface with sterile porous material, for
heated if necessary to not more than 409 C, or in exceptional example sterile gauze, to prevent the patches from sticking
cases to not more than 459C. Mix carefully and if necessary together, and transfer the patches to a suitable volume of the
maintain the temperature in a water-bath. Add sufficient of chosen diluent containing inactivators such as polysorbate 80
the pre-warmed chosen diluent to make a 1 in 10 dilution of and/or lecithin. Shake the preparation vigorously for at least
the original product. Mix carefully whilst maintaining the 30 minutes.
temperature for the shortest time necessary for the forma- 3.4.2. Inoculation and dilution
tion of an emulsion. Further serial tenfold dilutions may be Add to the sample prepared as described above (3.4.1.)
prepared using the chosen diluent containing a suitable con- and to a control (with no test material included) a sufficient
centration of sterile polysorbate 80 or another non-inhibito- volume of the microbial suspension to obtain an inoculum of
ry sterile surface-active reagent. not more than 100 CFU. The volume of the suspension of
(iv) Fluids or solids in aerosol form: Aseptically transfer the inoculum should not exceed 1 per cent of the volume of
the product into a membrane filter apparatus or a sterile diluted product.
container for further sampling. Use either the total contents To demonstrate acceptable microbial recovery from the
or a defined number of metered doses from each of the con- product, the lowest possible dilution factor of the prepared
tainers tested. sample must be used for the test. Where this is not possible
(v) Transdermal patches: Remove the protective cover due to antimicrobial activity or poor solubility, further
sheets (``release liner'') of the transdermal patches and place appropriate protocols must be developed. If inhibition of

Table 4.05-I-2 Common neutralizing agents for interfering substances

Interfering substance Potential neutralizing method

Glutaraldehyde, Mercurials Sodium hydrogen sulfite (Sodium bisulfite)

Phenolics, Alcohol, Aldehydes, Sorbate Dilution

Aldehydes Glycine

Quaternary Ammonium Compounds (QACs), Lecithin

Parahydroxybenzoates (Parabens),
Bis-biguanides

QAC, Iodine, Parabens Polysorbate

Mercurials Thioglycollate

Mercurials, Halogens, Aldehydes Thiosulfate

EDTA (edetate) Mg or Ca ions

growth by the sample cannot otherwise be avoided, the ali- membrane filter is used.
quot of the microbial suspension may be added after neu- Transfer a suitable amount of the sample prepared as
tralization, dilution or filtration. described under 3.4.1. to 3.4.3. (preferably representing 1 g
3.4.3. Neutralization/removal of antimicrobial activity of the product, or less if large numbers of CFU are expected)
The number of micro-organisms recovered from the pre- to the membrane filter, filter immediately and rinse the
pared sample diluted as described in 3.4.2. and incubated membrane filter with an appropriate volume of diluent.
following the procedure described in 3.4.4., is compared to For the determination of total aerobic microbial count
the number of micro-organisms recovered from the control (TAMC), transfer the membrane filter to the surface of
preparation. casein soya bean digest agar. For the determination of total
If growth is inhibited (reduction by a factor greater than combined yeasts/moulds count (TYMC) transfer the mem-
2), then modify the procedure for the particular enumeration brane to the surface of Sabouraud-dextrose agar. Incubate
test to ensure the validity of the results. Modification of the the plates as indicated in Table 4.05-I-1. Perform the count-
procedure may include, for example, (1) an increase in the ing.
volume of the diluent or culture medium, (2) incorporation 3.4.4.2. Plate-count methods
of a specific or general neutralizing agents into the diluent, Perform plate-count methods at least in duplicate for each
(3) membrane filtration or (4) a combination of the above medium and use the mean count of the result.
measures. (i) Pour-plate method: For Petri dishes 9 cm in diameter,
Neutralizing agents. Neutralizing agents may be used to add to the dish 1 mL of the sample prepared as described
neutralize the activity of antimicrobial agents (Table under 3.4.1. to 3.4.3. and 15 – 20 mL of casein soya bean
4.05-I-2). They may be added to the chosen diluent or the digest agar or Sabouraud-dextrose agar, both media being at
medium preferably before sterilization. If used, their effi- not more than 459C. If larger Petri dishes are used, the
cacy and their absence of toxicity for micro-organisms must amount of agar medium is increased accordingly. For each
be demonstrated by carrying out a blank with neutralizer of the micro-organisms listed in Table 4.05-I-1, at least 2
and without product. Petri dishes are used.
If no suitable neutralizing method can be found, it can be Incubate the plates as indicated in Table 4.05-I-1. Take the
assumed that the failure to isolate the inoculated organism is arithmetic mean of the counts per medium and calculate the
attributable to the microbicidal activity of the product. This number of CFU in the original inoculum.
information serves to indicate that the article is not likely to (ii) Surface-spread method: For Petri dishes 9 cm in di-
be contaminated with the given species of the micro-organ- ameter, add 15 – 20 mL of casein soya bean digest agar or
ism. However, it is possible that the product only inhibits Sabouraud-dextrose agar at about 459 C to each Petri dish
some of the micro-organisms specified herein, but does not and allow to solidify. If larger Petri dishes are used, the
inhibit others not included amongst the test strains or for volume of the agar is increased accordingly. Dry the plates,
which the latter are not representative. Then, perform the for example in a laminar-air-flow cabinet or in an incubator.
test with the highest dilution factor compatible with micro- For each of the micro-organisms listed in Table 4.05-I-1, at
bial growth and the specific acceptance criterion. least 2 Petri dishes are used. Spread a measured volume of
3.4.4. Recovery of micro-organism in the presence of not less than 0.1 mL of the sample prepared as described
product under 3.4.1. to 3.4.3. over the surface of the medium. Incu-
For each of the micro-organisms listed in Table 4.05-I-1, bate and count as prescribed under 3.4.4.2. (i).
separate tests are performed. Only micro-organisms of the 3.4.4.3. Most-probable-number (MPN) method
added test strain are counted. The precision and accuracy of the MPN method is less
3.4.4.1. Membrane filtration than that of the membrane filtration method or the plate-
Use membrane filters having a nominal pore size not count method. Unreliable results are obtained particularly
greater than 0.45 mm. The type of filter material is chosen in for the enumeration of moulds. For these reasons the MPN
such a way that the bacteria-retaining efficiency is not affect- method is reserved for the enumeration of TAMC in situa-
ed by the components of the sample to be investigated. For tions where no other method is available. If the use of the
each of the micro-organisms listed in Table 4.05-I-1, one method is justified, proceed as follows.

Prepare a series of at least 3 serial tenfold dilutions of the medium. Thus, if 3 levels of dilution are prepared 9 tubes
product as described under 3.4.1. to 3.4.3.. From each level are inoculated.
of dilution, 3 aliquots of 1 g or 1 mL are used to inoculate 3 Incubate all tubes at 30 – 359C for not more than 3 days.
tubes with 9 – 10 mL of casein soya bean digest broth. If If reading of the results is difficult or uncertain owing to the
necessary a surface-active agent such as polysorbate 80, or nature of the product to be examined, subculture in the same
an inactivator of antimicrobial agents may be added to the broth, or casein soya bean digest agar, for 1 – 2 days at the

same temperature and use these results. Determine the most 4.2.2. Plate-count methods
probable number of micro-organisms per gram or millilitre (i) Pour-plate method: Prepare the sample using a
of the product to be examined from Table 4.05-I-3. method that has been shown to be suitable as described in
3.5. Results and interpretation section 3. Prepare for each medium at least 2 Petri dishes for
When verifying the suitability of the membrane filtration each level of dilution. Incubate the plates of casein soya bean
method or the plate-count method, a mean count of any of digest agar at 30 – 359 C for 3 – 5 days and the plates of
the test organisms not differing by a factor greater than 2 Sabouraud-dextrose agar at 20 – 259C for 5 – 7 days. Select
from the value of the control defined in 3.4.2. in the absence the plates corresponding to a given dilution and showing the
of the product must be obtained. When verifying the suita- highest number of colonies less than 250 for TAMC and 50
bility of the MPN method the calculated value from the infor TYMC. Take the arithmetic mean per culture medium of
oculum must be within 95 per cent confidence limits of the the counts and calculate the number of CFU per gram or per
results obtained with the control. millilitre of product.
If the above criteria cannot be met for one or more of the (ii) Surface-spread method: Prepare the sample using a
organisms tested with any of the described methods, the method that has been shown to be suitable as described in
method and test conditions that come closest to the criteria section 3. Prepare at least 2 Petri dishes for each medium
are used to test the product. and each level of dilution. For incubation and calculation of
the number of CFU proceed as described for the pour-plate
4. Testing of Products
method.
4.1. Amount used for the test
4.2.3. Most-probable-number method
Unless otherwise prescribed, use 10 g or 10 mL of the
Prepare and dilute the sample using a method that has
product to be examined taken with the precautions referred
been shown to be suitable as described in section 3. Incubate
to above. For fluids or solids in aerosol form, sample 10
all tubes for 3 – 5 days at 30 – 359C. Subculture if necessary,
containers. For transdermal patches, sample 10 patches.
using the procedure shown to be suitable. Record for each
The amount to be tested may be reduced for active sub-
level of dilution the number of tubes showing microbial
stances that will be formulated in the following conditions:
growth. Determine the most probable number of micro-
the amount per dosage unit (e.g. tablet, capsule, injection) is
organisms per gram or millilitre of the product to be exa-
less than or equal to 1 mg or the amount per gram or mil-
mined from Table 4.05-I-3.
lilitre (for preparations not presented in dose units) is less
4.3. Interpretation of the results
than 1 mg. In these cases, the amount of sample to be tested
The total aerobic microbial count (TAMC) is considered
is not less than the amount present in 10 dosage units or 10 g
to be equal to the number of CFU found using casein soya
or 10 mL of the product.
bean digest agar; if colonies of fungi are detected on this me-
For materials used as active substances where sample
dium, they are counted as part of TAMC. The total com-
quantity is limited or batch size is extremely small (i.e. less
bined yeasts/mould count (TYMC) is considered to be equal
than 1000 mL or 1000 g), the amount tested shall be 1 per
to the number of CFU found using Sabouraud-dextrose
cent of the batch unless a lesser amount is prescribed or
agar; if colonies of bacteria are detected on this medium,
justified and authorised.
they are counted as part of TYMC. When the TYMC is ex-
For products where the total number of entities in a batch
pected to exceed the acceptance criterion due to the bacterial
is less than 200 (e.g. samples used in clinical trials), the sam-
growth, Sabouraud-dextrose agar containing antibiotics may
ple size may be reduced to 2 units, or 1 unit if the size is less
be used. If the count is carried out by the MPN method the
than 100.
calculated value is the TAMC.
Select the sample(s) at random from the bulk material or
When an acceptance criterion for microbiological quality
from the available containers of the preparation. To obtain
is prescribed it is interpreted as follows:
the required quantity, mix the contents of a sufficient num-
－101 CFU: maximum acceptable count ＝ 20,
ber of containers to provide the sample.
－102 CFU: maximum acceptable count ＝ 200,
4.2. Examination of the product
－103 CFU: maximum acceptable count ＝ 2000,
4.2.1. Membrane filtration
and so forth.
Use a filtration apparatus designed to allow the transfer of
The recommended solutions and media are described in II.
the filter to the medium. Prepare the sample using a method
Tests for specified micro-organisms.
that has been shown suitable as described in section 3 and
transfer the appropriate amount to each of 2 membrane
II. Microbiological Examination of Non-sterile Products:
filters and filter immediately. Wash each filter following the
Tests for Specified Micro-organisms
procedure shown to be suitable.
These tests are harmonized with the European Pharmaco-
For the determination of TAMC, transfer one of the
poeia and the U.S. Pharmacopeia.
membrane filters to the surface of casein soya bean digest
agar. For the determination of TYMC, transfer the other The tests described hereafter will allow determination of
membrane to the surface of Sabouraud-dextrose agar. Incu- the absence or limited occurrence of specified micro-
bate the plate of casein soya bean digest agar at 30 – 359 C organisms which may be detected under the conditions
for 3 – 5 days and the plate of Sabouraud-dextrose agar at described.
20 – 259 C for 5 – 7 days. Calculate the number of CFU per The tests are designed primarily to determine whether a
gram or per millilitre of product. substance or preparation complies with an established spe-
When examining transdermal patches, filter 10 per cent of cification for microbiological quality. When used for such
the volume of the preparation described under 3.4.1. sepa- purposes follow the instructions given below, including the
rately through each of 2 sterile filter membranes. Transfer number of samples to be taken and interpret the results as
one membrane to casein soya bean digest agar for TAMC stated below.
and the other membrane to Sabouraud-dextrose agar for Alternative microbiological procedures, including auto-
TYMC. mated methods may be used, provided that their equivalence
to the Pharmacopoeial method has been demonstrated.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
126 4.05 Microbiological Examination of Non-sterile Products / General Tests JP XVII
1. General Procedures 2.3. Growth promotion and inhibitory properties of the
The preparation of samples is carried out as described in I. media
Microbial enumeration tests. Test each batch of ready-prepared medium and each batch
If the product to be examined has antimicrobial activity, of medium prepared either from dehydrated medium or
this is insofar as possible removed or neutralized as from ingredients.
described in I. Microbial enumeration tests. Verify suitable properties of relevant media as described in
If surface-active substances are used for sample prepara- Table 4.05-II-1.
tion, their absence of toxicity for micro-organisms and their (i) Test for growth promoting properties, liquid media:
compatibility with inactivators used must be demonstrated inoculate a portion of the appropriate medium with a small
as described in I. Microbial enumeration tests. number (not more than 100 CFU) of the appropriate micro-
organism. Incubate at the specified temperature for not
2. Growth Promoting and Inhibitory Properties of the
more than the shortest period of time specified in the test.
Media, Suitability of the Test and Negative Controls
Clearly visible growth of the micro-organism comparable to
The ability of the test to detect micro-organisms in the
that previously obtained with a previously tested and ap-
presence of the product to be tested must be established.
proved batch of medium occurs.
Suitability must be confirmed if a change in testing per-
(ii) Test for growth promoting properties, solid media:
formance, or the product, which may affect the outcome of
perform surface-spread method, inoculating each plate with
the test is introduced.
a small number (not more than 100 CFU) of the appropriate
2.1. Preparation of test strains
micro-organism. Incubate at the specified temperature for
Use standardized stable suspensions of test strains or pre-
not more than the shortest period of time specified in the
pare as stated below. Seed lot culture maintenance tech-
test. Growth of the micro-organism comparable to that pre-
niques (seed-lot systems) are used so that the viable micro-
viously obtained with a previously tested and approved batch
organisms used for inoculation are not more than 5 passages
of medium occurs.
removed from the original master seed-lot.
(iii) Test for inhibitory properties, liquid or solid media:
2.1.1. Aerobic micro-organisms
inoculate the appropriate medium with at least 100 CFU of
Grow each of the bacterial test strains separately in con-
the appropriate micro-organism. Incubate at the specified
tainers containing casein soya bean digest broth or on casein
temperature for not less than the longest period of time spe-
soya bean digest agar at 30 – 359C for 18 – 24 hours. Grow
cified in the test. No growth of the test micro-organism oc-
the test strain for Candida albicans separately on
curs.
Sabouraud-dextrose agar or in Sabouraud-dextrose broth at
(iv) Test for indicative properties: perform surface-
20 – 259 C for 2–3 days.
spread method, inoculating each plate with a small number
Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
(not more than 100 CFU) of the appropriate micro-organ-
CIP 4.83 or NBRC 13276,
ism. Incubate at the specified temperature for a period of
Pseudomonas aeruginosa such as ATCC 9027, NCIMB
time within the range specified in the test. Colonies are com-
8626, CIP 82.118 or NBRC 13275,
parable in appearance and indication reactions to those pre-
Escherichia coli such as ATCC 8739, NCIMB 8545, CIP
viously obtained with a previously tested and approved batch
53.126 or NBRC 3972,
of medium.
Salmonella enterica subsp. enterica serovar Typhimurium
2.4. Suitability of the test method
such as ATCC 14028
For each product to be tested perform sample preparation
or, as an alternative,
as described in the relevant paragraph in section 3. Add each
Salmonella enterica subsp. enterica serovar Abony such as
test strain at the time of mixing, in the prescribed growth
NBRC 100797, NCTC 6017 or CIP 80.39,
medium. Inoculate the test strains individually. Use a num-
Candida albicans such as ATCC 10231, NCPF 3179, IP
ber of micro-organisms equivalent to not more than 100
48.72 or NBRC 1594.
CFU in the inoculated test preparation.
Use buffered sodium chloride-peptone solution pH 7.0 or
Perform the test as described in the relevant paragraph in
phosphate buffer solution pH 7.2 to make test suspensions.
section 3 using the shortest incubation period prescribed.
Use the suspensions within 2 hours or within 24 hours if
The specified micro-organisms must be detected with the
stored at 2 – 89C.
indication reactions as described in section 3.
2.1.2. Clostridia
Any antimicrobial activity of the product necessitates a
Use Clostridium sporogenes such as ATCC 11437 (NBRC
modification of the test procedure (see 3.4.3. of I. Microbial
14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC
enumeration tests).
532 or CIP 79.3). Grow the clostridial test strain under
If for a given product the antimicrobial activity with
anaerobic conditions in reinforced medium for Clostridia at
respect to a micro-organism for which testing is prescribed
30 – 359 C for 24 – 48 hours. As an alternative to preparing
cannot be neutralized, then it is to be assumed that the in-
and then diluting down a fresh suspension of vegetative cells
hibited micro-organism will not be present in the product.
of Cl. sporogenes, a stable spore suspension is used for test
inoculation. The stable spore suspension may be maintained 3. Testing of Products
at 2 – 89C for a validated period. 3.1. Bile-tolerant gram-negative bacteria
2.2. Negative control 3.1.1. Sample preparation and pre-incubation
To verify testing conditions a negative control is per- Prepare a sample using a 1 in 10 dilution of not less than
formed using the chosen diluent in place of the test prepara- 1 g of the product to be examined as described in I. Microbi-
tion. There must be no growth of micro-organisms. A nega- al enumeration tests, but using casein soya bean digest broth
tive control is also performed when testing the products as as the chosen diluent, mix and incubate at 20 – 259 C for a
described under 3. A failed negative control required an in- time sufficient to resuscitate the bacteria but not sufficient to
vestigation. encourage multiplication of the organisms (usually 2 hours
but not more than 5 hours).

Table 4.05-II-1 Growth promoting, inhibitory and indicative properties of media

Medium Property Test strains

Test for bile-tolerant gram-negative bacteria

Enterobacteria enrichment broth- Growth promoting E. coli

Mossel P. aeruginosa

Inhibitory S. aureus
Violet red bile glucose agar Growth promoting＋ E. coli
Indicative P. aeruginosa

Test for Escherichia coli

MacConkey broth Growth promoting E. coli

Inhibitory S. aureus

MacConkey agar Growth promoting＋ E. coli

Indicative

Test for Salmonella

Rappaport Vassiliadis Salmonella Growth promoting Salmonella enterica subsp. enterica serovar
enrichment broth Typhimurium or
Salmonella enterica subsp. enterica serovar
Abony

Inhibitory S. aureus

Xylose, lysine, deoxycholate agar Growth promoting＋ Salmonella enterica subsp. enterica serovar
Indicative Typhimurium or
Salmonella enterica subsp. enterica serovar
Abony

Test for Pseudomonas aeruginosa

Cetrimide agar Growth promoting P. aeruginosa

Inhibitory E. coli

Test for Staphylococcus aureus

Mannitol salt agar Growth promoting＋ S. aureus

Indicative

Inhibitory E. coli

Test for Clostridia

Reinforced medium for Clostridia Growth promoting Cl. sporogenes

Columbia agar Growth promoting Cl. sporogenes

Test for Candida albicans

Sabouraud-dextrose broth Growth promoting C. albicans

Sabouraud-dextrose agar Growth promoting＋ C. albicans

Indicative

3.1.2. Test for absence 3.1.3. Quantitative test

Unless otherwise prescribed use the volume corresponding 3.1.3.1. Selection and subculture
to 1 g of the product, as prepared in 3.1.1. to inoculate en- Inoculate suitable quantities of enterobacteria enrichment
terobacteria enrichment broth-Mossel. Incubate at 30 – 359 C broth-Mossel with the preparation as described under 3.1.1.
for 24 – 48 hours. Subculture on plates of violet red bile glu- and/or dilutions of it containing respectively 0.1 g, 0.01 g
cose agar. Incubate at 30 – 359C for 18 – 24 hours. and 0.001 g (or 0.1 mL, 0.01 mL and 0.001 mL) of the
The product complies with the test if there is no growth of product to be examined. Incubate at 30 – 359C for 24 – 48
colonies. hours. Subculture each of the cultures on a plate of violet
red bile glucose agar. Incubate at 30 – 359C for 18 – 24
hours.

Results for each quantity of product Probable number of bacteria

0.1 g or 0.1 mL 0.01 g or 0.01 mL 0.001 g or 0.001 mL per gram or mL of product

＋＋＋ more than 103

＋＋－ less than 103 and more than 102
＋－－ less than 102 and more than 10
－－－ less than 10

3.1.3.2. Interpretation filter the volume of sample corresponding to 1 patch of the

Growth of colonies constitutes a positive result. Note the preparation described in I. Microbial enumeration tests
smallest quantity of the product that gives a positive result (3.4.1.) through a sterile filter membrane and place in 100
and the largest quantity that gives a negative result. Deter- mL of casein soya bean digest broth. Incubate at 30 – 359C
mine from Table 4.05-II-2 the probable number of bacteria. for 18 – 24 hours.
3.2. Escherichia coli 3.4.2. Selection and subculture
3.2.1. Sample preparation and pre-incubation Subculture on a plate of cetrimide agar and incubate at
Prepare a sample using a 1 in 10 dilution of not less than 30 – 359C for 18 – 72 hours.
1 g of the product to be examined as described in I. Micro- 3.4.3. Interpretation
bial enumeration tests and use 10 mL or the quantity cor- Growth of colonies indicates the possible presence of
responding to 1 g or 1 mL to inoculate a suitable amount P. aeruginosa. This is confirmed by identification tests.
(determined as described under 2.4.) of casein soya bean The product complies with the test if colonies are not
digest broth, mix and incubate at 30 – 359 C for 18 – 24 present or if the confirmatory identification tests are nega-
hours. tive.
3.2.2. Selection and subculture 3.5. Staphylococcus aureus
Shake the container, transfer 1 mL of casein soya bean 3.5.1. Sample preparation and pre-incubation
digest broth to 100 mL of MacConkey broth and incubate at Prepare a sample using a 1 in 10 dilution of not less than
42 – 449 C for 24 – 48 hours. Subculture on a plate of Mac- 1 g of the product to be examined as described in I. Micro-
Conkey agar at 30 – 359C for 18 – 72 hours. bial enumeration tests and use 10 mL or the quantity cor-
3.2.3. Interpretation responding to 1 g or 1 mL to inoculate a suitable amount
Growth of colonies indicates the possible presence of (determined as described under 2.4.) of casein soya bean
E. coli. This is confirmed by identification tests. digest broth and homogenise. When testing transdermal
The product complies with the test if no colonies are patches, filter the volume of sample corresponding to 1
present or if the identification tests are negative. patch of the preparation described in I. Microbial enumera-
3.3. Salmonella tion tests (3.4.1.) through a sterile filter membrane and place
3.3.1. Sample preparation and pre-incubation in 100 mL of casein soya bean digest broth. Incubate at 30 –
Prepare the product to be examined as described in I. 359C for 18 – 24 hours.
Microbial enumeration tests and use the quantity cor- 3.5.2. Selection and subculture
responding to not less than 10 g or 10 mL to inoculate a suit- Subculture on a plate of mannitol salt agar and incubate at
able amount (determined as described under 2.4.) of casein 30 – 359C for 18 – 72 hours.
soya bean digest broth, mix and incubate at 30 – 359C for 3.5.3. Interpretation
18 – 24 hours. The possible presence of S. aureus is indicated by the
3.3.2. Selection and subculture growth of yellow/white colonies surrounded by a yellow
Transfer 0.1 mL of casein soya bean digest broth to 10 mL zone. This is confirmed by identification tests.
of Rappaport Vassiliadis Salmonella enrichment broth and The product complies with the test if colonies of the types
incubate at 30 – 359 C for 18 – 24 hours. Subculture on plates described are not present or if the confirmatory identifica-
of xylose, lysine, deoxycholate agar. Incubate at 30 – 359C tion tests are negative.
for 18 – 48 hours. 3.6. Clostridia
3.3.3. Interpretation 3.6.1. Sample preparation and heat treatment
The possible presence of Salmonella is indicated by the Prepare a sample using a 1 in 10 dilution (with a minimum
growth of well-developed, red colonies, with or without total volume of 20 mL) of not less than 2 g or 2 mL of the
black centres. This is confirmed by identification tests. product to be examined as described in I. Microbial enumer-
The product complies with the test if colonies of the types ation tests. Divide the sample into two portions of at least 10
described are not present or if the confirmatory identifica- mL. Heat 1 portion at 809C for 10 minutes and cool rapidly.
tion tests are negative. Do not heat the other portion.
3.4. Pseudomonas aeruginosa 3.6.2. Selection and subculture
3.4.1. Sample preparation and pre-incubation Use 10 mL or the quantity corresponding to 1 g or 1 mL of
Prepare a sample using a 1 in 10 dilution of not less than the product to be examined of both portions to inoculate
1 g of the product to be examined as described in I. Micro- suitable amounts (determined as described under 2.4.) of
bial enumeration tests and use 10 mL or the quantity cor- reinforced medium for Clostridia. Incubate under anaerobic
responding to 1 g or 1 mL to inoculate a suitable amount conditions at 30 – 359C for 48 hours. After incubation,
(determined as described under 2.4.) of casein soya bean make subcultures from each tube on Columbia agar and
digest broth and mix. When testing transdermal patches, incubate under anaerobic conditions at 30 – 359 C for 48 – 72

hours. Agar 15.0 g

3.6.3. Interpretation Purified water 1000 mL
The occurrence of anaerobic growth of rods (with or with- Adjust the pH so that after sterilization it is 7.3 ± 0.2 at
out endospores) giving a negative catalase reaction indicates 259C. Sterilize in an autoclave using a validated cycle.
the presence of Clostridia. This is confirmed by identifica-
tion tests. (v) Sabouraud-dextrose agar
The product complies with the test if colonies of the types Dextrose 40.0 g
described are not present or if the confirmatory identifica- Mixture of peptic digest of animal tissue and pan-
tion tests are negative. creatic digest of casein (1:1) 10.0 g
3.7. Candida albicans Agar 15.0 g
3.7.1. Sample preparation and pre-incubation Purified water 1000 mL
Prepare the product to be examined as described in I. Adjust the pH so that after sterilization it is 5.6 ± 0.2 at
Microbial enumeration tests and use 10 mL or the quantity 259C. Sterilize in an autoclave using a validated cycle.
corresponding to not less than 1 g or 1 mL to inoculate 100
mL of Sabouraud-dextrose broth and mix. Incubate at 30 – (vi) Potato dextrose agar
359 C for 3-5 days. Infusion from potatoes 200 g
3.7.2. Selection and subculture Dextrose 20.0 g
Subculture on a plate of Sabouraud-dextrose agar and in- Agar 15.0 g
cubate at 30 – 359 C for 24 – 48 hours. Purified water 1000 mL
3.7.3. Interpretation Adjust the pH so that after sterilization it is 5.6 ± 0.2 at
Growth of white colonies may indicate the presence of C. 259C. Sterilize in an autoclave using a validated cycle.
albicans. This is confirmed by identification tests.
The product complies with the test if such colonies are not (vii) Sabouraud-dextrose broth
present or if the confirmatory identification tests are nega- Dextrose 20.0 g
tive. Mixture of peptic digest of animal tissue and pan-
creatic digest of casein (1:1) 10.0 g
The following section is given for information.
Purified water 1000 mL
4. Recommended Solutions and Culture Media Adjust the pH so that after sterilization it is 5.6 ± 0.2 at
The following solutions and culture media have been 259C. Sterilize in an autoclave using a validated cycle.
found satisfactory for the purposes for which they are
prescribed in the test for microbial contamination in the (viii) Enterobacteria enrichment broth-Mossel
Pharmacopoeia. Other media may be used provided that Pancreatic digest of gelatin 10.0 g
their suitability can be demonstrated. Glucose monohydrate 5.0 g
Dehydrated ox bile 20.0 g
(i) Phosphate buffer solution pH 7.2 Potassium dihydrogen phosphate 2.0 g
Prepare a mixture of water and stock buffer solution (800:1 Disodium hydrogen phosphate dihydrate 8.0 g
V/V) and sterilize. Brilliant green 15 mg
Purified water 1000 mL
Stock buffer solution. Transfer 34 g of potassium dihydro-
Adjust the pH so that after heating it is 7.2 ± 0.2 at 259C.
gen phosphate to a 1000 mL volumetric flask, dissolve in 500
Heat at 1009 C for 30 minutes and cool immediately.
mL of purified water, adjust to pH 7.2 to ±0.2 with sodium
hydroxide, add purified water to volume and mix. Dispense
(ix) Violet red bile glucose agar
in containers and sterilize. Store at a temperature of 2 – 89C.
Yeast extract 3.0 g
Pancreatic digest of gelatin 7.0 g
(ii) Buffered sodium chloride-peptone solution pH 7.0
Bile salts 1.5 g
Potassium dihydrogen phosphate 3.6 g
Sodium chloride 5.0 g
Disodium hydrogen phosphate dihydrate 7.2 g
Glucose monohydrate 10.0 g
(equivalent to 0.067 mol phosphate)
Agar 15.0 g
Sodium chloride 4.3 g
Neutral red 30 mg
Peptone (meat or casein) 1.0 g
Crystal violet 2 mg
Water 1000 mL
Purified water 1000 mL
Sterilize in an autoclave using a validated cycle.
Adjust the pH so that after heating it is 7.4 ± 0.2 at 259C.
Heat to boiling; do not heat in an autoclave.
(iii) Casein soya bean digest broth
Pancreatic digest of casein 17.0 g
(x) MacConkey broth
Papaic digest of soya bean 3.0 g
Pancreatic digest of gelatin 20.0 g
Sodium chloride 5.0 g
Lactose monohydrate 10.0 g
Dipotassium hydrogen phosphate 2.5 g
Dehydrated ox bile 5.0 g
Glucose monohydrate 2.5 g
Bromocresol purple 10 mg
Purified water 1000 mL
Purified water 1000 mL
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at
259 C. Sterilize in an autoclave using a validated cycle.
259C. Sterilize in an autoclave using a validated cycle.
(iv) Casein soya bean digest agar
(xi) MacConkey agar
Panceratic digest of casein 15.0 g
Pancreatic digest of gelatin 17.0 g
Papaic digest of soya bean 5.0 g
Peptones (meat and casein) 3.0 g
Sodium chloride 5.0 g
Lactose monohydrate 10.0 g

Yeast extract (water-soluble) 5.0 g Table 4.06-1. Strains of the test micro-organisms suitable
Pancreatic digest of casein 15.0 g for use in the Growth Promotion Test and the Method
Sodium thioglycollate or 0.5 g suitability Test
Thioglycollic acid 0.3 mL
Resazurin sodium solution (1 in 1000), freshly Aerobic bacteria ATCC 6538, CIP 4.83,
prepared 1.0 mL Staphylococcus aureus NCTC 10788, NCIMB 9518,
Water 1000 mL NBRC 13276
(pH after sterilization 7.1 ± 0.2) Bacillus subtilis ATCC 6633, CIP 52.62,
Mix the L-cystine, agar, sodium chloride, glucose, water- NCIMB 8054, NBRC 3134
soluble yeast extract and pancreatic digest of casein with Pseudomonas aeruginosa ATCC 9027, NCIMB 8626,
water, and heat until solution is effected. Dissolve the so-
CIP 82.118, NBRC 13275
dium thioglycollate or thioglycollic acid in the solution and,
if necessary, add sodium hydroxide TS so that, after sterili- Anaerobic bacterium ATCC 19404, CIP 79.3,
zation, the solution will have a pH of 7.1 ± 0.2. If filtration Clostridium sporogenes NCTC 532 or ATCC 11437,
is necessary, heat the solution again without boiling and NBRC 14293
filter while hot through moistened filter paper. Add the
resazurin sodium solution (1 in 1000), mix and place the me- Fungi ATCC 10231, IP 48.72,
dium in suitable vessels which provide a ratio of surface to Candida albicans NCPF 3179, NBRC 1594
depth of medium such that not more than the upper half of Aspergillus brasiliensis ATCC 16404, IP 1431.83,
the medium has undergone a color change indicative of oxy- IMI 149007, NBRC 9455
gen uptake at the end of the incubation period. Sterilize
using a validated process. If the medium is stored, store at a
temperature between 29C and 259C in a sterile, tight con-
tainer. If more than the upper one-third of the medium has
3. Suitability of the culture medium
acquired a pink color, the medium may be restored once by
The media used comply with the following tests, carried
heating the containers in a water-bath or in free-flowing
out before or in parallel with the test on the product to be
steam until the pink color disappears and cooling quickly,
examined.
taking care to prevent the introduction of non-sterile air into
3.1. Sterility
the container. Do not use the medium for a longer storage
Incubate portions of the media for 14 days. No growth of
period than has been validated.
micro-organisms occurs.
Fluid thioglycollate medium is to be incubated at 30 –
3.2. Growth promotion test of aerobes, anaerobes and
359 C. For products containing a mercurial preservative that
fungi
cannot be tested by the membrane-filtration method, fluid
Test each batch of ready-prepared medium and each batch
thioglycollate medium incubated at 20 – 259C may be used
of medium prepared either from dehydrated medium or
instead of soya-bean casein digest medium provided that it
from ingredients. Suitable strains of micro-organisms are
has been validated as described in growth promotion test.
indicated in Table 4.06-1.
Where prescribed or justified and authorized, the follow-
Inoculate portions of fluid thioglycollate medium with a
ing alternative thioglycollate medium might be used. Prepare
small number (not more than 100 CFU) of the following
a mixture having the same composition as that of the fluid
micro-organisms, using a separate portion of medium for
thioglycollate medium, but omitting the agar and the resazu-
each of the following species of micro-organism:
rin sodium solution (1 in 1000), sterilize as directed above.
Clostridium sporogenes,
The pH after sterilization is 7.1 ± 0.2. Heat in a water bath
Pseudomonas aeruginosa,
prior to use and incubate at 30 – 359C under anaerobic con-
Staphylococcus aureus.
ditions.
Inoculate portions of soya-bean casein digest medium with
(ii) Soya-bean casein digest medium
a small number (not more than 100 CFU) of the following
Pancreatic digest of casein 17.0 g
micro-organisms, using a separate portion of medium for
Papaic digest of soya-bean meal 3.0 g
each of the following species of micro-organism:
Sodium chloride 5.0 g
Aspergillus brasiliensis,
Dipotassium hydrogen phosphate 2.5 g
Bacillus subtilis,
Glucose monohydrate/anhydrous 2.5/2.3 g
Candida albicans.
Water 1000 mL
Incubate for not more than 3 days in the case of bacteria
(pH after sterilization 7.3 ± 0.2)
and not more than 5 days in the case of fungi.
Dissolve the solids in water, warming slightly to effect so- Seed lot culture maintenance techniques (seed-lot systems)
lution. Cool the solution to room temperature. Add sodium are used so that the viable micro-organisms used for inocula-
hydroxide TS, if necessary, so that after sterilization the so- tion are not more than five passages removed from the origi-
lution will have a pH of 7.3 ± 0.2. Filter, if necessary, to nal master seed-lot.
clarify, distribute into suitable vessels and sterilize using a The media are suitable if a clearly visible growth of the
validated process. Store at a temperature between 29C and micro-organisms occurs.
259 C in a sterile tight container, unless it is intended for im-
4. Method suitability test
mediate use. Do not use the medium for a longer storage
Carry out a test as described below under 5. Test for steril-
period than has been validated.
ity of the product to be examined using exactly the same
Soya-bean casein digest medium is to be incubated at 20 –
methods except for the following modifications.
259 C.
(i) Membrane filtration: After transferring the content
of the container or containers to be tested to the membrane
add an inoculum of a small number of viable micro-organ-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
132 4.06 Sterility Test / General Tests JP XVII
isms (not more than 100 CFU) to the final portion of sterile Table 4.06-2. Minimum quantity to be used for each medi-
diluent used to rinse the filter. um
(ii) Direct inoculation: After transferring the contents of
the container or containers to be tested to the culture me- Quantity per container Minimum quantity to be used
dium add an inoculum of a small number of viable micro-or- for each medium unless other-
ganisms (not more than 100 CFU) to the medium. wise justified and authorized
In both cases use the same micro-organisms as those Liquids
described above under 3.2. Growth promotion test of aer- —less than 1 mL: The whole contents of each
obes, anaerobes and fungi. Perform a growth promotion test container
as a positive control. Incubate all the containers containing —1 – 40 mL: Half the contents of each con-
medium for not more than 5 days. tainer but not less than 1 mL
If clearly visible growth of micro-organisms is obtained —greater than 40 mL 20 mL
after the incubation, visually comparable to that in the con- and not greater than
trol vessel without product, either the product possesses no 100 mL
antimicrobial activity under the conditions of the test or such —greater than 100 mL: 10z of the contents of the
activity has been satisfactorily eliminated. The test for sterili- container but not less than 20
ty may then be carried out without further modification. mL
If clearly visible growth is not obtained in the presence of Antibiotic liquids 1 mL
the product to be tested, visually comparable to that in the
control vessels without product, the product possesses anti- Insoluble preparations, Use the contents of each con-
microbial activity that has not been satisfactorily eliminated creams and ointments to tainer to provide not less than
under the conditions of the test. Modify the conditions in be suspended or emulsified 200 mg
order to eliminate the antimicrobial activity and repeat the
Solids
method suitability test.
—less than 50 mg The whole contents of each
This method suitability is performed:
container
a) when the test for sterility has to be carried out on a
—50 mg or more but Half the contents of each con-
new product;
less than 300 mg tainer but not less than 50 mg
b) whenever there is a change in the experimental condi-
—300 mg – 5 g 150 mg
tions of the test.
—greater than 5 g 500 mg
The method suitability may be performed simultaneously
with the Test for sterility of the product to be examined.
5. Test for sterility of the product to be examined
The test may be carried out using the technique of mem-
tested to the membrane or membranes, if necessary after
brane filtration or by direct inoculation of the culture media
diluting to the volume used in the method suitability test
with the product to be examined. Appropriate negative con-
with the chosen sterile diluent but in any case using not less
trols are included. The technique of membrane filtration is
than the quantities of the product to be examined prescribed
used whenever the nature of the product permits, that is, for
in Table 4.06-2. Filter immediately. If the product has anti-
filterable aqueous preparations, for alcoholic or oily prepa-
microbial properties, wash the membrane not less than three
rations and for preparations miscible with or soluble in
times by filtering through it each time the volume of the
aqueous or oily solvents provided these solvents do not have
chosen sterile diluent used in the method suitability test. Do
an antimicrobial effect in the conditions of the test.
not exceed a washing cycle of 5 times 100 mL per filter, even
5.1. Membrane filtration
if during method suitability it has been demonstrated that
Use membrane filters having a nominal pore size not
such a cycle does not fully eliminate the antimicrobial ac-
greater than 0.45 mm whose effectiveness to retain micro-
tivity. Transfer the whole membrane to the culture medium
organisms has been established. Cellulose nitrate filters, for
or cut it aseptically into two equal parts and transfer one
example, are used for aqueous, oily and weakly alcoholic so-
half to each of two suitable media. Use the same volume of
lutions and cellulose acetate filters, for example, for strongly
each medium as in the method suitability test. Alternatively,
alcoholic solutions. Specially adapted filters may be needed
transfer the medium onto the membrane in the apparatus.
for certain products, e.g. for antibiotics.
Incubate the media for not less than 14 days.
The technique described below assumes that membranes
(ii) Soluble solids: Use for each medium not less than the
about 50 mm in diameter will be used. If filters of a different
quantity prescribed in Table 4.06-2 of the product dissolved
diameter are used the volumes of the dilutions and the wash-
in a suitable solvent such as the solvent provided with the
ings should be adjusted accordingly. The filtration apparatus
preparation, water for injection, saline or a 1 g/L neutral so-
and membrane are sterilized by appropriate means. The ap-
lution of meat or casein peptone and proceed with the test as
paratus is designed so that the solution to be examined can
described above for aqueous solutions using a membrane ap-
be introduced and filtered under aseptic conditions; it per-
propriate to the chosen solvent.
mits the aseptic removal of the membrane for transfer to the
(iii) Oils and oily solutions: Use for each medium not
medium or it is suitable for carrying out the incubation after
less than the quantity of the product prescribed in Table
adding the medium to the apparatus itself.
4.06-2. Oils and oily solutions of sufficiently low viscosity
(i) Aqueous solutions: If appropriate, transfer a small
may be filtered without dilution through a dry membrane.
quantity of a suitable, sterile diluent such as a 1 g/L neutral
Viscous oils may be diluted as necessary with a suitable
solution of meat or casein peptone pH 7.1 ± 0.2 onto the
sterile diluent such as isopropyl myristate shown not to have
membrane in the apparatus and filter. The diluent may con-
antimicrobial activity in the conditions of the test. Allow the
tain suitable neutralizing substances and/or appropriate in-
oil to penetrate the membrane by its own weight then filter,
activating substances for example in the case of antibiotics.
applying the pressure or suction gradually. Wash the mem-
Transfer the contents of the container or containers to be
brane at least three times by filtering through it each time

about 100 mL of a suitable sterile solution such as 1 g/L neu- Table 4.06-3. Minimum number of items to be tested
tral meat or casein peptone containing a suitable emulsifying
agent at a concentration shown to be appropriate in the Minimum number of items
method suitability of the test, for example polysorbate 80 at Number of items to be tested for each medi-
a concentration of 10 g/L. Transfer the membrane or mem- in the batch* um, unless otherwise justi-
branes to the culture medium or media or vice versa as fied and authorized**
described above for aqueous solutions, and incubate at the Parenteral preparations
same temperatures and for the same times. —Not more than 100 con- 10z or 4 containers which-
(iv) Ointments and creams: Use for each medium not less tainers ever is the greater
than the quantities of the product prescribed in Table 4.06-2. —More than 100 but not 10 containers
Ointments in a fatty base and emulsions of the water-in-oil more than 500 contain-
type may be diluted to 1 per cent in isopropyl myristate as ers
described above, by heating, if necessary, to not more than —More than 500 contain- 2z or 20 containers (10
409 C. In exceptional cases it may be necessary to heat to not ers containers for parenterals
more than 449C. Filter as rapidly as possible and proceed as with a nominal volume of
described above for oils and oily solutions. 100 mL or more) which-
5.2. Direct inoculation of the culture medium ever is the less
Transfer the quantity of the preparation to be examined
prescribed in Table 4.06-2 directly into the culture medium Ophthalmic and other non-
so that the volume of the product is not more than 10 per injectable preparations
cent of the volume of the medium, unless otherwise —Not more than 200 con- 5z or 2 containers which-
prescribed. If the product to be examined has antimicrobial tainers ever is the greater
activity, carry out the test after neutralizing this with a suita- —More than 200 contain- 10 containers
ble neutralizing substance or by dilution in a sufficient quan- ers
tity of culture medium. When it is necessary to use a large —If the product is pre-
volume of the product it may be preferable to use a concen- sented in the form of
trated culture medium prepared in such a way that it takes single-dose containers,
account of the subsequent dilution. Where appropriate the apply the scheme shown
concentrated medium may be added directly to the product above for preparations
in its container. for parenteral use
(i) Oily liquids: Use media to which have been added a
Bulk solid products
suitable emulsifying agent at a concentration shown to be
—Up to 4 containers Each container
appropriate in the method suitability of the test, for example
—More than 4 containers 20z or 4 containers which-
polysorbate 80 at a concentration of 10 g/L.
but not more than 50 ever is the greater
(ii) Ointments and creams: Prepare by diluting to about
containers
1 in 10 by emulsifying with the chosen emulsifying agent in a
—More than 50 containers 2z or 10 containers which-
suitable sterile diluent such as a 1 g/L neutral solution of
ever is the greater
meat or casein peptone. Transfer the diluted product to a
medium not containing an emulsifying agent. * If the batch size is not known, use the maximum number of
Incubate the inoculated media for not less than 14 days. items prescribed
Observe the cultures several times during the incubation ** If the contents of one container are enough to inoculate the
period. Shake cultures containing oily products gently each two media, this column gives the number of containers needed
day. However when fluid thioglycollate medium is used for for both the media together.
the detection of anaerobic micro-organisms keep shaking or
mixing to a minimum in order to maintain anaerobic condi-
tions.
(ii) a review of the testing procedure used during the test
6. Observation and interpretation of results
in question reveals a fault;
At intervals during the incubation period and at its conclu-
(iii) microbial growth is found in the negative controls;
sion, examine the media for macroscopic evidence of
(iv) after determination of the identity of the micro-
microbial growth. If the material being tested renders the
organisms isolated from the test, the growth of this species
medium turbid so that the presence or absence of microbial
or these species may be ascribed unequivocally to faults with
growth cannot be readily determined by visual examination,
respect to the material and/or the technique used in conduct-
14 days after the beginning of incubation transfer portions
ing the sterility test procedure.
(each not less than 1 mL) of the medium to fresh vessels of
If the test is declared to be invalid it is repeated with the
the same medium and then incubate the original and transfer
same number of units as in the original test. If no evidence
vessels for not less than 4 days.
of microbial growth is found in the repeat test the product
If no evidence of microbial growth is found, the product
examined complies with the test for sterility. If microbial
to be examined complies with the test for sterility. If evi-
growth is found in the repeat test the product examined does
dence of microbial growth is found the product to be exa-
not comply with the test for sterility.
mined does not comply with the test for sterility, unless it
can be clearly demonstrated that the test was invalid for 7. Application of the test to parenteral preparations, oph-
causes unrelated to the product to be examined. The test may thalmic and other non-injectable preparations required to
be considered invalid only if one or more of the following comply with the test for sterility
conditions are fulfilled: When using the technique of membrane filtration, use,
(i) the data of the microbiological monitoring of the whenever possible, the whole contents of the container, but
sterility testing facility show a fault; not less than the quantities indicated in Table 4.06-2, dilut-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
134 5.01 Crude Drugs Test / General Tests JP XVII
ing where necessary to about 100 mL with a suitable sterile drop of mounting agent, and put a cover glass on it in the
solution, such as 1 g/L neutral meat or casein peptone. same manner as above.
When using the technique of direct inoculation of media, Unless otherwise specified, use a mixture of glycerin and
use the quantities shown in Table 4.06-2, unless otherwise water (1:1) or a mixture of water, ethanol (95) and glycerin
justified and authorized. The tests for bacterial and fungal (1:1:1) as the mounting agent and swelling agent.
sterility are carried out on the same sample of the product to 3.3. Observation of components in the Description
be examined. When the volume or the quantity in a single In each monograph, description is usually given of the
container is insufficient to carry out the tests, the contents of outer portion and the inner portion of a section in this order,
two or more containers are used to inoculate the different followed by a specification of cell contents. Observation
media. should be made in the same order. In the case of a powdered
sample, description is given of a characteristic component or
8. Minimum number of items to be tested
a matter present in large amount, rarely existing matter, and
The minimum number of items to be tested in relation to
cell contents in this order. Observation should be made in
the size of the batch is given in Table 4.06-3.
the same order.
4. Purity
4.1 Heavy metals
5. Tests for Crude Drugs There are two ways to specify the heavy metals, one is to
specify with the total amount of heavy metals and the other
is with individual amount of particular metal(s). Heavy
5.01 Crude Drugs Test metals for crude drugs are usually specified with the total
amount of heavy metals according to Heavy Metals Limit
Crude Drugs Test is applied to the crude drugs mentioned
Test <1.07> described in monographs. However, rarely there
in the General Rules for Crude Drugs.
is the case where the test cannot be carried out due to getting
1. Sampling turbid or such of the test solution. In these cases, the accep-
Unless otherwise specified, sample should be taken by the tance can be evaluated by determining individual amount of
following methods. If necessary, preserve the samples in particular metal(s) using Atomic Absorption Spectropho-
tight containers. tometry <2.23> or Inductively Coupled Plasma-Atomic Emis-
(i) When crude drugs to be sampled are small-sized, cut sion Spectrometry and Inductively Coupled Plasma-Mass
or powdered, 50 to 250 g of sample should be taken after Spectrometry <2.63>.
mixing thoroughly. 4.2. Foreign matter
(ii) When crude drugs to be sampled are large-sized, 250 Unless otherwise specified, weigh 25 to 500 g of the sam-
to 500 g of sample should be taken after mixing thoroughly. ple, spread out in a thin layer, and separate the foreign mat-
(iii) When the mass of each single piece of the crude ter by inspecting with the naked eye or with the use of a mag-
drugs is not less than 100 g, not less than 5 pieces should be nifying glass of 10 magnifications. Weigh, and determine the
taken for a sample, or not less than 500 g of the sample percentage of foreign matter.
should be taken after cutting to a suitable size and mixing 4.3. Total BHC's and total DDT's
thoroughly. Sodium chloride, anhydrous sodium sulfate and synthetic
magnesium silicate for column chromatography used in this
2. Preparation of the test sample for analysis
procedure are used after drying by heating at about 1309 C
Preparations are to be made by mixing the sample well.
for more than 12 hours and cooling in a desiccator (silica
Powdered drugs should be used as they are, and in the case
gel). Chromatographic column is prepared as follows: Place
of unpowdered drugs, unless otherwise specified, grind the
20 g of synthetic magnesium silicate for column chromatog-
sample into powder. If the sample cannot be ground into
raphy in a 200-mL flask, add 50 mL of hexane for Purity of
powder, reduce it as finely as possible, spread it out in a thin
crude drug, shake vigorously, and immediately pour the mix-
layer, and withdraw a typical portion for analysis. If neces-
ture into a chromatographic tube about 2 cm in inside diam-
sary, preserve the test sample in a tight container.
eter and about 30 cm in length. Drip until the depth of
3. Micropic examination hexane layer at the upper part is about 5 cm, introduce 8 g of
3.1. Apparatus anhydrous sodium sulfate from the top, and further drip
Use an optical microscope with objectives of 10 and 40 until a small quantity of hexane is left at the upper part.
magnifications, and an ocular of 10 magnifications. Weigh accurately about 5 g of pulverized sample, place in
3.2. Preparation for microscopic examination a glass-stoppered centrifuge tube, add 30 mL of a mixture of
(i) Section: To a section on a slide glass add 1 to 2 drops acetone for Purity of crude drug and water (5:2), stopper
of a mounting agent, and put a cover glass on it, taking tightly, shake for 15 minutes, centrifuge, and separate the
precaution against inclusion of bubbles. Usually use a sec- supernatant liquid. Repeat the same procedure twice with the
tion 10 to 20 mm in thickness. residue using two 30-mL portions of the mixture of acetone
(ii) Powder: Place about 1 mg of powdered sample on a for Purity of crude drug and water (5:2). Combine all the su-
slide glass, add 1 to 2 drops of a swelling agent, stir well with pernatant liquids, and concentrate under reduced pressure at
a small rod preventing inclusion of bubbles, and allow to a temperature not higher than 409C until the order of ace-
stand for a while to swell the sample. Add 1 drop of the tone is faint. Transfer the concentrated solution to a separa-
mounting agent, and put a cover glass on it so that the tissue tor containing 100 mL of sodium chloride TS, and shake
sections spread evenly without overlapping each other, twice with two 50-mL portions of hexane for Purity of crude
taking precaution against inclusion of bubbles. In the case drug for 5 minutes each. Combine the hexane layers, trans-
where the tissue sections are opaque, place about 1 mg of fer to a separator containing 50 mL of sodium chloride TS,
powdered sample on a slide glass, add 1 to 2 drops of chloral and shake for 5 minutes. Take the hexane layer, dry with
hydrate TS, heat to make the tissues clear while stirring with 30 g of anhydrous sodium sulfate, and filter. Wash the
a small glass rod to prevent boiling. After cooling, add 1 residue on the filter paper with 20 mL of hexane for Purity

of crude drug. Combine the filtrate and the washings, and M: Amount (g) of pulverized sample
concentrate under reduced pressure at a temperature not
Content (ppm) of total BHC's
higher than 409 C to about 5 mL. Transfer this solution to
＝ content (ppm) of a-BHC ＋ content (ppm) of
the chromatographic column and allow to pass with 300 mL
b-BHC ＋ content (ppm) of g-BHC ＋
of a mixture of hexane for Purity of crude drug and diethyl
content (ppm) of d-BHC
ether for Purity of crude drug (17:3) at a rate of not more
than 5 mL per minute. After concentrating the eluate under Content (ppm) of total DDT's
reduced pressure at a temperature not higher than 409C, add ＝ content (ppm) of o, p?-DDT ＋ content (ppm) of
hexane for Purity of crude drug to make exactly 5 mL. p, p?-DDT ＋ content (ppm) of p, p?-DDD ＋
Transfer this solution to a glass-stoppered test tube, add 1 content (ppm) of p, p?-DDE
mL of sulfuric acid, and shake carefully. Take 4 mL of the
Operating conditions—
upper layer, transfer to a separate glass-stoppered test tube,
Detector: An electron capture detector.
add 2 mL of water, and shake gently. Take 3 mL of the
Sample injection system: A splitless injection system.
upper layer so obtained, transfer to a glass-stoppered centri-
Column: A fused silica capillary column about 0.3 mm in
fuge tube, dry with 1 g of anhydrous sodium sulfate, centri-
inside diameter and about 30 m in length, coated the inside
fuge, and use the supernatant liquid as the sample solution.
wall with 7z cyanopropyl-7z phenylmethylsilicone poly-
Separately, weigh accurately about 10 mg each of a-BHC,
mer for gas chromatography in a thickness of 0.25 to 1.0
b-BHC, g-BHC, d-BHC, o, p?-DDT, p, p?-DDT, p, p?-DDD
mm.
and p, p?-DDE, dissolve in 5 mL of acetone for Purity of
Column temperature: Maintain the temperature at 609C
crude drug, and add hexane for Purity of crude drug to
for 2 minutes after injection, program to increase the tem-
make exactly 100 mL. Pipet 10 mL of this solution, and add
perature at a rate of 109 C per minute to 2009C, and then
hexane for Purity of crude drug to make exactly 100 mL.
program to increase the temperature at a rate of 29 C per
Pipet 1 mL of this solution, add hexane for Purity of crude
minute to 2609 C.
drug to make exactly 100 mL, and use this solution as the
Carrier gas: Helium.
standard solution. Perform the test with 1 mL each of the
Flow rate: Adjust so that the retention times of the objec-
sample solution and the standard solution as directed under
tive compounds are between 10 and 30 minutes.
Gas Chromatography <2.02> according to the following con-
System suitability—
ditions, and determine the peak areas corresponding to a-
Test for required detectability: To exactly 1 mL of the
BHC, b-BHC, g-BHC, d-BHC, o, p?-DDT, p, p?-DDT, p, p?-
standard solution add hexane to make exactly 10 mL. Con-
DDD and p, p?-DDE, ATA and ASA; ATB and ASB; ATC and
firm that the peak area of each objective compound ob-
ASC; ATD and ASD; ATE and ASE; ATF and ASF; ATG and ASG;
tained with 1 mL of this solution is equivalent to 5 to 15z of
ATH and ASH. Calculate the content of each of a-BHC,
that of corresponding compound with 1 mL of the standard
b-BHC, g-BHC, d-BHC, o, p?-DDT, p, p?-DDT, p, p?-DDD
solution.
and p, p?-DDE by means of the following equations.
System performance: When the procedure is run with 1 mL
Content (ppm) of a-BHC of the standard solution under the above operating condi-
tions, the peaks of the object compounds separate com-
amount (g) of a － BHC ATA
＝ × × 50 pletely each other.
M ASA
System repeatability: Repeat the test 6 times with 1 mL of
Content (ppm) of b-BHC the standard solution under the above operating conditions,
the relative standard deviation of the peak area of each ob-
amount (g) of b － BHC ATB
＝ × × 50 ject compound is not more than 10z.
M ASB
5. Loss on drying
Content (ppm) of g-BHC
Unless otherwise specified, transfer 2 to 6 g of the test
amount (g) of g － BHC ATC sample for analysis to a tared weighing bottle, and weigh ac-
＝ × × 50
M ASC curately. Dry at 1059C for 5 hours, allow to cool in a desic-
cator (silica gel), and weigh accurately. Continue the drying
Content (ppm) of d-BHC
at 1059C, and weigh accurately at 1-hour intervals. When
amount (g) of d － BHC ATD the mass of the sample becomes constant, the loss of mass
＝ × × 50
M ASD represents the percentage of loss on drying (z). When the
period of time for drying is specified, weigh accurately after
Content (ppm) of o,p?-DDT
drying for the period of time specified, and determine the
amount (g) of o, p? － DDT ATE loss on drying (z).
＝ × × 50
M ASE
6. Total ash
Content (ppm) of p, p?-DDT Ignite previously a crucible of platinum, quartz or por-
celain between 5009C and 5509C for 1 hour. Cool, and
amount (g) of p, p? － DDT ATF
＝ × × 50 weigh accurately the crucible. Unless otherwise specified,
M ASF
weigh accurately 2 to 4 g of the test sample for analysis in
Content (ppm) of p, p?- DDD this crucible, take off the lid or keep it open a little if neces-
sary, heat the crucible at a low temperature at first, then
amount (g) of p, p? － DDD ATG
＝ × × 50 gradually heat to a temperature between 5009C and 5509 C,
M ASG
ignite to incinerate the residue for more than 4 hours until no
Content (ppm) of p, p?-DDE carbonized substance remains in the ash, cool, and weigh ac-
curately the ash. Incinerate repeatedly to constant mass,
amount (g) of p, p? － DDE ATH
＝ × × 50 cool, weigh accurately, and determine the amount (z) of
M ASH
total ash. If a carbonized substance remains and a constant

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
136 5.01 Crude Drugs Test / General Tests JP XVII
mass cannot be obtained in the above-mentioned method,
extract the charred mass with hot water, collect the insoluble
residue on filter paper for assay, and incinerate the residue
and filter paper until no carbonized substance remains in the
ash. Then add the filtrate, evaporate it to dryness, and incin-
erate. Cool, weigh accurately, and determine the mass (z)
of the total ash. If a carbon-free ash cannot be obtained even
in this way, moisten the ash with a small amount of ethanol
(95), break up the ash with a glass rod, wash the rod with a
small amount of ethanol (95), evaporate carefully, and deter-
mine the mass of the total ash as described above. A desicca-
tor (silica gel) is used for cooling.
7. Acid-insoluble ash
Add carefully 25 mL of dilute hydrochloric acid to the
total ash, boil gently for 5 minutes, collect the insoluble mat-
ter on filter paper for assay, and wash thoroughly with hot
water. Dry the residue together with the filter paper, and
ignite to incinerate in a tared crucible of platinum, quartz or
porcelain for 3 hours. Cool in a desiccator (silica gel), weigh,
and determine the amount (z) of acid-insoluble ash. When
the amount determined exceeds the limit specified, incinerate
repeatedly to a constant mass.
8. Extract content
The test for the extract content in crude drugs is per-
Fig. 5.01-1 Fig. 5.01-2
formed as directed in the following methods:
8.1. Dilute ethanol-soluble extract
Unless otherwise specified, weigh accurately about 2.3 g of
the sample for analysis, extract with 70 mL of dilute ethanol
in a suitable flask with occasional shaking for 5 hours, and of it, and heat the content of the flask in an oil bath between
allow to stand for 16 to 20 hours. Filter, and wash the flask 1309C and 1509C to boiling. The graduated tube of the ap-
and residue with small portions of dilute ethanol until the fil- paratus is to be previously filled with water to the standard
trate measures 100 mL. Evaporate a 50 mL aliquot of the fil- line, and 2.0 mL of xylene is added to the graduated tube.
trate to dryness, dry at 1059C for 4 hours, and cool in a Unless otherwise specified, continue boiling for 5 hours,
desiccator (silica gel). Weigh accurately the amount, multi- allow to stand for some time, and open the stopper of the
ply it by 2, and determine the amount of dilute ethanol-solu- apparatus. Draw off the water slowly until the surface of the
ble extract. Calculate the extract content (z) with respect to oil layer corresponds to the preparation line, and allow it to
the amount of the sample on the dried basis, obtained under stand for more than 1 hour at ordinary temperature. Then
the loss on drying. lower the surface of the oil layer to the zero line, and read
8.2. Water-soluble extract the volume (mL) of the oil at ordinary temperature. Subtract
Proceed as directed in 8.1., using water instead of dilute the volume (mL) of xylene from the volume of the total oil.
ethanol, weigh accurately the amount, multiply by 2, and de-
10. Assay of Marker Compounds for the Assay of Crude
termine the amount of water-soluble extract. Calculate the
Drugs and Extracts of Kampo Formulations Utilizing
extract content (z) with respect to the amount of the sample
Nuclear Magnetic Resonance (NMR) Spectroscopy
on the dried basis, obtained under the loss on drying.
10.1. Principle of Quantitative Analytical Technique
8.3. Diethyl ether-soluble extract
Utilizing Nuclear Magnetic Resonance (NMR) Spectroscopy
Unless otherwise specified, dry the test sample for analysis
The spectra obtained by proton nuclear magnetic reso-
in a desiccator (silica gel) for 48 hours, weigh accurately
nance (1H-NMR) spectroscopy after dissolving the substance
about 2 g of it, and place in a suitable flask. Add 70 mL of
to be measured in a solution, are frequently used as a power-
diethyl ether, attach a reflux condenser to the flask, and boil
ful analytical method for determining the chemical structure
gently on a water bath for 4 hours. Cool, filter, and wash the
of the substance from the following reasons: the resonance
flask and the residue with small portions of diethyl ether
signals appear at different chemical shifts depending on the
until the filtrate measures 100 mL. Evaporate a 50 mL ali-
chemical structure of the substance measured; the signals are
quot of the filtrate to dryness on a water bath, dry in a desic-
split by spin-spin interactions through chemical bonds
cator (silica gel) for 24 hours, weigh accurately the amount,
mainly depending on the number of 1H bonded to adjacent
multiply it by 2, determine the amount of diethyl ether-solu-
carbon atoms; the signal intensities (areas) are proportional
ble extract, and calculate the extract content (z).
to the number of 1H resonating at the same frequency; etc.
9. Essential oil content In the 1H-NMR spectra, the proton nuclei (1Hs) in differ-
The test of essential oil content in crude drugs is per- ent chemical environments within the same molecule are
formed as directed in the following method. observed as the separate signals having different chemical
9.1. Essential oil determination shifts depending on their resonance frequencies. Accord-
Weigh the quantity of the test sample for analysis directed ingly, we can compare the intensities of 2 signals having
in the monograph in a 1-L hard glass-stoppered flask, and different chemical shifts each other. The intensity of the
add from 5 to 10 times as much water as the drug. Set up an signal Si would be given by the following equation (1);
apparatus for essential oil determination (Fig. 5.01-1), in-
m 1 － e－Tr/T1i
serting a reflux condenser (Fig. 5.01-2) in the upper mouth Si 1 N i p sin b M0 (1)
VM 1 － e－Tr/T1i cos b

where Ni is the number of resonating 1H which gives the System of Units (SI) is guaranteed for the standard material,
signal, V is the volume of the sample solution, m is the mass purity of the analyte compound can be calculated indirectly
of the sample, M is the molecular mass of the substance as the SI traceable value by using the standard material as
measured, p is the purity of the sample, b is the excitation the superior standard. In such a measurement, it is necessary
pulse angle, T1i is the spin-lattice relaxation time of 1H which to dissolve the sample and the standard material in a solu-
gives the signal, Tr is the repetition time, M0 is the equilibri- tion. Thus, it is practically important for precise evaluation
um magnetization and the subscript i indicates the indepen- of the purity of analyte compound that both of the sample
dent signal. The relaxation time of a 1H is different depend- and the standard material should be weighed accurately, and
ing on the environments of the 1Hs. Since the sensitivity of dissolved in a solvent for NMR measurement.
NMR is not so good, the signal-to-noise ratio (SN ratio) of 10.2. Supply of Reference Materials and Software for
signals should generally be improved by measuring it repeat- Quantitative NMR
edly and averaging noises. When the NMR measurement is From among certified reference materials supplied from
performed under the condition with the repetition time Tr public institutions (NMIJ CRM), those with SI traceable
sufficiently longer than the longest T1 among the T1s of the pricing have been marketed as internal reference materials.
signals observed for the analyte compound, the condition of Easy-to-use solid-state compounds include 1,4-bis(trimethyl-
1 － e－Tr/T1 § 1 for all of the signals of the analyte com- silyl)benzene-d4 (BTMSB-d4), methanol, and dimethylsul-
pounds would be satisfied and quantitative analysis utilizing foxide for organic solvent use and 3-(trimethylsilyl)-1-
NMR (quantitative NMR) can be performed. On the other propanesulfonic acid-d6-sodium salt (DSS-d6), maleic acid
hand, when NMR is used for the structural determination, and dimethyl sulfone for aquatic use which both exhibit a
priority is given to improve detection sensitivity, and the sharp peak for specific chemical shifts in 1H-NMR. In addi-
condition for increasing the SN ratio of signals by using tion, such measurement software capable of performing
repeated measurements is usually used. Under this condi- quantification (qNMR) easier based on the above-mentioned
tion, since the repetition time is not long enough to ensure principle is also supplied by NMR manufacturers.
quantitative NMR, the proportion of signal intensity to the 10.3. Marker Compounds for Assay and Reference Sub-
number of each equivalent 1H nuclei in the measured molec- stances for Quantitative Analysis of Crude Drugs and Ex-
ule is not obtained precisely. tracts of Kampo Formulations in the JP
However, when NMR is measured under the conditions, If it is possible to price a reagent used as a quantitative
which ensure quantitative performance, the signal intensity index component in a crude medicine with a correct content
ratio proportional to each number of equivalent molecule is using qNMR based on the above-mentioned principle, it also
obtained. becomes possible to use the reagent as a reference substance
When the intensity of two signals having different chemi- for analysis with assured metrological traceability. Accord-
cal shifts in the same molecule are compared under the quan- ing to a result of a validation experiment, in case of a com-
titative conditions which ensure quantitative performance, pound with molecular mass of around 300 to be measured, it
the following equation (2) is obtained and the signal intensi- is possible to perform pricing at an ordinary laboratory level
ties Si and Sj are found to be proportional to the number of by using about 10 mg of the compound for the measurement
resonating 1Hs. while ensuring two significant figures even if including errors
between used devices. As content of quantitative index com-
Si Ni
＝ (2) ponent in a crude medicine is just a few percent at most in
Sj Nj
general and the minimum unit of regulation value is 0.1z,
This proportionality between the signal area and number two significant figures is believed to be enough to ensure
of resonating 1H can be applied to the signals from 2 differ- accuracy of content of reference substance for quantitative
ent molecules. In this case, since it is considered that the ex- analysis in consideration of variation for each crude medi-
citation pulse angle and the volume of the sample solution cine as a natural substance.
used for the measurement can be kept constant independent Such reagents priced with SI traceable quantitative value
of the substance measured, the following equation (3), in (degree of purity) by qNMR that have been defined in a
which the observed signal area S is proportional only to the paragraph for reagent and test solution are available as
purity, molecular mass and mass used for the measurement Japanese Pharmacopoeia reagents for quantitative analysis.
of analyte compound, can be obtained. (a and s indicate the Further, in cases where a reagent priced by qMNR is used as
signals of the analyte compound and a reference substance a reference substance for quantitative analysis such as HPLC
(internal standard), respectively.) and involved in a calculation of quantitative value of subject
compound after converting degree of purity (z) of the
S a N s M a ms
pa ＝ ps (3) priced reagent, it becomes possible to use the resulting quan-
Ss Na M s m a
titative value as a SI traceable value. In addition, in cases
Although there are some prerequisites to be met, such that where a reagent priced by qNMR is used as a reference mate-
each molecule should not interact (such as react) with other rial for a quantitative analysis based on HPLC, condition of
molecules in the solution and the molecule should have sepa- the quantitative analysis is based on an assumption that no
rate signals at different chemical shifts from others, we will impurity is recognized at any peak of a component of the
be able to evaluate the purity of the analyte compound by reagent to be quantified, which is required to be confirmed
measuring its 1H-NMR under the conditions which ensure separately by a device such as photodiode array detector or
quantitative performance, if we have a standard material mass spectrometer.
with known purity and use it as an internal standard for the 10.4. Precautions for Performing qNMR
measurement. In other words, if a standard material whose In order to perform qNMR, such device is required that is
molecular mass and accurate purity are known would be capable of gated decoupling for 13C-NMR with higher ac-
provided as the superior standard, we can evaluate the purity curacy in a magnetic field with a resonance frequency of at
of the substances coexisting in the solution of the standard least 400 MHz or higher for 1H-NMR in consideration of
material by measuring 1H-NMR of the solution. In this case, resolution required for separation of impurities from peaks
when traceability of the measurement to the International and even detection sensitivity as well. Further, it is also

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
138 5.02 Microbial Limit Test for Crude Drugs and Preparations containing Crude Drugs as Main Ingredient / General Tests JP XVII
required to perform measurement under a condition that I. Total Viable Aerobic Count
receiving sensitivity of the receiver is appropriate with The tests described hereafter will allow quantitative
optimally adjusted probe tuning and shim. enumeration of mesophilic bacteria and fungi which may
In terms of reagents for quantification for which qNMR is grow under aerobic conditions.
performed, amounts of reagents and internal reference mate- The tests are designed primarily to determine whether a
rials to be taken are defined in paragraph 9.41 Reagents, substance or preparation complies with an established speci-
Test Solutions. As high accuracy is required for both of fication for microbiological quality. When used for such
them, it is required to use an ultramicro balance to take the purposes follow the instructions given below, including the
amounts by the minimum weight of the balance or higher. number of samples to be taken and interpret the results as
Defined amounts to be taken for both of them are those de- stated below.
scribed as validated realistic minimum amounts. Therefore, Alternative microbiological procedures, including rapid
in cases where both of them are completely dissolved, SN methods, may be used, provided that they give a result equal
ratio of spectrum is improved when measured after increas- to or better than that of the Pharmacopoeial methods.
ing the amounts while keeping the quantitative ratio, result-
1. General Procedures
ing in measurements with higher accuracy in most cases.
Carry out the determination under conditions designed to
Even though SN ratio is even more improved when a meas-
avoid extrinsic microbial contamination of the product to be
urement is performed by integrating as many times as possi-
examined. The precautions taken to avoid contamination
ble resulting in a measurement result with higher accuracy, it
must be such that they do not affect any micro-organisms
is required to consider stability of the magnetic field and the
which are to be revealed in the test.
devices if the measurement lasts more than a few hours. Sen-
If the product to be examined has antimicrobial activity,
sitivity is also improved, albeit a little, by using deuterated
this is insofar as possible removed or neutralised. If inactiva-
solvent with higher deuteration rate. In some cases, impurity
tors are used for this purpose their efficacy and their absence
signal may be detected on spectrum which has not been ob-
of toxicity for micro-organisms must be demonstrated.
served before, by further improving SN ratio. In cases where
If surface-active substances are used for sample prepara-
any existence of signal derived from such impurities has been
tion, their absence of toxicity for micro-organisms and their
made clear, the range of chemical shift where such signal ex-
compatibility with inactivators used must be demonstrated.
ists should not be integrated. In addition, as signals of small
amount of impurities have been observed also in deuterated 2. Enumeration Methods
solvent for NMR measurement or BTMSB-d4 or DSS-d6 as The choice of a method is based on factors such as the na-
internal reference materials, it is important to recognize the ture of the product and the required limit of micro-organ-
range of these impurities signals before qNMR meas- isms. The method chosen must allow testing of a sufficient
urement. Moreover, qNMR measurement should be per- sample size to judge compliance with the specification. The
formed immediately after sample preparation, since impurity suitability of the chosen method must be established.
signals have been confirmed to increase albeit a little by little
3. Growth Promotion Test, Suitability of the Counting
if samples are kept in the solvent for measurement for long
Method and Negative Controls
hours. Even though it is not necessary to measure NMR
The ability of the test to detect micro-organisms in the
under qNMR condition for confirming impurity signal, it is
presence of product to be tested must be established.
easier to distinguish it from satellite signal by performing a
Suitability must be confirmed if a change in testing per-
measurement under a condition of decoupling of 13C NMR
formance, or the product, which may affect the outcome of
without performing spinning. While BTMSB-d4 and DSS-d6,
the test is introduced.
which are used for qNMR as internal reference materials,
3.1. Preparation of test strains
have chemical shift values at around 0.2 ppm and 0.1 ppm
Use standardised stable suspensions of test strains or pre-
respectively when tetramethylsilane (in organic solvent) or
pare as stated below.
DDS (in deuterated water) is used as the reference for chemi-
Seed lot culture maintenance techniques (seed-lot systems)
cal shifts (d), chemical shifts of other signals are indicated by
are used so that the viable micro-organisms used for inocula-
regarding the chemical shifts of these internal reference ma-
tion are not more than 5 passages removed from the original
terials as 0 ppm for convenience when measuring qNMR.
master seed-lot. Grow each of the bacterial and fungal test
strains separately as described in Table 5.02-I-1.
Use Buffered Sodium Chloride-Peptone Solution (pH 7.0)
5.02 Microbial Limit Test for or Phosphate Buffer (pH 7.2) to make test suspensions; to
suspend Aspergillus brasiliensis spores, 0.05z of polysor-
Crude Drugs and Preparations bate 80 may be added to the buffer. Use the suspensions
containing Crude Drugs as within 2 h or within 24 h if stored at 2 – 89 C. As an alterna-
tive to preparing and then diluting a fresh suspension of
Main Ingredient vegetative cells of Aspergillus brasiliensis or Bacillus subtilis,
a stable spore suspension is prepared and then an appropri-
Microbial limit test for crude drugs and preparations con-
ate volume of the spore suspension is used for test inocula-
taining crude drugs as main ingredient (crude drug prepara-
tion. The stable spore suspension may be maintained at 2 –
tions) includes microbial enumeration tests and tests for spe-
89 C for a validated period of time.
cified micro-organisms. For the test, use a mixture of several
3.2. Negative control
portions selected at random from the bulk or from the con-
To verify testing conditions, a negative control is per-
tents of a sufficient number of containers. If test specimens
formed using the chosen diluent in place of the test prepara-
are diluted with fluid medium, the test should be performed
tion. There must be no growth of micro-organisms. A nega-
quickly. In performing the test, precautions must be taken to
tive control is also performed when testing the products as
prevent biohazard.
described under 4. Testing of Products. A failed negative
control requires an investigation.

3.3. Growth promotion of the media more than 100 CFU) of the micro-organisms indicated in
Test each batch of ready-prepared medium and each batch Table 5.02-I-1, using a separate plate of medium for each.
of medium, prepared either from dehydrated medium or Incubate in the conditions described in Table 5.02-I-1.
from the ingredients described. For solid media, growth obtained must not differ by a fac-
Inoculate portions/plates of Fluid Soybean-Casein Digest tor greater than 2 from the calculated value for a stand-
Medium and Soybean-Casein Digest Agar Medium with a ardized inoculum. For a freshly prepared inoculum, growth
small number (not more than 100 CFU) of the microorgan- of the micro-organisms comparable to that previously ob-
isms indicated in Table 5.02-I-1, using a separate por- tained with a previously tested and approved batch of me-
tion/plate of medium for each. Inoculate plates of dium occurs.
Sabouraud Glucose Agar Medium with a small number (not Liquid media are suitable if clearly visible growth of the

Table 5.02-I-1 Preparation and use of test micro-organisms

Suitability of counting method

Growth promotion
Preparation of in the presence of the product
Micro-organism
test strain Total aerobic Total yeasts and Total aerobic Total yeasts and
microbial count moulds count microbial count moulds count

Staphylococcus Soybean-Casein Soybean-Casein Soybean-Casein

aureus Digest Agar Medi- Digest Agar Medi- Digest Agar
such as ATCC um or Fluid Soy- um* and Fluid Medium/MPN
6538, NCIMB bean-Casein Digest Soybean-Casein Fluid Soybean-
9518, CIP 4.83 or Medium Digest Medium Casein Digest
NBRC 13276 30 – 359C ≦ 100 CFU Medium
18 – 24 h 30 – 359C ≦ 100 CFU
≦ 3 days 30 – 359C
≦ 5 days

Pseudomonas aer- Soybean-Casein Soybean-Casein Soybean-Casein

uginosa Digest Agar Medi- Digest Agar Medi- Digest Agar
such as ATCC um or Fluid Soy- um* and Fluid Medium/MPN
9027, NCIMB bean-Casein Digest Soybean-Casein Fluid Soybean-
8626, CIP 82.118 Medium Digest Medium Casein Digest
or NBRC 13275 30 – 359C ≦ 100 CFU Medium
18 – 24 h 30 – 359C ≦ 100 CFU
≦ 3 days 30 – 359C
≦ 5 days

Bacillus subtilis Soybean-Casein Soybean-Casein Soybean-Casein

such as ATCC Digest Agar Medi- Digest Agar Medi- Digest Agar
6633, NCIMB um or Fluid Soy- um* and Fluid Medium/MPN
8054, CIP 52.62 or bean-Casein Digest Soybean-Casein Fluid Soybean-
NBRC 3134 Medium Digest Medium Casein Digest
30 – 359C ≦ 100 CFU Medium
18 – 24 h 30 – 359C ≦ 100 CFU
≦ 3 days 30 – 359C
≦ 5 days

Candida albicans Sabouraud Glucose Soybean-Casein Sabouraud Glucose Soybean-Casein Sabouraud Glucose
such as ATCC Agar Medium or Digest Agar Medi- Agar Medium with Digest Agar Medi- Agar Medium with
10231, NCPF 3179, Fluid Sabouraud um* antibiotics um antibiotics
IP 48.72 or NBRC Glucose Medium ≦ 100 CFU ≦ 100 CFU ≦ 100 CFU ≦ 100 CFU
1594 20 – 259C 30 – 359C 20 – 259C 30 – 359C 20 – 259C
2 – 3 days ≦ 5 days ≦ 5 days ≦ 5 days ≦ 5 days
MPN: not applicable

Aspergillus Sabouraud Glucose Soybean-Casein Sabouraud Glucose Soybean-Casein Sabouraud Glucose

brasiliensis Agar Medium or Digest Agar Medi- Agar Medium with Digest Agar Medi- Agar Medium with
such as ATCC Potato Dextrose um* antibiotics um antibiotics
16404, IMI 149007, Agar Medium ≦ 100 CFU ≦ 100 CFU ≦ 100 CFU ≦ 100 CFU
IP 1431.83 or 20 – 259C 30 – 359C 20 – 259C 30 – 359C 20 – 259C
NBRC 9455 5 – 7 days, or until ≦ 5 days ≦ 5 days ≦ 5 days ≦ 5 days
good sporulation is MPN: not applicable
achieved
* In the case where TTC TS or amphotericin B TS is added, check with the media with these additives. When amphotericin B
TS is added, the check for C. albicans and A. brasiliensis is not required.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
140 5.02 Microbial Limit Test for Crude Drugs and Preparations containing Crude Drugs as Main Ingredient / General Tests JP XVII
micro-organisms comparable to that previously obtained inhibit others not included amongst the test strains or for
with a previously tested and approved batch of medium oc- which the latter are not representative. Then, perform the
curs. test with the highest dilution factor compatible with micro-
3.4. Suitability of the counting method in the presence of bial growth and the specific acceptance criterion.
product 3.4.4. Recovery of micro-organism in the presence of
3.4.1. Preparation of the sample product
The method for sample preparation depends on the physi- For each of the micro-organisms listed in Table 5.02-I-1,
cal characteristics of the product to be tested. If none of the separate tests are performed. Only micro-organisms of the
procedures described below can be demonstrated to be satis- added test strain are counted.
factory, an alternative procedure must be developed. 3.4.4.1. Membrane filtration method
Buffered Sodium Chloride-Peptone Solution (pH 7.0), Use membrane filters having a nominal pore size not
Phosphate Buffer (pH 7.2) or Fluid Soybean-Casein Digest greater than 0.45 mm. The type of filter material is chosen in
Medium is used to suspend or dilute the test specimen. Un- such a way that the bacteria-retaining efficiency is not affect-
less otherwise specified, usually take 10 g or 10 mL of the ed by the components of the sample to be investigated. For
test specimen, and suspend or dissolve it in 90 mL of the each of the micro-organisms listed in Table 5.02-I-1, one
buffer or fluid medium specified. A test specimen as a sus- membrane filter is used.
pension must be shaken for 10 minutes. If necessary, for the Transfer a suitable amount of the sample prepared as de-
product to be tested which recovery rate of microorganisms scribed under 3.4.1. to 3.4.3. (preferably representing 1 g of
is low, repeat the same method and use this as the test fluid. the product, or less if large numbers of CFU are expected) to
A different quantity or volume may be used if the nature of the membrane filter, filter immediately and rinse the mem-
the test specimen requires it. A surface-active agent such as 1 brane filter with an appropriate volume of diluent.
g/L of polysorbate 80 may be added to assist the suspension For the determination of total aerobic microbial count
of poorly wettable substances. If necessary adjust to pH 6 – (TAMC), transfer the membrane filter to the surface of Soy-
8. Further dilutions, where necessary, are prepared with the bean-Casein Digest Agar Medium. For the determination of
same diluent. total combined yeasts/moulds count (TYMC) transfer the
3.4.2. Inoculation and dilution membrane to the surface of Sabouraud Glucose Agar Medi-
Add to the sample prepared as described above (3.4.1.) um with antibiotics. For Soybean-Casein Digest Agar Medi-
and to a control (with no test material included) a sufficient um that is suffused with fungi or when the TAMC is ex-
volume of the microbial suspension to obtain an inoculum of pected to exceed the acceptance criterion due to the fungus
not more than 100 CFU. The volume of the suspension of growth, amphotericin B TS as an antimycotic may be added
the inoculum should not exceed 1 per cent of the volume of to the agar. Incubate the plates as indicated in Table
diluted product. 5.02-I-1. Perform the counting.
To demonstrate acceptable microbial recovery from the 3.4.4.2. Plate-count methods
product, the lowest possible dilution factor of the prepared Perform plate-count methods at least in duplicate for each
sample must be used for the test. Where this is not possible medium and use the mean count of the result.
due to antimicrobial activity or poor solubility, further ap- (i) Pour-plate method: For Petri dishes 9 cm in diameter,
propriate protocols must be developed. add to the dish 1 mL of the sample prepared as described
If inhibition of growth by the sample cannot otherwise be under 3.4.1. to 3.4.3. and 15 – 20 mL of Soybean-Casein
avoided, the aliquot of the microbial suspension may be Digest Agar Medium or Sabouraud Glucose Agar Medium
added after neutralization, dilution or filtration. with antibiotics, both media being warmed at not more than
3.4.3. Neutralization/removal of antimicrobial activity 459C. For Soybean-Casein Digest Agar Medium, TTC TS
The number of micro-organisms recovered from the pre- may be added to distinct the colonies from the fragments of
pared sample diluted as described in 3.4.2. and incubated crude drugs existed in specimens, and when the agar medium
following the procedure described in 3.4.4., is compared to is suffused with fungi or the TAMC is expected to exceed the
the number of micro-organisms recovered from the control acceptance criterion due to the fungus growth, amphotericin
preparation. B TS as an antimycotic may be added to the medium. In the
If growth is inhibited (reduction by a factor greater than case of Sabouraud Glucose Agar Medium with antibiotics
2), then modify the procedure for the particular enumeration that is suffused with fungi, Rose Bengal TS may be added. If
test to ensure the validity of the results. Modification of the larger Petri dishes are used, the amount of agar medium is
procedure may include, for example, (1) an increase in the increased accordingly. For each of the micro-organisms list-
volume of the diluent or culture medium, (2) incorporation ed in Table 5.02-I-1, at least 2 Petri dishes are used.
of a specific or general neutralizing agents into the diluent, Incubate the plates as indicated in Table 5.02-I-1. Take the
(3) membrane filtration or (4) a combination of the above arithmetic mean of the counts per medium and calculate the
measures. number of CFU in the original inoculum.
Neutralizing agents—Neutralizing agents may be used to (ii) Surface-spread method: For Petri dishes 9 cm in di-
neutralize the activity of antimicrobial agents. They may be ameter, add 15 – 20 mL of Soybean-Casein Digest Agar
added to the chosen diluent or the medium preferably before Medium or Sabouraud Glucose Agar Medium with antibiot-
sterilization. If used, their efficacy and their absence of tox- ics at about 459 C to each Petri dish and allow to solidify.
icity for micro-organisms must be demonstrated by carrying Dry the plates, for example in a laminar-air-flow cabinet or
out a blank with neutralizing agents, without product. in an incubator. The test solutions to be added to the agar
If no suitable neutralizing method can be found, it can be medium are the same as described in (i) Pour-plate method.
assumed that the failure to isolate the inoculated organism is If larger Petri dishes are used, the volume of the agar is in-
attributable to the microbicidal activity of the product. This creased accordingly. For each of the micro-organisms listed
information serves to indicate that the article is not likely to in Table 5.02-I-1, at least 2 Petri dishes are used. Spread a
be contaminated with the given species of the micro-organ- measured volume of not less than 0.1 mL of the sample pre-
ism. However, it is possible that the product only inhibits pared as described under 3.4.1. to 3.4.3. over the surface of
some of the micro-organisms specified herein, but does not the medium. Incubate and count as prescribed under 3.4.4.2.

(i). ate amount to each of 2 membrane filters and filter immedi-

3.4.4.3. Most-probable-number (MPN) method ately. Wash each filter following the procedure shown to be
The precision and accuracy of the MPN method is less suitable.
than that of the membrane filtration method or the plate- For the determination of TAMC, transfer one of the
count method. Unreliable results are obtained particularly membrane filters to the surface of Soybean-Casein Digest
for the enumeration of moulds. For these reasons the MPN Agar Medium. For the determination of TYMC, transfer the
method is reserved for the enumeration of TAMC in situa- other membrane to the surface of Sabouraud Glucose Agar
tions where no other method is available. If the use of the Medium with antibiotics. Incubate the plate of Soybean-
method is justified, proceed as follows. Casein Digest Agar Medium at 30 – 359C for 5 – 7 days and
Prepare a series of at least 3 serial tenfold dilutions of the the plate of Sabouraud Glucose Agar Medium with antibiot-
product as described under 3.4.1. to 3.4.3. From each level ics at 20 – 259C for 5 – 7 days.
of dilution, 3 aliquots of 1 g or 1 mL are used to inoculate 3 Calculate the number of CFU per gram or per millilitre of
tubes with 9 – 10 mL of Fluid Soybean-Casein Digest Medi- product.
um. If necessary a surface-active agent such as polysorbate 4.2.2. Plate-count methods
80, or an inactivator of antimicrobial agents may be added (i) Pour-plate method: Prepare the sample using a
to the medium. Thus, if 3 levels of dilution are prepared 9 method that has been shown to be suitable as described in
tubes are inoculated. section 3. Prepare for each medium at least 2 Petri dishes for
Incubate all tubes at 30 – 359 C for not more than 3 days. each level of dilution. Incubate the plates of Soybean-Casein
If reading of the results is difficult or uncertain owing to the Digest Agar Medium at 30 – 359C for 5 – 7 days and the
nature of the product to be examined, subculture in the same plates of Sabouraud Glucose Agar Medium with antibiotics
broth, or Soybean-Casein Digest Agar Medium, for 1 – 2 at 20 – 259C for 5 – 7 days. Select the plates corresponding
days at the same temperature and use these results. Deter- to a given dilution and showing the highest number of colo-
mine the most probable number of micro-organisms per nies less than 250 for TAMC and 50 for TYMC. Take the
gram or millilitre of the product to be examined from Table arithmetic mean per culture medium of the counts and calcu-
5.02-I-2. late the number of CFU per gram or per millilitre of
3.5. Results and interpretation product.
When verifying the suitability of the membrane filtration (ii) Surface-spread method: Prepare the sample using a
method or the plate-count method, a mean count of any of method that has been shown to be suitable as described in
the test organisms not differing by a factor greater than 2 section 3. Prepare at least 2 Petri dishes for each medium
from the value of the control defined in 3.4.2. in the absence and each level of dilution. For incubation and calculation of
of the product must be obtained. When verifying the the number of CFU proceed as described for the pour-plate
suitability of the MPN method the calculated value from the method.
inoculum must be within 95 per cent confidence limits of the 4.2.3. Most-probable-number method
results obtained with the control. Prepare and dilute the sample using a method that has
If the above criteria cannot be met for one or more of the been shown to be suitable as described in section 3. Incubate
organisms tested with any of the described methods, the all tubes for 3 – 5 days at 30 – 359C. Subculture if necessary,
method and test conditions that come closest to the criteria using the procedure shown to be suitable. Record for each
are used to test the product. level of dilution the number of tubes showing microbial
growth.
4. Testing of Products
Determine the most probable number of micro-organisms
4.1. Sampling and preparation of the test specimens
per gram or millilitre of the product to be examined from
Select the sample(s) at random from the bulk material or
Table 5.02-I-2.
from the available containers of the preparation. To obtain
4.3. Interpretation of the results
the required quantity, mix the contents of a sufficient num-
The total aerobic microbial count (TAMC) is considered
ber of containers to provide the sample.
to be equal to the number of CFU found using Soybean-
Unless otherwise specified, samples should be taken by the
Casein Digest Agar Medium; if colonies of fungi are detect-
following methods.
ed on this medium, they are counted as part of TAMC. The
(i) When crude drugs to be sampled are small-sized, cut
total combined yeasts/mould count (TYMC) is considered to
or powdered, 50 to 250 g of sample should be taken after
be equal to the number of CFU found using Sabouraud Glu-
mixing thoroughly.
cose Agar Medium with antibiotics; if colonies of bacteria
(ii) When crude drugs to be sampled are large-sized, 250
are detected on this medium, they are counted as part of
to 500 g of sample should be taken after mixing thoroughly
TYMC. In the case here the bacterial growth is expected not
and cutting.
to give any affection to the interpretation of the result,
(iii) When the mass of each single piece of the crude drug
Sabouraud Glucose Agar Medium containing no antibiotics
is not less than 100 g, not less than 5 pieces should be taken
may be used. If the count is carried out by the MPN method
for a sample, or not less than 500 g of the sample should be
the calculated value is the TAMC.
taken after cutting to a suitable size and mixing thoroughly.
The recommended solutions and media are described in
If necessary, cut more for use.
``II. Tests for Specified Micro-organisms''.
(iv) When crude drugs to be sampled are in the form of a
solution or a preparation, the sample should be taken after II. Tests for Specified Micro-organisms
mixing thoroughly. The tests described hereafter will allow determination of
4.2. Examination of the product the absence of, or limited occurrence of specified micro-
4.2.1. Membrane filtration method organisms which may be detected under the conditions de-
Use a filtration apparatus designed to allow the transfer of scribed.
the filter to the medium. The tests are designed primarily to determine whether a
Prepare the sample using a method that has been shown substance or preparation complies with an established speci-
suitable as described in section 3 and transfer the appropri- fication for microbiological quality. When used for such

Table 5.02-II-1 Growth promoting, inhibitory and indica- proved batch of medium occurs.
tive properties of media (ii) Test for growth promoting properties, solid media:
perform surface-spread method, inoculating each plate with
Medium Property Test strains
a small number (not more than 100 CFU) of the appropriate
Test for bile-tolerant gram-negative bacteria micro-organism. Incubate at the specified temperature for
not more than the shortest period of time specified in the
Enterobacteria enrich- Growth promoting E. coli
ment broth-Mossel P. aeruginosa test. Growth of the micro-organism comparable to that pre-
viously obtained with a previously tested and approved batch
Inhibitory S. aureus of medium occurs.
VRB (Violet/Red/Bile) Growth promoting E. coli (iii) Test for inhibitory properties, liquid or solid media:
Agar with glucose ＋ Indicative P. aeruginosa inoculate the appropriate medium with at least 100 CFU of
the appropriate micro-organism. Incubate at the specified
Test for Escherichia coli
temperature for not less than the longest period of time
Fluid MacConkey Growth promoting E. coli specified in the test. No growth of the test micro-organism
Broth Medium occurs.
Inhibitory S. aureus
(iv) Test for indicative properties, solid media: perform
MacConkey Agar Growth promoting E. coli surface-spread method, inoculating each plate with a small
Medium ＋ Indicative number (not more than 100 CFU) of the appropriate micro-
Enzyme substrate Growth promoting E. coli organism. Incubate at the specified temperature for a period
medium for E. coli ＋ Indicative of time within the range specified in the test. Colonies are
comparable in indication reactions to those previously ob-
Test for Salmonella tained with a previously tested and approved batch of me-
Fluid Rappaport Vas- Growth promoting Salmonella enterica dium.
siliadis Salmonella En- subsp. enterica sero- (v) Test for indicative properties, liquid media: inoculate
richment Broth Medium var Typhimurium or a portion of the appropriate medium with a small number
Salmonella enterica (not more than 100 CFU) of the appropriate micro-organ-
subsp. enterica sero- ism. Incubate at the specified temperature for a period of
var Abony time within the range specified in the test. Colonies are com-
Inhibitory S. aureus parable in indication reactions to those previously obtained
with a previously tested and approved batch of medium.
XLD (Xylose-Lysine- Growth promoting Salmonella enterica
2.4. Suitability of the test method
Deoxycholate) Agar ＋ Indicative subsp. enterica sero-
For each product to be tested perform sample preparation
Medium var Typhimurium or
Salmonella enterica as described in the relevant paragraph in section 3. Add each
subsp. enterica sero- test strain at the time of mixing, in the prescribed growth
var Abony medium. Inoculate the test strains individually. Use a num-
ber of micro-organisms equivalent to not more than 100
Enzyme substrate Growth promoting Salmonella enterica
CFU in the inoculated test preparation.
medium for Salmonella ＋ Indicative subsp. enterica sero-
Perform the test as described in the relevant paragraph in
var Typhimurium or
Salmonella enterica section 3 using the shortest incubation period prescribed.
subsp. enterica sero- The specified micro-organisms must be detected with the
var Abony indication reactions as described in section 3.
Any antimicrobial activity of the product necessitates a
Test for Staphylococcus aureus
modification of the test procedure (see 3.4.3. of I. Total
Fluid Soybean-Casein Growth promoting S. aureus Viable Aerobic Count).
Digest Medium with If for a given product the antimicrobial activity with
7.5z Sodium Chloride respect to a micro-organism for which testing is prescribed
Vogel-Johnson Agar Growth promoting S. aureus cannot be neutralised, then it is to be assumed that the in-
Medium ＋ Indicative hibited micro-organism will not be present in the product.

Inhibitory E. coli 3. Testing of Products

3.1. Bile-tolerant gram-negative bacteria
Baird-Parker Agar Growth promoting S. aureus 3.1.1. Sample preparation and pre-incubation
Medium ＋ Indicative
Prepare a sample using a 1 in 10 dilution of not less than
Inhibitory E. coli 1 g of the product to be examined as described in I. Total
viable aerobic count, but using Fluid Soybean-Casein Digest
Mannitol Salt Agar Growth promoting S. aureus
Medium ＋ Indicative Medium as the chosen diluent, mix and incubate at 20 –
259C for a time sufficient to resuscitate the bacteria but not
Inhibitory E. coli sufficient to encourage multiplication of the organisms (usu-
ally 2 hours but not more than 5 hours).
3.1.2. Selection and subculture
Inoculate to a suitable amount of Fluid Enterobacteria
Enrichment Broth Mossel Medium successive three dilutions
inoculate a portion of the appropriate medium with a small
which are chosen according to an objective acceptable limit
number (not more than 100 CFU) of the appropriate micro-
from four dilutions of the sample preparation described
organism. Incubate at the specified temperature for not
under 3.1.1. and/or the dilutions of it, containing respec-
more than the shortest period of time specified in the test.
tively 0.1 g, 0.01 g, 0.001 g and 0.0001 g (or 0.1 mL, 0.01
Clearly visible growth of the micro-organism comparable to
mL, 0.001 mL and 0.0001 mL) of the product to be exam-
that previously obtained with a previously tested and ap-

Results for each quantity of product Probable number Results for each quantity of product Probable number
of bacteria per of microorganisms
0.1 g or 0.01 g or 0.001 g or 0.0001 g or gram or mL of 0.1 g or 0.01 g or 0.001 g or
0.1 mL 0.01 mL 0.001 mL per gram or mL
0.1 mL 0.01 mL 0.001 mL 0.0001 mL product
＋＋＋＋ more than 104 ＋＋＋ more than 103

less than 104 and less than 103 and

＋＋＋－＋＋－
more than 103 more than 102

less than 103 and less than 102 and

＋＋－－＋－－
more than 102 more than 10

less than 102 and －－－ less than 10

＋－－－
more than 10
－－－－ less than 10
18 – 24 hours.
3.2.2.2. Selection and subculture
Shake the container, transfer 1 mL of Fluid Soybean-
ined. Incubate at 30 – 359 C for 24 – 48 hours. Subculture Casein Digest Medium to 10 mL of Fluid MacConkey Broth
each of the cultures on a plate of VRB (Violet/Red/Bile) Medium and incubate at 44 ± 0.59 C for 24 – 48 hours. Sub-
Agar with glucose. Incubate at 30 – 359C for 18 – 24 hours. culture on a plate of MacConkey Agar Medium at 30 – 359 C
3.1.3. Interpretation for 18 – 72 hours. An appropriate enzyme substrate medium
Generally, growth of colonies constitutes a positive result. for E. coli such as CHE Agar Medium or ESC Medium may
Note the smallest quantity of the product that gives a posi- be used instead of MacConkey Agar Medium. When such
tive result and the largest quantity that gives a negative enzyme substrate media are used, incubate under the condi-
result. Determine from Table 5.02-II-2 the probable number tions indicated for each medium.
of bile-tolerant gram-negative bacteria. 3.2.2.3. Interpretation
3.2. Escherichia coli Growth of brick-red colonies surrounded by a reddish
3.2.1. Qualitative test precipitation line on MacConkey Agar Medium or of colo-
3.2.1.1. Sample preparation and pre-incubation nies showing aspects or responses corresponding to E. coli
Prepare a sample using a 1 in 10 dilution of not less than on an enzyme substrate medium for E. coli indicates the pos-
1 g of the product to be examined as described in ``I. Total sible presence of E. coli. This is confirmed by identification
viable aerobic count'' and use 10 mL or the quantity corre- tests.
sponding to 1 g or 1 mL to inoculate a suitable amount Note the smallest quantity of the product that gives a posi-
(determined as described under 2.4.) of Fluid Soybean- tive result and the largest quantity that gives a negative
Casein Digest Medium, mix and incubate at 30 – 359C for result, and determine the probable number of E. coli from
18 – 24 hours. Table 5.02-II-3.
3.2.1.2. Selection and subculture 3.3. Salmonella
Shake the container, transfer 1 mL of Fluid Soybean- 3.3.1. Sample preparation and pre-incubation
Casein Digest Medium to 10 mL of Fluid MacConkey Broth Inoculate 10 g or 10 mL of the product to be examined to
Medium and incubate at 44 ± 0.59C for 24 – 48 hours. Sub- a suitable amount (determined as described under 2.4.) of
culture on a plate of MacConkey Agar Medium at 30 – 359 C Fluid Soybean-Casein Digest Medium, mix and incubate at
for 18 – 72 hours. An appropriate enzyme substrate medium 30 – 359C for 18 – 24 hours.
for E. coli such as CHE Agar Medium or ESC Medium may 3.3.2. Selection and subculture
be used instead of MacConkey Agar Medium. When such Inoculate 0.1 mL of Fluid Soybean-Casein Digest Medium
enzyme substrate media are used, incubate under the condi- to 10 mL of Fluid Rappaport Vassiliadis Salmonella Enrich-
tions indicated for each medium. ment Broth Medium and incubate at 42 ± 0.59 C for 18 – 24
3.2.1.3. Interpretation hours. Transfer on plates of XLD (Xylose-Lysine-Desoxy-
Growth of brick-red colonies surrounded by a reddish cholate) Agar Medium, and incubate at 30 – 359C for 18 – 48
precipitation line on MacConkey Agar Medium or of colo- hours. An appropriate enzyme substrate medium such as
nies showing aspects or responses corresponding to E. coli CHS Agar Medium or ES II Agar Medium may be used in-
on an enzyme substrate medium for E. coli indicates the pos- stead of XLD Agar Medium. When such enzyme substrate
sible presence of E. coli. This is confirmed by identification media are used, incubate under the conditions indicated for
tests. each medium.
The product complies with the test if no colonies showing 3.3.3. Interpretation
such aspects or responses are present or if the identification The possible presence of Salmonella is indicated by the
tests are negative. growth of well-developed, red colonies, with or without
3.2.2. Quantitative test black centers or of colonies showing aspects or responses
3.2.2.1. Sample preparation and pre-incubation corresponding to Salmonella. This is confirmed by identifi-
Inoculate to a suitable amount (determined as described cation tests.
under 2.4) of Fluid Soybean-Casein Digest Medium three The product complies with the test if colonies of the types
amounts of a 1 in 10 dilution, prepared as directed in I. described are not present or if the confirmatory identifica-
Total Viable Aerobic Count, respectively equivalent to 0.1 g, tion tests are negative.
0.01 g and 0.001 g (or 0.1 mL, 0.01 mL and 0.001 mL) of the
product to be examined, mix, and incubate at 30 – 359 C for

Table 5.02-II-4 Morphologic characteristics of Staphylo- Soybean peptone 3.0 g

coccus aureus on selective agar media Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Medium Colonial characteristic Glucose monohydrate 2.5 g
Vogel-Johnson Agar Medium Black surrounded by a yel- Water 1000 mL
low zone Adjust the pH so that after sterilization it is 7.1 – 7.5 at
259C. Sterilize in an autoclave using a validated cycle.
Baird-Parker Agar Medium Black, shiny, surrounded by
(iv) Soybean-Casein Digest Agar Medium
a clear zone
Casein peptone 15.0 g
Mannitol-Salt Agar Medium Yellow colonies surrounded Soybean peptone 5.0 g
by a yellow zone Sodium chloride 5.0 g
Agar 15.0 g
The following section is given for information. Water 1000 mL
Adjust the pH so that after sterilization it is 7.1 – 7.5 at
259C. Sterilize in an autoclave using a validated cycle.
3.4. Staphylococcus aureus (v) Sabouraud Glucose Agar Medium
3.4.1. Sample preparation and pre-incubation Glucose 40.0 g
Prepare a sample using a 1 in 10 dilution of not less than Peptones (animal tissue and casein 1:1) 10.0 g
1 g of the product to be examined as described in I. Total Agar 15.0 g
Viable Aerobic Count and use 10 mL or the quantity corre- Water 1000 mL
sponding to 1 g or 1 mL to inoculate a suitable amount (de- Adjust the pH so that after sterilization it is 5.4 – 5.8 at
termined as described under 2.4.) of Fluid Soybean-Casein 259C. Sterilize in an autoclave using a validated cycle. Just
Digest Medium and homogenise. prior to use, add 0.10 g of benzylpenicillin potassium and
Incubate at 30 – 359C for 24 – 48 hours. 0.10 g of tetracycline per liter of medium as sterile solutions
3.4.2. Selection and enrichment subculture or, alternatively, add 50 mg of chloramphenicol per liter of
To 9 mL of Fluid Soybean-Casein Digest Medium with medium.
7.5z Sodium Chloride add 1 mL of Fluid Soybean-Casein
(vi) Potato Dextrose Agar Medium
Digest Medium and incubate at 30 – 359C for 24 – 48 hours.
Infusion from potatoes 200 g
3.4.3. Selection and subculture
Glucose 20.0 g
If the growth is apparent, take a portion of the culture
Agar 15.0 g
fluid by means of an inoculating loop and streak it on the
Water 1000 mL
surface of either Vogel-Johnson Agar Medium, Baird-
Adjust the pH so that after sterilization it is 5.4 – 5.8 at
Parker Agar Medium or Mannitol-Salt Agar Medium, and
259C. Sterilize in an autoclave using a validated cycle.
incubate at 30 – 359C for 24 – 48 hours.
3.4.4. Interpretation (vii) Fluid Sabouraud Glucose Medium
The product complies with the test if colonies of the char- Glucose 20.0 g
acteristics described in Table 5.02-II-4 are not present or if Peptones (animal tissue and casein 1:1) 10.0 g
the confirmatory identification tests are negative. Water 1000 mL
Adjust the pH so that after sterilization it is 5.4 – 5.8 at
4. Recommended Solutions, Culture Media and Test Solu-
259C. Sterilize in an autoclave using a validated cycle.
tions
The following solutions, culture media and test solutions (viii) Fluid Enterobacteria Enrichment Broth Mossel
have been found satisfactory for the purposes for which they Medium
are prescribed in the test for microbial contamination in the Gelatin peptone 10.0 g
Pharmacopoeia. Other media may be used provided that Glucose monohydrate 5.0 g
their suitability can be demonstrated. Bile salts 20.0 g
Potassium dihydrogen phosphate 2.0 g
(i) Phosphate Buffer (pH 7.2)
Disodium hydrogen phosphate dihydrate 8.0 g
Prepare a mixture of water and stock buffer solution (800:1
Brilliant green 15 mg
V/V ) and sterilize.
Water 1000 mL
Stock buffer solution: Transfer 34 g of potassium dihydro-
Adjust the pH so that after heating it is 7.0 – 7.4 at 259C.
gen phosphate to a 1000 mL volumetric flask, dissolve in 500
Heat at 1009 C for 30 min and cool immediately.
mL of water, adjust to pH 7.0 to 7.4 with sodium hydroxide
TS, add water to volume and mix. Dispense in containers (ix) VRB (Violet/Red/Bile) Agar with glucose
and sterilize. Store at a temperature of 2 – 89C. Yeast extract 3.0 g
Gelatin peptone 7.0 g
(ii) Buffered Sodium Chloride-Peptone Solution (pH
Bile salts 1.5 g
7.0)
Sodium chloride 5.0 g
Potassium dihydrogen phosphate 3.6 g
Glucose monohydrate 10.0 g
Disodium hydrogen phosphate dihydrate 7.2 g
Agar 15.0 g
(equivalent to 0.067 mol phosphate)
Neutral red 30 mg
Sodium chloride 4.3 g
Crystal violet 2 mg
Peptone (meat or casein) 1.0 g
Water 1000 mL
Water 1000 mL
Adjust the pH so that after heating it is 7.2 – 7.6 at 259C.
Sterilize in an autoclave using a validated cycle.
Heat to boiling; do not heat in autoclave.
(iii) Fluid Soybean-Casein Digest Medium
Casein peptone 17.0 g

5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside 0.1 g
4-Methylumbelliferyl-b-D-glucuronide 0.1 g 6. Tests for Preparations
Water 1000 mL
Adjust the pH so that after sterilization it is 6.9 – 7.3 at
259C. Sterilize in an autoclave using a validated cycle. 6.01 Test for Metal Particles in
Enzyme substrate media for Salmonella Ophthalmic Ointments
Use the media shown below as examples and that have
been validated their capabilities. Test of Metal Particles in Ophthalmic Ointments is a
method to test the existence of foreign metal particles in the
(xx) CHS Agar Medium
ophthalmic ointments described in General Rules for Prepa-
Peptone 5.0 g
rations.
Yeast extract 2.0 g
Sodium chloride 0.8 g 1. Preparation of test sample
Other salts 7.2 g The test should be carried out in a clean place. Take 10
A mixture of selecting agent and particular enzyme substrate ophthalmic ointments to be tested, and extrude 5 g each of
4.9 g their contents into separate flat-bottomed petri dishes 60 mm
Ager 15.0 g in diameter. Cover the dishes, and heat between 859C and
Water 1000 mL 1109C for 2 hours to dissolve bases completely. Allow the
Adjust the pH so that after heating it is 7.4 – 7.8 at 259C. samples to cool to room temperature without agitation to
Heat to boiling, cool to 509C and pour into Petri dishes. Do solidify the contents. When the amount of the content is less
not heat in autoclave. than 5 g, extrude the contents as completely as practicable,
and proceed in the same manner as described above.
(xxi) ESII Agar Medium
Peptone 10.0 g 2. Procedure
Yeast extract 1.0 g Invert each dish on the stage of a suitable microscope pre-
Sodium chloride 5.0 g viously adjusted to provide not less than 40 times magnifica-
Disodium hydrogen phosphate 1.0 g tions and equipped with an eyepiece micrometer disk. Each
Sodium thiosulfate 1.0 g dish is illuminated from above 459relative to the plane of
Sodium deoxycholate 1.0 g the dish. Examine the entire bottom of each dish for metal
D-Mannitol 15.0 g particles, and record the total number of particles, measur-
Neutral red 30 mg ing 50 mm or more in any dimension.
Synthetic enzyme substract 0.45 g Note: Use petri dishes with a clean bottom and free from
Novobiocin 0.02 g foams and scratches, and if possible, the walls are at right
Ager 15.0 g angles with the bottom.
Water 1000 mL
3. Evaluation
Adjust the pH so that after sterilization it is 6.9 – 7.3 at
The preparation complies with the test if the total number
259 C. Sterilize in an autoclave using a validated cycle. Cool
of metal particles of a size equal to or greater than 50 mm
to 509C and pour into Petri dishes.
found in 10 units tested, is not more than 50, and also the
(xxii) Amphotericin B TS: Dissolve 22.5 mg of am- number of dishes containing more than 8 particles is not
photericin B powder in 9 mL of sterile purified water. more than 1. If this requirement is not met, repeat the test
Amphotericin B powder: Amphotericin B that is added with a further 20 units in the same manner, and if the total
sodium deoxycholate, sterilized by g-ray. number of the particles found in the total of 30 units is not
more than 150, and also the number of dishes containing
(xxiii) Rose bengal TS: Dissolve 1 g of rose bengal in
more than 8 particles is not more than 3, the preparation
water to make 100 mL.
complies with the test.
(xxiv) TTC TS: Dissolve 0.8 g of 2,3,5-triphenyl-2H-
tetrazolium chloride in water to make 100 mL, divide into
small tubes, and sterilize in an autoclave using a validated
cycle. Store protected from light. 6.02 Uniformity of Dosage Units
Preparation method This test is harmonized with the European Pharmacopoeia
(i) Ager medium with TTC: Just prior to use, add 2.5 to and the U. S. Pharmacopeia. The parts of the text that are
5 mL of TTC TS per liter (20 to 40 mg/L) of sterile ager me- not harmonized are marked with symbols ( ).
dium and mix.
The term ``Uniformity of dosage unit'' is defined as the
(ii) Ager medium with amphotericin B: Just prior to use,
degree of uniformity in the amount of the drug substance
add 2 mL of amphotericin B TS per liter (5 mg/L) of ager
among dosage units. Therefore, the requirements of this
medium previously sterilized in an autoclave using a validat-
chapter apply to each drug substance being comprised in
ed cycle, and mix.
dosage units containing one or more drug substances, unless
(iii) Ager medium with rose bengal: Add 5 mL of rose
otherwise specified elsewhere in this Pharmacopoeia.
bengal TS per liter (50 mg/L) of agar medium, mix, and
To ensure the consistency of dosage units, each unit in a
sterilize in an autoclave using a validated cycle.
batch should have a drug substance content within a narrow
range around the label claim. Dosage units are defined as
dosage forms containing a single dose or a part of a dose of
a drug substance in each dosage unit. The Uniformity of
Dosage Units specification is not intended to apply to sus-
pensions, emulsions, or gels in unit-dose containers intended
for external, cutaneous administration.

Table 6.02-1 Application of Content Uniformity (CU) and Mass Variation (MV) Test for dosage forms
Dose and ratio of drug substance
Dosage Forms Type Sub-type
25 mg & 25z ＜25 mg or ＜25z

Tablets uncoated MV CU
coated Film MV CU

Others CU CU

Capsules hard MV CU
soft Sus., eml., gel CU CU

Solutions MV MV
Solids in single-dose Single component MV MV
packages (divided
forms, lyophilized
forms, et al.) Multiple components Solution freeze-dried MV MV
in final container
Others CU CU
(perfectly
Solutions MV MV
dissolved) enclosed in
unit-dose containers

Others CU CU

Sus.: suspension; eml.: emulsion;

Variable Definition Conditions Value

X̃ Mean of individual contents (x1, x2, ..., xn)
expressed as a percentage of the label claim

x1, x2, ..., xn Individual contents of the dosage units tested,

expressed as a percentage of the label claim
n Sample size (number of dosage units in a sample)

k Acceptability constant If n ＝ 10, then 2.4

If n ＝ 30, then 2.0
n
s Sample standard deviation
∑
i＝
(xi － X̃ )2
1
n－1

RSD Relative standard deviation (the sample standard 100s

deviation expressed as a percentage of the mean) X̃
M (case 1) Reference value If 98.5z ≦ X̃ ≦ 101.5z, M ＝ X̃
then (AV ＝ ks)

To be applied If X̃ º 98.5z, then M ＝ 98.5z

when T ≦ 101.5 (AV ＝ 98.5 － X̃ ＋ ks)
If X̃ À 101.5z, then M ＝ 101.5z
(AV ＝ X̃ －101.5 ＋ ks)

M (case 2) Reference value If 98.5z ≦ X̃ ≦ T, then M ＝ X̃

(AV ＝ ks)

To be applied If X º 98.5z, then M ＝ 98.5z

when T À 101.5 (AV ＝ 98.5 － X̃ ＋ ks)
If X̃ À T, then M ＝ Tz
(AV ＝ X̃ － T ＋ ks)

Acceptance General formula:

Value (AV) |M － X̃| ＋ ks
[Calculations are specified above for
the different cases.]

L1 Maximum allowed acceptance value L1 ＝ 15.0

unless otherwise specified.
L2 Maximum allowed range for deviation of each On the low side, no dosage L2 ＝ 25.0
dosage unit tested from the calculated value of M unit result can be less than unless otherwise specified.
0.75M while on the high
side, no dosage unit result
can be greater than 1.25M
(This is based on an L2
value of 25.0.)

T Target content per dosage unit at time of

manufacture, expressed as the percentage of the
label claim. Unless otherwise stated, T is
100.0z, or T is the manufacturer's approved
target content per dosage unit.

value. avoid uptake or loss of moisture. Weigh the individual

(ii) Hard Capsules: Accurately weigh 10 capsules indi- shells, and calculate the net contents. Calculate the drug
vidually, taking care to preserve the identity of each capsule. substance content in each capsule from the mass of product
Remove the contents of each capsule by suitable means. Ac- removed from the individual capsules and the result of the
curately weigh the emptied shells individually, and calculate assay. Calculate the acceptance value.
for each capsule the net mass of its contents by subtracting (iv) Solid dosage forms other than tablets and capsules:
the mass of the shell from the respective gross mass. Calcu- Proceed as directed for Hard Capsules, treating each dosage
late the drug substance content of each capsule from the unit as described therein. Calculate the acceptance value.
mass of the individual capsules and the result of the assay. (v) Liquid dosage forms: Accurately weigh the amount
Calculate the acceptance value. of liquid that is removed from each of 10 individual contain-
(iii) Soft Capsules: Accurately weigh the 10 intact cap- ers in conditions of normal use. If necessary, compute the
sules individually to obtain their gross masses, taking care to equivalent volume after determining the density. Calculate
preserve the identity of each capsule. Then cut open the cap- the drug substance content in each container from the mass
sules by means of a suitable clean, dry cutting instrument of product removed from the individual containers and the
such as scissors or a sharp open blade, and remove the con- result of the assay. Calculate the acceptance value.
tents by washing with a suitable solvent. Allow the occluded 2.1. Calculation of Acceptance Value
solvent to evaporate from the shells at room temperature Calculate the acceptance value as shown in Content
over a period of about 30 minutes, taking precautions to Uniformity, except that the value of X̃ is replaced with A,

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
150 6.03 Particle Size Distribution Test for Preparations / General Tests JP XVII
and that the individual contents of the dosage units are to Powders in the General Rules for Preparations: may be
replaced with the individual estimated contents defined used, without any treatment, as the sample. Preparations in
below. dose-unit packages: weigh accurately the content of not less
than 20 packages, calculate the average mass of the content
x1, x2 ... xn: individual estimated contents of the dosage
for a daily dose, mix uniformly, and use the mixture as the
units tested
sample. Granules in dose-unit packages and other solid
A preparations which do not conform to Powders in the
xi ＝ wi ×
W̃ General Rules for Preparations: weigh accurately the content
of not less than 20 packages, calculate the average mass of
w1, w2 ... wn: Individual masses of the dosage units tested
the content for a daily dose, powder it, and use as the sam-
A: Content of drug substance (z of label claim) obtained
ple. Granules not in dose-unit packages and other solid
using an appropriate analytical method
preparations which do not conform to Powders in the
W̃: Mean of individual masses (w1, w2 ..., wn)
General Rules for Preparations: take not less than 20 doses,
3. Criteria powder it, and use as the sample. Capsules and tablets: take
Apply the following criteria, unless otherwise specified. not less than 20 doses, weigh accurately, calculate the
(i) Solid, Semi-Solid and Liquid dosage forms: The re- average mass for a daily dose, powder it, and use as the sam-
quirements for dosage uniformity are met if the acceptance ple. Liquid preparations: shake well, and use as the sample.
value of the first 10 dosage units is less than or equal to L1
2. Procedure
z. If the acceptance value is greater than L1z, test the next
Take an amount of the sample so that `a' in the equation
20 dosage units and calculate the acceptance value. The re-
falls between 20 mL and 30 mL, and perform the test.
quirements are met if the final acceptance value of the 30
Accurately weigh the sample of the crude material or
dosage units is less than or equal to L1z and no individual
preparation, and place it in a glass-stoppered, 200-mL flask.
content of the dosage unit is less than (1 － L2 × 0.01)M nor
Add exactly 100 mL of 0.1 mol/L hydrochloric acid VS,
more than (1 ＋ L2 × 0.01)M in Calculation of Acceptance
stopper tightly, shake at 37 ± 29C for 1 hour, and filter.
Value under Content Uniformity or under Mass Variation.
Take precaution against gas to be generated on the addition
Unless otherwise specified, L1 is 15.0 and L2 is 25.0.
of 0.1 mol/L hydrochloric acid VS, and stopper tightly. Af-
ter cooling, filter the solution again, if necessary. Pipet 50 mL
of the filtrate, and titrate <2.50> the excess hydrochloric acid
6.03 Particle Size Distribution with 0.1 mol/L sodium hydroxide VS (pH Determination
<2.54>, end point: pH 3.5). Perform a blank determination.
Test for Preparations For liquid preparations, pipet the sample in a 100-mL
volumetric flask, add water to make 45 mL, then add exactly
Particle Size Distribution Test for Preparations is a
50 mL of 0.1 mol/L hydrochloric acid VS while shaking.
method to determine the particle size distribution of prepara-
Add water again to make the solution 100 mL. Transfer the
tions described in General Rules for Preparations.
solution to a glass-stoppered, 200-mL flask, wash the residue
1. Procedure with 20.0 mL of water, stopper tightly, shake at 37 ± 29C
The test is performed employing No. 18 (850 mm) and No. for 1 hour, and filter. Pipet 60 mL of the filtrate, and titrate
30 (500 mm) sieves with the inside diameter of 75 mm. <2.50> the excess hydrochloric acid with 0.1 mol/L sodium
Weigh accurately 10.0 g of sample to be tested, and place hydroxide VS (pH Determination <2.54>, end point: pH 3.5).
on the uppermost sieve which is placed on the other sieves Perform a blank determination.
described above and a close-fitting receiving pan and is co-
Acid-neutralizing capacity (amount of 0.1 mol/L hydrochlo-
vered with a lid. Shake the sieves in a horizontal direction
ric acid VS consumed per g or daily dose) (mL)
for 3 minutes, and tap slightly at intervals. Weigh the
＝ (b － a) f × 2 × (t/s)
amount remaining on each sieve and in the receiving pan.
a: Amount (mL) of 0.1 mol/L sodium hydroxide VS con-
sumed
b: Amount (mL) of 0.1 mol/L sodium hydroxide VS con-
6.04 Test for Acid-neutralizing sumed in the blank determination
Capacity of Gastrointestinal f: The molarity coefficient of 0.1 mol/L sodium hydrox-
ide VS
Medicines t: 1000 mg of crude material or daily dose of preparation
(in mg of solid preparation, mL of liquid preparation)
Test for Acid-neutralizing Capacity of Gastrointestinal
s: Amount of the sample (in mg of crude material and
Medicines is a test to determine the acid-neutralizing capaci-
solid preparation, mL of liquid preparation)
ty of a medicine, as a crude material or preparation, which
reacts with the stomach acid and exercises an acid control ac-
tion in the stomach. When performing the test according to
the following procedure, the acid-neutralizing capacity of a 6.05 Test for Extractable Volume
crude material is expressed in terms of the amount (mL) of
0.1 mol/L hydrochloric acid VS consumed per g of the mate- of Parenteral Preparations
rial, and that of a preparation is expressed by the amount
This test is harmonized with the European Pharmacopoeia
(mL) of 0.1 mol/L hydrochloric acid VS consumed per dose
and the U. S. Pharmacopeia. The parts of the text that are
per day (when the daily dose varies, the minimum dose is
not harmonized are marked with symbols ( ).
used).

Test for Extractable Volume of Parenteral Preparations
1. Preparation of sample
is performed to confirm that a slightly excess volume is filled
A crude material and a solid preparation which conforms
for the nominal volume to be withdrawn. Injections may be

supplied in single-dose containers such as ampoules or plas-

tic bags, or in multi-dose containers filled with a volume of 6.06 Foreign Insoluble Matter
injection which is sufficient to permit administration of the
nominal volume declared on the label. The excess volume is Test for Injections
determined by the characteristics of the product.
Foreign Insoluble Matter Test for Injections is a test
Suspensions and emulsions must be shaken before with-
method to examine foreign insoluble matters in injections.
drawal of the contents and before the determination of the
density. Oily and viscous preparations may be warmed ac- 1. Method 1.
cording to the instructions on the label, if necessary, and This method is applied to either injections in solution, sus-
thoroughly shaken immediately before removing the con- pension or emulsion, and vehicles for solid injections to be
tents. The contents are then cooled to 20 – 259C before dissolved or suspended before use.
measuring the volume. Clean the exterior of containers, and inspect against both
a white and a black background for 5 seconds each time with
1. Single-dose containers
the unaided eyes at a position of light intensity of 2000 to
Select one container if the volume is 10 mL or more, 3
3750 lx under a white light source: Injections or vehicles
containers if the nominal volume is more than 3 mL and less
must be free from readily detectable foreign insoluble mat-
than 10 mL, or 5 containers if the nominal volume is 3 mL
ters. As to Injections in plastic containers for aqueous injec-
or less. Take up individually the total contents of each con-
tions, the inspection should be performed with the unaided
tainer selected into a dry syringe of a capacity not exceeding
eyes at a position of light intensity of approximately 8000 to
three times the volume to be measured, and fitted with a 21-
10,000 lx, with a white light source at appropriate distances
gage needle not less than 2.5 cm in length. Expel any air bub-
above and below the container. The inspection time should
bles from the syringe and needle, then discharge the contents
be extended accordingly if the inspection is not easy.
of the syringe without emptying the needle into a stand-
ardised dry cylinder (graduated to contain rather than to 2. Method 2.
deliver the designated volumes) of such size that the volume This method is applied to solid injections to be dissolved
to be measured occupies at least 40z of its graduated or suspended before use.
volume. Alternatively, the volume of the contents in mil- Clean the exterior of containers, and dissolve or suspend
liliters may be calculated as the mass in grams divided by the the contents with vehicles attached to the preparations or
density. with Water for Injection carefully, avoiding any contamina-
For containers with a nominal volume of 2 mL or less the tion with extraneous foreign substances. The solution thus
contents of a sufficient number of containers may be pooled constituted must be free from foreign insoluble matters that
to obtain the volume required for the measurement provided is clearly detectable when inspected against both a white and
that a separate, dry syringe assembly is used for each con- a black background for 5 seconds each time with the unaided
tainer. The contents of containers holding 10 mL or more eyes at a position of light intensity of 2000 to 3750 lx under a
may be determined by opening them and emptying the con- white light source. The inspection time should be extended
tents directly into the graduated cylinder or tared beaker. accordingly if the inspection is not easy.
The volume is not less than the nominal volume in case of
containers examined individually, or, in case of containers
with a nominal volume of 2 mL or less, is not less than the
sum of the nominal volumes of the containers taken collec- 6.07 Insoluble Particulate Matter
tively. Test for Injections
2. Multi-dose containers
This test is harmonized with the European Pharmacopoeia
For injections in multidose containers labeled to yield a
and the U. S. Pharmacopeia. The parts of the text that are
specific number of doses of a stated volume, select one con-
not harmonized are marked with symbols ( ).
tainer and proceed as directed for single-dose containers
using the same number of separate syringe assemblies as the Insoluble particulate matters in injections and parenteral
number of doses specified. The volume is such that each infusions consist of extraneous, mobile undissolved parti-
syringe delivers not less than the stated dose. cles, other than gas bubbles, that are unintentionally present
in the solutions.
3. Cartridges and pre-filled syringes
For the determination of particulate contamination, 2
Select one container if the volume is 10 mL or more, 3
procedures, Method 1 (Light Obscuration Particle Count
containers if the nominal volume is more than 3 mL and less
Test) and Method 2 (Microscopic Particle Count Test), are
than 10 mL, or 5 containers if the nominal volume is 3 mL
specified hereinafter. When examining injections and paren-
or less. If necessary, fit the containers with the accessories
teral infusions for sub-visible particles, Method 1 is prefera-
required for their use (needle, piston, syringe) and transfer
bly applied. However, it may be necessary to test some
the entire contents of each container without emptying the
preparations by Method 1 followed by Method 2 to reach a
needle into a dry tared beaker by slowly and constantly
conclusion on conformance to the requirements.
depressing the piston. Determine the volume in milliliters
Not all parenteral preparations can be examined for sub-
calculated as the mass in grams divided by the density.
visible particles by one or both of these methods. When
The volume measured for each of the containers is not less
Method 1 is not applicable, e.g. in case of preparations hav-
than the nominal volume.
ing reduced clarity or increased viscosity, the test should be
4. Parenteral infusions carried out according to Method 2. Emulsions, colloids, and
Select one container. Transfer the contents into a dry liposomal preparations are examples. Similarly, products
measuring cylinder of such a capacity that the volume to be that produce air or gas bubbles when drawn into the sensor
determined occupies at least 40z of the nominal volume of may also require microscopic particle count testing. If the
the cylinder. Measure the volume transferred. viscosity of the preparation to be tested is sufficiently high so
The volume is not less than the nominal volume. as to preclude its examination by either test method, a quan-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
152 6.07 Insoluble Particulate Matter Test for Injections / General Tests JP XVII
titative dilution with an appropriate diluent may be made to 1.1.4. Sensor
decrease viscosity, as necessary, to allow the analysis to be There is a possibility of changes of particle size resolution
performed. and counting rate of particle-detecting sensor in each sensor
The results obtained in examining a discrete unit or group by assembling accuracy and parts accuracy even in the same
of units for particulate contamination cannot be extrapo- type sensor. The threshold accuracy also needs to be con-
lated with certainty to other units that remain untested. firmed. Testing should accordingly be performed for each of
Thus, statistically sound sampling plans must be developed if particle size resolution, accuracy in counting and in
valid inferences are to be drawn from observed data to threshold setting, using Particle Count Reference Standard
characterise the level of particulate contamination in a large Suspension (PSL spheres having mean diameter of approxi-
group of units. mately 10 mm, of a concentration at 1000 particles/mL
±10z, not more than 5z of CV value).
1. Method 1. Light Obscuration Particle Count Test
During measurement, stirring should be made for assuring
1.1. Apparatus
the uniformity in sample concentration.
Use a suitable apparatus based on the principle of light
1.1.4.1. Sensor resolution (Particle size resolution of appa-
blockage which allows an automatic determination of the
ratus)
size of particles and the number of particles according to
Measurement should be made by either one of the follow-
size. It is necessary to perform calibration, as well as to
ing methods. The difference between the threshold of par-
demonstrate the sample volume accuracy, sample flow rate,
ticle size counting 16z and 84z of the total counts and the
particle size response curve, sensor resolution, and counting
test-particle size should be within 10z, whereas, electronic
accuracy, at least once a year.
 method and automated method should be both validated for
1.1.1. Calibration
obtaining the same result as that of the manual method.
Particles to be used for calibration should be subject to
(i) Manual method to obtain the spread of histogram
particle size sensitivity measurement, using spherical polysty-
prepared from the counting value of the apparatus.
rene particles having at least 5, 10 and 25 mm in diameter
(ii) Electronic method to obtain the spread of histogram
(PSL particles) in mono-dispersed suspension. The PSL par-
of the classification of system-responding signal by using the
ticles should have either a domestic or international tracea-
multichannel peak height analyzer.
bility in terms of length, with a level of uncertainly at not
(iii) Automated method to obtain the spread of histo-
greater than 3z. The particles to be used for calibration
gram of responsive signal of the test-particle by using the
should be dispersed in particle-free water.
software prepared by the manufacturer or the user.
1.1.1.1. Manual method
1.1.4.2. Particle counting accuracy
The particle size response of the system to be applied
Data obtained by counting particles of 5 mm and greater
should be determined using at least 3 channels for threshold-
should be 763 to 1155 particles per mL.
voltage setting, according to the half counting method of
1.1.4.3. Threshold accuracy
window moving type. The threshold-voltage window should
Particle size calculated from a threshold corresponding to
be ±20z of the measuring particle size. After measuring the
50z counts for particles of 5 mm and greater should fall
sensitivity of response for the designated particle size, the
within ±5z of the mean diameter of the test particles.
size response curve is prepared by the method indicated by
1.2. General precautions
the manufacturer from particle-response measuring point,
The test is carried out under conditions limiting particulate
and threshold-voltage of 5, 10 and 25 mm of the apparatus is
contamination, preferably in a laminar-flow cabinet.
obtained.
Very carefully wash the glassware and filtration equip-
1.1.1.2. Electronic method
ment used, except for the membrane filters, with a warm de-
In the use of multichannel peak height analyzer, the parti-
tergent solution and rinse with abundant amounts of water
cle size response is measured by half-count method of mov-
to remove all traces of detergent. Immediately before use,
ing window system same as the manual method, and the
rinse the equipment from top to bottom, outside and then in-
particle size response curve is prepared by the method desig-
side, with particle-free water.
nated by the instrument manufacturer, then, the threshold-
Take care not to introduce air bubbles into the prepara-
voltage of 5, 10 and 25 mm of the apparatus is obtained. In
tion to be examined, especially when fractions of the prepa-
this case, the instrument manufacturer or the user should
ration are being transferred to the container in which the de-
validate the obtainability of the same result as that of the
termination is to be carried out.
manual method.
In order to check that the environment is suitable for the
1.1.1.3. Automated method
test, that the glassware is properly cleaned and that the water
The particle size response curve of the apparatus may be
to be used is particle-free, the following test is carried out:
obtained by using the software developed by the user or
determine the particulate contamination of 5 samples of par-
supplied by the instrument manufacturer, whereas the manu-
ticle-free water, each of 5 mL, according to the method
facturer or the user should validate the obtainability of the
described below. If the number of particles of 10 mm or
same result as that of the manual method.
greater size exceeds 25 for the combined 25 mL, the precau-
1.1.2. Sample volume accuracy
tions taken for the test are not sufficient. The preparatory
Sample volume accuracy should fall within 5z of the
steps must be repeated until the environment, glassware and
measuring value in case the decrease of test solution is
water are suitable for the test.
measured by the mass method after measuring the test solu-
1.3. Method
tion of 10 mL.
Mix the contents of the sample by slowly inverting the
1.1.3. Sample flow rate
container 20 times successively. If necessary, cautiously re-
The flow rate of the sample indicated into the sensor
move the sealing closure. Clean the outer surfaces of the
should be calculated from the observed sample volume and
container opening using a jet of particle-free water and re-
time, and should be conformed within the range of the
move the closure, avoiding any contamination of the con-
manufacturer's specification for sensor used.
tents. Eliminate gas bubbles by appropriate measures such as
allowing to stand for 2 minutes or sonicating.

For large-volume parenterals or for small-volume paren-

terals having a volume of 25 mL or more, single units are
tested. For small-volume parenterals less than 25 mL in
volume, the contents of 10 or more units are combined in a
cleaned container to obtain a volume of not less than 25 mL;
where justified and authorised, the test solution may be pre-
pared by mixing the contents of a suitable number of vials
and diluting to 25 mL with particle-free water or with an ap-
propriate solvent without contamination of particles when
particle-free water is not suitable.
Powders for parenteral use are reconstituted with particle-
free water or with an appropriate solvent without contami-
nation of particles when particle-free water is not suitable.
The number of test specimens must be adequate to provide
a statistically sound assessment. For large-volume parenter-
als or for small-volume parenterals having a volume of 25
mL or more, fewer than 10 units may be tested, based on an
appropriate sampling plan.
Fig. 6.07-1 Circular diameter graticule
Remove 4 portions, each of not less than 5 mL, and count
the number of particles equal to or greater than 10 mm and
25 mm. Disregard the result obtained for the first portion,
and calculate the mean number of particles for the prepara-
tion to be examined. membrane filter.
1.4. Evaluation The membrane filter is of suitable size, black or dark gray
If the average number of particles exceeds the limits, test in color, non-gridded or gridded, and 1.0 mm or finer in
the preparation by Method 2 (Microscopic Particle Count nominal pore size.
Test). 2.2. General precautions
Test 1.A—Solutions for injection supplied in containers with The test is carried out under conditions limiting particulate
a nominal content of equal to or more than 100 mL contamination, preferably in a laminar-flow cabinet.
The preparation complies with the test if the average number Very carefully wash the glassware and filter assembly
of particles present in the units tested does not exceed 25 per used, except for the membrane filter, with a warm detergent
milliliter equal to or greater than 10 mm and does not exceed solution and rinse with abundant amounts of water to re-
3 per milliliter equal to or greater than 25 mm. move all traces of detergent. Immediately before use, rinse
Test 1.B—Solutions for injection supplied in containers with both sides of the membrane filter and the equipment from
a nominal content of less than 100 mL top to bottom, outside and then inside, with particle-free
The preparation complies with the test if the average number water.
of particles present in the units tested does not exceed 6000 In order to check that the environment is suitable for the
per container equal to or greater than 10 mm and does not test, that the glassware and the membrane filter are properly
exceed 600 per container equal to or greater than 25 mm. cleaned and that the water to be used is particle-free, the fol-
lowing test is carried out: determine the particulate contami-
2. Method 2. Microscopic Particle Count Test
nation of a 50 mL volume of particle-free water according to
2.1. Apparatus
the method described below. If more than 20 particles 10 mm
Use a suitable binocular microscope, filter assembly for
or larger in size or if more than 5 particles 25 mm or larger in
retaining particulate contamination and membrane filter for
size are present within the filtration area, the precautions
examination.
taken for the test are not sufficient. The preparatory steps
The microscope is equipped with an ocular micrometer
must be repeated until the environment, glassware, mem-
calibrated with an objective micrometer, a mechanical stage
brane filter and water are suitable for the test.
capable of holding and traversing the entire filtration area of
2.3. Method
the membrane filter, two suitable illuminators to provide
Mix the contents of the samples by slowly inverting the
episcopic illumination in addition to oblique illumination,
container 20 times successively. If necessary, cautiously re-
and is adjusted to 100 ± 10 magnifications. The ocular
move the sealing closure. Clean the outer surfaces of the
micrometer is a circular diameter graticule (see Fig. 6.07-1)
container opening using a jet of particle-free water and re-
and consists of a large circle divided by crosshairs into quad-
move the closure, avoiding any contamination of the con-
rants, transparent and black reference circles 10 mm and 25
tents.
mm in diameter at 100 magnifications, and a linear scale
For large-volume parenterals, single units are tested. For
graduated in 10 mm increments. It is calibrated using a stage
small-volume parenterals less than 25 mL in volume, the
micrometer that is certified by either a domestic or interna-
contents of 10 or more units is combined in a cleaned con-
tional standard institution. A relative error of the linear scale
tainer; where justified and authorised, the test solution may
of the graticule within ± 2z is acceptable. The large circle is
be prepared by mixing the contents of a suitable number of
designated the graticule field of view (GFOV).
vials and diluting to 25 mL with particle-free water or with
Two illuminators are required. One is an episcopic bright-
an appropriate solvent without contamination of particles
field illuminator internal to the microscope, the other is an
when particle-free water is not suitable. Small-volume paren-
external, focussable auxiliary illuminator adjustable to give
terals having a volume of 25 mL or more may be tested indi-
reflected oblique illumination at an angle of 109to 209.
vidually.
The filter assembly for retaining particulate contamination
Powders for parenteral use are constituted with particle-
consists of a filter holder made of glass or other suitable ma-
free water or with an appropriate solvent without contami-
terial, and is equipped with a vacuum source and a suitable
nation of particles when particle-free water is not suitable.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
154 6.08 Insoluble Particulate Matter Test for Ophthalmic Solutions / General Tests JP XVII
The number of test specimens must be adequate to provide
a statistically sound assessment. For large-volume parenter- 6.08 Insoluble Particulate Matter
als or for small-volume parenterals having a volume of 25
mL or more, fewer than 10 units may be tested, based on an Test for Ophthalmic Solutions
appropriate sampling plan.
Insoluble Particulate Matter Test for Ophthalmic Solu-
Wet the inside of the filter holder fitted with the mem-
tions is to examine for the size and the number of insoluble
brane filter with several milliliter of particle-free water.
particulate matter in Ophthalmic Solutions.
Transfer to the filtration funnel the total volume of a solu-
tion pool or of a single unit, and apply vacuum. If needed 1. Apparatus
add stepwise a portion of the solution until the entire volume Use a microscope, filter assembly for retaining insoluble
is filtered. After the last addition of solution, begin rinsing particulate matter and membrane filter for determination.
the inner walls of the filter holder by using a jet of particle- (i) Microscope: The microscope is equipped with a
free water. Maintain the vacuum until the surface of the micrometer system, a mobile stage and an illuminator, and is
membrane filter is free from liquid. Place the filter in a petri adjusted to 100 magnifications.
dish and allow the filter to air-dry with the cover slightly (ii) Filter assembly for retaining insoluble particulate
ajar. After the filter has been dried, place the petri dish on matter: The filter assembly for retaining insoluble particu-
the stage of the microscope, scan the entire membrane filter late matter consists of a filter holder made of glass or a
under the reflected light from the illuminating device, and proper material uncapable of causing any trouble in testing,
count the number of particles that are equal to or greater and a clip. The unit is capable of fitting with a membrane
than 10 mm and the number of particles that are equal to or filter 25 mm or 13 mm in diameter and can be used under
greater than 25 mm. Alternatively, partial filter count and reduced pressure.
determination of the total filter count by calculation is (iii) Membrane filter for testing: The membrane filter is
allowed. Calculate the mean number of particles for the white in color, 25 mm or 13 mm in diameter, not more than
preparation to be examined. 10 mm in nominal pore size and is imprinted with about 3
The particle sizing process with the use of the circular di- mm grid marks. Upon preliminary testing, the insoluble par-
ameter graticule is carried out by transforming mentally the ticulate matter equal to or greater than 25 mm in size should
image of each particle into a circle and then comparing it to not be found on the filter. When necessary, wash the filter
the 10 mm and 25 mm graticule reference circles. Thereby the with water for particulate matter test.
particles are not moved from their initial locations within the
2. Reagents
graticule field of view and are not superimposed on the refer-
(i) Water for particulate matter test: Water prepared be-
ence circles for comparison. The inner diameter of the trans-
fore use by filtering through a membrane filter with a pore
parent graticule reference circles is used to size white and
size not exceeding 0.45 mm. It contains not more than 10 par-
transparent particles, while dark particles are sized by using
ticles of 10 mm or grater size in 100 mL.
the outer diameter of the black opaque graticule reference
circles. 3. Procedure
In performing the microscopic particle count test (Method 3.1. Aqueous ophthalmic solutions
2) do not attempt to size or enumerate amorphous, semi- Carry out all operations carefully in clean equipment and
liquid, or otherwise morphologically indistinct materials that facilities which are low in dust. Fit the membrane filter onto
have the appearance of a stain or discoloration on the mem- the membrane filter holder, and fix them with the clip.
brane filter. These materials show little or no surface relief Thoroughly rinse the holder inside with water for particulate
and present a gelatinous or film-like appearance. In such matter test, and filter under reduced pressure with 200 mL of
cases the interpretation of enumeration may be aided by test- water for particulate matter test at a rate of 20 to 30 mL per
ing a sample of the solution by Method 1. minute. Apply the vacuum until the surface of the mem-
2.4. Evaluation brane filter is free from water, and remove the membrane
Test 2.A—Solutions for injection supplied in containers with filter. Place the filter in a flat-bottomed petri dish with the
a nominal content of equal to or more than 100 mL cover slightly ajar, and dry the filter fully at a temperature
The preparation complies with the test if the average number not exceeding 509 C. After the filter has been dried, place the
of particles present in the units tested does not exceed 12 per petri dish on the stage of the microscope. Under a down-
milliliter equal to or greater than 10 mm and does not exceed light from an illuminating device, adjust the grid of the
2 per milliliter equal to or greater than 25 mm. membrane filter to the coordinate axes of the microscope,
Test 2.B—Solutions for injection supplied in containers with adjust the microscope so as to get the best view of the insolu-
a nominal content of less than 100 mL ble particulate matter, then count the number of particles
The preparation complies with the test if the average number that are equal to or greater than 150 mm within the effective
of particles present in the units tested does not exceed 3000 filtering area of the filter, moving the mobile stage, and
per container equal to or greater than 10 mm and does not ascertain that the number is not more than 1. In this case the
exceed 300 per container equal to or greater than 25 mm. particle is sized on the longest axis.
3. Fit another membrane filter to the filtration device, and
Reagents
fix them with the clip, then wet the inside of the filter holder
Particle-free water: The filtered water through a mem-
with several mL of water for particulate matter test. Clean
brane filter with a pore size not exceeding 0.45 mm, contain-
the outer surface of the container, and mix the sample solu-
ing not more than 5 particles of 10 mm or greater size, and
tion gently by inverting the container several times. Remove
not more than 2 particles of 25 mm or greater size in 10 mL
the cap, clean the outer surface of the nozzle, and pour the
of the insoluble particle number measured by the light ob-
sample solution into a measuring cylinder which has been
scuration particle counter.
rinsed well with water for particulate matter test. Repeat the
process to prepare 25 mL of the test solution. Pour the test
solution into the filter holder along the inner wall of the hol-
der. Apply the vacuum and filter mildly so as to keep the so-

lution always on the filter. As for viscous sample solution, bly become submerged. The time required for the upward
dilute suitably with water for particulate matter test or suita- stroke is equal to the time required for the downward stroke,
ble diluent and then filter as described above. When the and the change in stroke direction is a smooth transition,
amount of the solution on the filter becomes small, add 30 rather than an abrupt reversal of motion. The basket-rack
mL of water for particulate matter test or suitable diluent in assembly moves vertically along its axis. There is no appreci-
such manner as to wash the inner wall of the filter holder. able horizontal motion or movement of the axis from the
Repeat the process 3 times with 30 mL of the water. Apply vertical.
the vacuum gently until the surface of the membrane filter is (i) Basket-rack assembly: The basket-rack assembly con-
free from water. Place the filter in a petri dish, and dry the sists of six open-ended transparent tubes, each 77.5 ± 2.5
filter at a temperature below 509C with the cover slightly mm long and having an inside diameter of 20.7 to 23 mm
ajar. After the filter has been dried, place the petri dish on and a wall 1.0 to 2.8 mm thick; the tubes are held in a verti-
the stage of the microscope, and count the number of parti- cal position by two plates, each 88 to 92 mm in diameter and
cles which are equal to or larger than 300 mm within the ef- 5 to 8.5 mm in thickness, with six holes, each 22 to 26 mm in
fective filtering area of the filter according to the same diameter, equidistant from the center of the plate and
procedure of the microscope as described above. In this case equally spaced from one another. Attached to the under sur-
the particle is sized on the longest axis. face of the lower plate is a woven stainless steel wire cloth,
3.2. Ophthalmic solutions which are dissolved before use which has a plain square weave with 1.8- to 2.2-mm aper-
Proceed as directed in Aqueous Ophthalmic Solutions tures and with a wire diameter of 0.57 to 0.66 mm. The parts
after dissolving the sample with the constituted solution. of the apparatus are assembled and rigidly held by means of
3.3. Suspension type ophthalmic solutions three bolts passing through the two plates. A suitable means
Proceed as directed in Aqueous Ophthalmic Solutions. is provided to suspend the basket-rack assembly from the
Take 25 mL of the sample in a vessel, which has been rinsed raising and lowering device using a point on its axis. The
well with water for particulate matter test, add a suitable basket-rack assembly conforms to the dimensions found in
amount of a suspension-solubilizing solvent or an adequate Fig. 6.09-1. The design of the basket-rack assembly may be
solvent, shake to dissolve the suspending particles, and use varied somewhat provided the specifications for the glass
this solution as the sample solution. Use a membrane filter tubes and the screen mesh size are maintained: for exam-
which is not affected by the solvent to be used. ple, in order to secure the glass tubes and the upper and the
3.4. Ophthalmic solutions contained in a single-dose con- lower plastic plates in position at the top or the bottom, an
tainer acid-resistant metal plate, 88 – 92 mm in diameter and 0.5 –
Proceed as directed in Aqueous Ophthalmic Solutions, 1 mm in thickness, having 6 perforations, each about 22 to
using 10 samples for the test. A 13-mm diameter membrane 26 mm in diameter, may be used which coincide with those
filter and a 4-mm diameter filter holder for retaining insolu- of the upper plastic plate and upper open ends of the glass
ble particulate matter are used. tubes.
(ii) Disks: The use of disks is permitted only where speci-
4. Evaluation
fied or allowed. Each tube is provided with a cylindrical disk
The preparation complies with the test if the calculated
9.5 ± 0.15 mm thick and 20.7 ± 0.15 mm in diameter. The
number per mL of insoluble particles of a size equal to or
disk is made of a suitable, transparent plastic material hav-
greater than 300 mm is not more than 1.
ing a specific gravity of between 1.18 and 1.20. Five parallel
2 ± 0.1 mm holes extend between the ends of the cylinder.
One of the holes is centered on the cylindrical axis. The other
6.09 Disintegration Test holes are centered 6 ± 0.2 mm from the axis on imaginary
lines perpendicular to the axis and parallel to each other.
This test is harmonized with the European Pharmacopoeia Four identical trapezoidal-shaped planes are cut into the wall
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols ( ).
Disintegration Test is provided to determine whether
tablets, capsules, granules, dry syrups or pills disintegrate
within the prescribed time when placed in a liquid medium at
the experimental conditions presented below.
For the purposes of this test, disintegration does not imply
complete solution of the unit or even of its active con-
stituent.
1. Apparatus
The apparatus consists of a basket-rack assembly, a
1000-mL, low-form beaker, 138 to 160 mm in height and
having an inside diameter of 97 to 115 mm for the immersion
fluid, a thermostatic arrangement for heating the fluid be-
tween 359and 399 , and a device for raising and lowering the
basket in the immersion fluid at a constant frequency rate
between 29 and 32 cycles per minute through a distance of
not less than 53 mm and not more than 57 mm. The volume
of the fluid in the vessel is such that at the highest point of
the upward stroke the wire mesh remains at least 15 mm
below the surface of the fluid and descends to not less than
25 mm from the bottom of the vessel on the downward
stroke. At no time should the top of the basket-rack assem- Fig. 6.09-1 Disintegration apparatus

mm, and its center lies at a depth of 2.6 ± 0.1 mm from the minutes in the same manner, using 1st fluid for disintegra-
cylinder's circumference. All surfaces of the disk are tion test as the immersion fluid. When any residue of the
smooth. If the use of disks is specified, add a disk to each unit is observed, carry out the test successively for 60
tube, and operate the apparatus as directed under Proce- minutes, using 2nd fluid for disintegration test.
dure. The disks conform to dimensions found in Fig. 6.09-1. In case of granules and dry syrups, shake preparations

The use of automatic detection employing modified disks is on a No. 30 (500 mm) sieve as directed in Particle Size Distri-
permitted where the use of disks is specified or allowed. Such bution Test for Preparations <6.03>, transfer 0.10 g of the
disks must comply with the requirements for density and residue on the sieve to each of the 6 auxiliary tubes, secure
dimension given in this chapter. the 6 tubes to the bottom of the basket tightly, and operate
(iii) Auxiliary tube: The auxiliary tube, as illustrated in the apparatus, using water as the immersion fluid, main-
Fig. 6.09-2, consists of a plastic tube D, 12 ± 0.2 mm in in- tained at 37 ± 29 C as the immesion fluid, unless otherwise
side diameter, 17 ± 0.2 mm in outside diameter, 20 ± 1 mm specified. Observe the samples after 30 minutes of operation
in length, having both outside ends screw-cut, and two plas- for plain granules and after 60 minutes for coated granules,
tic rings A, each 12 ± 0.2 mm in inside diameter, 17 ± 0.2 unless otherwise specified. Complete disintegration is de-
mm in outside diameter, 2.5 – 4.5 mm in length, having one fined as that state in which any residue of the granules, ex-
inside end screw-cut. Acid-resistant woven wire gauze having cept fragments of insoluble coating in the auxiliary tube, is a
0.42-mm openings and 0.29-mm wire diameter is placed in soft mass having no palpably firm core. The test is met if all
each plastic ring and the rings are attached by screws to each of 6 samples in the auxiliary tubes have disintegrated com-
end of the plastic tube. The distance between two wire pletely. If 1 or 2 samples fail to disintegrate, repeat the test
gauzes is 20 ± 1 mm. A handle of an acid-resistant wire, 1 on 12 additional samples. The test is met if not less than 16
mm in diameter and 80 ± 5 mm in length, is attached to the of the total of 18 samples tested are disintegrated.
mid portion of the plastic tube. The auxiliary tube is used for 2.2. Enteric coated preparations
the test of granules and capsules containing enteric coated Unless otherwise specified, perform the following two
granules. tests, (a) the test with 1st fluid for disintegration test and (b)
the test with the 2nd fluid for disintegration test, separately.
2. Procedure
2.2.1. Enteric coated tablets and capsules
2.1. Immediate-release preparations
(i) The test with 1st fluid for disintegration test: Carry
In case of tablets, capsules and pills (except for pills con-
out the test for 120 minutes, using 1st fluid for disintegration
taining crude drugs), place 1 dosage unit in each of the six
test according to the procedure described in immediate
tubes of the basket, and if prescribed add a disk. Unless
release preparations. Disintegration is defined as that state in
otherwise specified, operate the apparatus, using water as
which the tablet or capsule is broken or the enteric coating
the immersion fluid, maintained at 37 ± 29 C as the immer-
film is ruptured or broken. The test is met if none of six
sion fluid. Unless otherwise specified, carry out the test for
dosage units is disintegrated. If 1 or 2 dosage units are disin-
20 minutes for capsules, 30 minutes for plain tablets, and 60
tegrated, repeat the test on additional 12 dosage units. The
minutes for coated tablets and pills. Lift the basket from
test is met if not less than 16 of the total of 18 dosage units
the fluid, and observe the dosage units. Complete disinte-
tested are not disintegrated.
gration is defined as that state in which any residue of the
(ii) The test with 2nd fluid for disintegration test: Ac-
cording to the procedure described in immediate-release
preparations, carry out the test with new dosage units for 60
minutes, using 2nd fluid for disintegration test and deter-
mine if the test is met or not.
2.2.2. Enteric coated granules and capsules containing the
enteric coated granules
Shake granules or contents taken out from capsules on a
No. 30 (500 mm) sieve as directed in Particle Size Distribu-
tion Test for Preparations <6.03>, transfer 0.10 g of the
residue on the sieve to each of the 6 auxiliary tubes, secure
the 6 tubes to the bottom of the basket tightly, and operate
the apparatus, using the 1st and 2nd fluids for disintegration
test.
(i) The test with 1st fluid for disintegration test: Accord-
ing to the procedure described in immediate-release prepara-
tions, carry out the test for 60 minutes, using 1st fluid for
disintegration test. The test is met if particles fallen from the
openings of the wire gauze number not more than 15.
(ii) The test with 2nd fluid for disintegration test: Ac-
cording to the procedure described in immediate-release
Fig. 6.09-2 Auxiliary tube preparations, carry out the test with new samples for 30

minutes, using 2nd fluid for disintegration test and deter-

mine if test is met or not.

6.10 Dissolution Test

This test is harmonized with the European Pharmacopoeia
and the U. S. Pharmacopeia. The parts of the text that are
not harmonized are marked with symbols ( ).
Dissolution Test is provided to determine compliance with
the dissolution requirements for dosage forms administered
orally. This test also aims at preventing significant bioine-
quivalence. In this test, a dosage unit is defined as 1 tablet
or 1 capsule or the amount specified equivalent to minimum
dose.
1. Apparatus
1.1. Apparatus for Basket Method (Apparatus 1)
The assembly consists of the following: a vessel, which
may be covered, made of glass or other inert, transparent
material*1; a motor; a drive shaft; and a cylindrical basket.
The vessel is partially immersed in a suitable water bath of
any convenient size or heated by a suitable device such as a
heating jacket. The water bath or heating device permits
holding the temperature inside the vessel at 37 ± 0.59C
during the test and keeping the bath fluid in constant,
smooth motion. No part of the assembly, including the
environment in which the assembly is placed, contributes
significant motion, agitation, or vibration beyond that due
to the smoothly rotating stirring element. Make the appa-
ratus to permit observation of the specimen and stirring
element during the test. The vessel is cylindrical, with a Fig. 6.10-1 Apparatus 1, Basket stirring element
hemispherical bottom and a capacity of 1 liter. Its height is
160 mm to 210 mm and its inside diameter is 98 mm to 106
mm. Its sides are flanged at the top. Use a fitted cover to
retard evaporation.*2 The shaft is positioned so that its axis 
usually allowed to sink to the bottom of the vessel before
is not more than 2 mm at any point from the vertical axis of
rotation of the blade is started. A small, loose piece of non-
the vessel and rotates smoothly and without significant wob-
reactive material, such as not more than a few turns of wire
ble that could affect the results. Adjust a speed-regulating
helix or such one shown in Fig. 6.10-2a, may be attached to
device to maintaine the shaft rotation speed at a specified
the dosage unit that would otherwise float. Other validated
rate, within ± 4z.
sinker devices may also be used. If the use of sinker is
Shaft and basket components of the stirring element
specified, unless otherwise specified, use the sinker device
shown in Fig. 6.10-1 are fabricated of stainless steel
shown in Fig. 6.10-2a.
(SUS316) or other inert material. A basket having a gold
1.3. Apparatus for Flow-Through Cell Method (Apparatus
coating of about 0.0001 inch (2.5 mm) thick may be used.
3)
The dosage unit is placed in a dry basket at the beginning of
The assembly consists of a reservoir and a pump for the
each test. The distance between the inside bottom of the ves-
dissolution medium; a flow-through cell; a water bath that
sel and the bottom of the basket is maintained at 25 ± 2 mm
maintains the dissolution medium at 37 ± 0.59 C. Use the
during the test.
cell size specified in the individual monograph.
1.2. Apparatus for Paddle Method (Apparatus 2)
The pump forces the dissolution medium upwards through
Use the assembly from Apparatus 1, except that a paddle
the flow-through cell. The pump has a delivery range be-
formed from a blade and a shaft is used as the stirring ele-
tween 4 and 16 mL per minute, with standard flow rates of
ment. The shaft is positioned so that its axis is not more than
4, 8, and 16 mL per minute. It must deliver a constant flow
2 mm from the vertical axis of the vessel, at any point, and
(±5z of the nominal flow rate); the flow profile should be
rotates smoothly without significant wobble that could af-
sinusoidal with a pulsation of 120 ± 10 pulses per minute. A
fect the results. The vertical center line of the blade passes
pump without the pulsation may also be used. Dissolution
through the axis of the shaft so that the bottom of the blade
test procedure using the flow-through cell must be character-
is flush with the bottom of the shaft. The paddle conforms
ized with respect to rate and any pulsation.
to the specifications shown in Fig. 6.10-2. The distance of
The flow-through cell (see Figures 6.10-3 and 6.10-4), of
25 ± 2 mm between the bottom of the blade and the inside
transparent and inert material, is mounted vertically with a
bottom of the vessel is maintained during the test. The
filter system (specified in the individual monograph) that
metallic or suitably inert, rigid blade and shaft comprise a
prevents escape of undissolved particles from the top of the
single entity. A suitable two-part detachable design may be
cell; standard cell diameters are 12 and 22.6 mm; the bottom
used provided the assembly remains firmly engaged during
cone is usually filled with small glass beads of about 1-mm
the test. The paddle blade and shaft may be coated with a
diameter with one bead of about 5 mm positioned at the
suitable coating so as to make them inert. The dosage unit is
apex to protect the fluid entry tube; a tablet holder (see

Fig. 6.10-2 Apparatus 2, Paddle stirring element

Fig. 6.10-3 Apparatus 3

Large cell for tablets and capsules (top); tablet holder for the
large cell (bottom)
(All dimensions are expressed in mm unless otherwise noted.)

Fig. 6.10-2a Alternative sinker

ture of the dissolution medium, rotation speed (Basket
Method and Paddle Method), and flow rate of medium
(Flow-Through Cell Method).
Figures 6.10-3 and 6.10-4) is available for positioning of spe- Determine the acceptable performance of the dissolution
cial dosage forms. The cell is immersed in a water bath, and test assembly periodically.
the temperature is maintained at 37 ± 0.59C.
3. Procedure
The apparatus uses a clamp mechanism of two O-rings to
3.1. Basket Method or Paddle Method
assemble the cell. The pump is separated from the dissolu-
3.1.1. Immediate-release Dosage Forms
tion unit in order to shield the latter against any vibrations
(i) Procedure: Place the stated volume of the dissolution
originating from the pump. The position of the pump should
medium (±1z) in the vessel of the specified apparatus, as-
not be on a level higher than the reservoir flasks. Tube con-
semble the apparatus, equilibrate the dissolution medium to
nections are as short as possible. Use suitably inert tubing,
37 ± 0.59 C, and remove the thermometer. Place 1 dosage
such as polytef, with about 1.6-mm inner diameter and inert
unit in the apparatus, taking care to exclude air bubbles
flanged-end connections.
from the surface of the dosage unit, and immediately oper-
2. Apparatus Suitability ate the apparatus at the specified rate. Within the time inter-
The determination of suitability of a test assembly to per- val specified, or at each of the times stated, withdraw a
form dissolution testing must include conformance to the specimen from a zone midway between the surface of the
dimensions and tolerances of the apparatus as given above. Dissolution Medium and the top of the rotating basket or
In addition, critical test parameters that have to be moni- blade, not less than 10 mm from the vessel wall.
tored periodically during use include volume and tempera- [NOTE—Where multiple sampling times are specified,

withdrawn only at the stated times, within a tolerance of

± 2z .
3.1.2. Extended-release Dosage Forms
(i) Procedure: Proceed as described for Immediate-
Release Dosage Forms.
(ii) Dissolution Medium: Proceed as directed under Im-
mediate-Release Dosage Forms.
(iii) Time: The test-time points, generally three, are ex-
pressed in hours.
3.1.3. Delayed-release Dosage Forms
(i) Procedure: Unless otherwise specified, proceed the
acid stage test and buffer stage test separately as described
for Immediate-Release Dosage Forms.
(ii) Dissolution Medium: Acid stage: Unless 1st fluid
for dissolution test is used, proceed as directed under Im-
mediate-Release Dosage Forms. Buffer stage: Unless 2nd
fluid for dissolution test is used, proceed as directed under
Immediate-Release Dosage Forms.
(iii) Time: Acid stage: Generally, test time is 2 hours
for tablets and capsules, and 1 hour for granules. Buffer
stage: The same as directed under Immediate-Release
Dosage Forms. All test times stated are to be observed
within a tolerance of ±2z, unless otherwise specified.
3.2. Flow-Through Cell Method
3.2.1. Immediate-release Dosage Forms
(i) Procedure: Place the glass beads into the cell specified
in the individual monograph. Place 1 dosage unit on top of
the beads or, if specified, on a wire carrier. Assemble the
filter head and fix the parts together by means of a suitable
clamping device. Introduce by the pump the dissolution me-
dium warmed to 37 ± 0.59C through the bottom of the cell
to obtain the flow rate specified and measured with an ac-
curacy of 5z. Collect the eluate by fractions at each of the
times stated. Perform the analysis as directed. Repeat the
test with additional dosage units.
(ii) Dissolution Medium: Proceed as directed under Im-
Fig. 6.10-4 Apparatus 3 mediate-Release Dosage Forms under Basket Method and
Small cell for tablets and capsules (top); tablet holder for the Paddle Method.
small cell (bottom) (iii) Time: Proceed as directed under Immediate-Release
(All dimensions are expressed in mm unless otherwise noted.) Dosage Forms under Basket Method and Paddle Method.
3.2.2. Extended-release Dosage Forms
(i) Procedure: Proceed as described for Immediate-
Release Dosage Forms under Flow-Through Cell Method.
replace the aliquots withdrawn for analysis with equal (ii) Dissolution Medium: Proceed as described for Im-
volumes of fresh Dissolution Medium at 379 C or, where it mediate-Release under Flow-Through Cell Method.
can be shown that replacement of the medium is not neces- (iii) Time: The test-time points, generally three, are ex-
sary, correct for the volume change in the calculation. Keep pressed in hours.
the vessel covered for the duration of the test, and verify the
4. Interpretation
temperature of the mixture under test at suitable times.]
4.1. Immediate-release Dosage Forms
Perform the analysis using an indicated assay method.*3 Follow Interpretation 1 when the value Q is specified in
Repeat the test with additional dosage units.
the individual monograph, otherwise follow Interpretation
If automated equipment is used for sampling or the appa-
2.
ratus is otherwise modified, verification that the modified
4.1.1. Interpretation 1
apparatus will produce results equivalent to those obtained
Unless otherwise specified, the requirements are met if the
with the standard apparatus described in this chapter, is nec-
quantities of active ingredient dissolved from the dosage
essary.
units tested conform to Acceptance Table 6.10-1. Continue
(ii) Dissolution Medium: An appropriate dissolution me-
testing through the three stages unless the results conform at
dium is used. The volume specified refers to measurements
either S1 or S2. The quantity, Q, is the specified amount of
mode between 209 C and 259C. If the dissolution medium is
dissolved active ingredient, expressed as a percentage of the
a buffered solution, adjust the solution so that its pH is
labeled content of the dosage unit; the 5z, 15z, and 25z
within 0.05 unit of the specified pH. [NOTE—Dissolved
values in the Acceptance Tables are percentage of the labeled
gases can cause bubbles to form, which may change the
content so that three values and Q are in the same terms.
results of the test. If dissolved gases influence the dissolution
4.1.2. Interpretation 2
results, remove dissolved gases prior testing.*4]
Unless otherwise specified, perform the test on 6 dosage
(iii) Time: Where a single time specification is given, the
forms: if the individual dissolution rate meet the require-
test may be concluded in a shorter period if the requirement
ments specified in the individual monograph, the dosage
for minimum amount dissolved is met. Specimens are to be

Stage Number Acceptance Criteria Level Number Criteria

Tested Tested
S1 6 Each value is not less than Q＋5z. A1 6 No individual value exceeds 10z dissolved.
S2 6 Average value of the 12 dosage units (S1＋S2) A2 6 The average value of the 12 dosage units (A1＋
is equal to or greater than Q, and no value is A2) is not more than 10z dissolved, and no
less than Q－15z. value is greater than 25z dissolved.
S3 12 Average value of the 24 dosage units (S1＋S2＋ A3 12 The average value of the 24 dosage units (A1＋
S3) is equal to or greater than Q, not more than A2＋A3) is not more than 10z dissolved, and
2 values are less than Q－15z, and no value is no value is greater than 25z dissolved.
less than Q－25z.

Acceptance Table 6.10-4

Acceptance Table 6.10-2
Level Number Criteria
Number Tested
Level Criteria
Tested
B1 6 No value is less than Q＋5z.
L1 6 No individual value lies outside each of the B2 6 The average value of the 12 dosage units (B1＋
stated ranges and no individual value is less B2) is equal to or greater than Q, and no value
than the stated amount at the final test time. is less than Q－15z.
L2 6 The average value of the 12 dosage units (L1＋ B3 12 The average value of the 24 dosage units (B1＋
L2) lies within each of the stated ranges and is B2＋B3) is equal to or greater than Q, not more
not less than the stated amount at the final test than 2 values are less than Q－15z, and no
time; no value is more than 10z of labeled value is less than Q－25z.
content outside each of the stated ranges; and
no value is more than 10z of labeled content
below the stated amount at the final test time.
L3 12 The average value of the 24 dosage units (L1＋
L2＋L3) lies within each of the stated ranges, ply individually to each range.
and is not less than the stated amount at the 4.3. Delayed-release Dosage Forms
final test time; not more than 2 of the 24 values Follow Interpretation 1 when the value Q is specified in
are more than 10z of labeled content outside the test using 2nd fluid for dissolution test in the individual
each of the stated ranges; not more than 2 of
monograph, otherwise follow Interpretation 2.
the 24 values are more than 10z of labeled
content below the stated amount at the final
4.3.1. Interpretation 1
test time; and no value is more than 20z of la- (i) Test using 1st fulid for dissolution test: Unless other-
beled content outside each of the stated ranges wise specified, the requirements of this portion of the test are
or more than 20z of labeled content below the met if the quantities, based on the percentage of the labeled
stated amount at the final test time. content, of active ingredient dissolved from the units tested
conform to Acceptance Table 6.10-3. Continue testing
through the three levels unless the result conforms at A2.
(ii) Test using 2nd fluid for dissolution test: Unless
forms conform to the test. When individual dissolution rates otherwise specified, the requirements are met if the quanti-
of 1 or 2 dosage forms fail to meet the requirements, repeat ties of active ingredient dissolved from the units tested con-
the test on another 6 dosage forms: if individual dissolution form to Acceptance Table 6.10-4. Continue testing through
rates of not less than 10 dosage forms out of 12 meet the re- the three levels unless the results of both stages conform at
quirements, the dosage forms conform to the test. an earlier level. The value of Q in Acceptance Table 6.10-4 is
4.2. Extended-release Dosage Forms the amount specified in monograph of active ingredient
4.2.1. Interpretation 1 dissolved, expressed as a percentage of the labeled content.
Unless otherwise specified, the requirements are met if the The 5z and 15z and 25z values in Acceptance Table
quantities of active ingredient dissolved from the dosage 6.10-4 are percentages of the labeled contest so that these
units tested conform to Acceptance Table 6.10-2. Continue values and Q are in the same terms.
testing through the three levels unless the results conform at 4.3.2. Interpretation 2
either L1 or L2. Limits on the amounts of active ingredient Unless otherwise specified, both the tests using 1st fluid
dissolved are expressed in terms of the percentage of labeled for dissolution test and 2nd fluid for dissolution test in acid
content. The limits embrace each value of Qi, the amount and buffer stages, perform the test on 6 dosage forms: if the
dissolved at each specified fractional dosing interval. Where individual dissolution rate meet the requirements specified in
more than one range is specified, the acceptance criteria ap- the individual monograph, the dosage forms conform to the
ply individually to each range. test. When individual dissolution rates of 1 or 2 dosage
4.2.2. Interpretation 2 forms fail to meet the requirements, repeat the test on
Unless otherwise specified, perform the test on 6 dosage another 6 dosage forms: if individual dissolution rates of not
forms: if the individual dissolution rate meet the require- less than 10 dosage forms out of 12 meet the requirements,
ments specified in the individual monograph, the dosage the dosage forms conform to the test.
forms conform to the test. When individual dissolution rates *1The materials should not sorb, react, or interfere with the speci-
of 1 or 2 dosage forms fail to meet the requirements, repeat
men being tested.
the test on another 6 dosage forms: if individual dissolution *2If a cover is used, it provides sufficient openings to allow ready in-
rates of not less than 10 dosage forms out of 12 meet the re-
sertion of the thermometer and withdrawal of specimens.
quirements, the dosage forms conform to the test. Where *3Test specimens are filtered immediately upon sampling unless
more than one range is specified, the acceptance criteria ap-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
JP XVII General Tests / 6.12 Methods of Adhesion Testing 161
filtration is demonstrated to be unnecessary. Use an inert filter cleaning solvent, and additionally, clean with the method de-
that does not cause adsorption of the ingredient or contain ex- scribed above before use.
tractable substances that would interfere with the analysis.
*4One method of deaeration is as follows: Heat the medium, while
3. Measurement methods
3.1. Peel adhesion testing
stirring gently, to about 419C, immediately filter under vacuum
The peel adhesion test measures the force required to re-
using a filter having a porosity of 0.45 mm or less, with vigorous
move (peel away) the sample adhered to the testing plate at a
stirring, and continue stirring under vacuum for about 5 minutes.
180 or 90-degree angle.
Other validated deaeration techniques for removal of dissolved
3.1.1. Equipment
gases may be used.
The equipment consists of an application device and a ten-
sile tester. The application device (Figures of equipment
6.12-1a and 6.12-1b) is structured in such a way that only the
6.11 Foreign Insoluble Matter mass of the roller is applied to the sample as pressure when
the sample is crimped. The roller should be made of steel or
Test for Ophthalmic Liquids equivalent covered with the rubber for crimping rollers
and Solutions whose material is prescribed in the Japanese Industrial
Standard Z 0237:2009 (a diameter of 85 ± 2.5 mm, a width
Foreign Insoluble Matter Test for Ophthalmic Liquids and of 45 ± 1.5 mm, a thickness of about 6 mm). In addition,
Solutions is a test method to examine foreign insoluble mat- the shape must be exactly cylindrical, and without irregulari-
ters in ophthalmic liquids and solutions. ties on the surface. Set the mass of the roller to 2000 ± 100 g
When inspect with the unaided eyes at a position of lu- or 1000 ± 50 g.
minous intensity of 3000 – 5000 lx under an incandescent With regard to the testing plate for adhesion testing, the
lamp after cleaning the exterior of containers, Ophthalmic plate prescribed in the Japanese Industrial Standard Z
Solutions must be clear and free from readily detectable for- 0237:2009 or equivalent should be used unless otherwise
eign insoluble matters. specified. Use a tensile tester with a relative pointing error of
± 1.0z. The representation of measurements may be any
one of the analog type, digital type, digital recording system,
and chart recording system.
6.12 Methods of Adhesion Testing 3.1.2. Operation procedures
Prepare the sample in such a way that it has a chucking
These are testing methods to measure the adhesion of
allowance on one end, and adhere it to the testing plate using
patches. The methods include peel adhesion testing, inclined
the application device after exposing the adhesive side within
ball tack testing, rolling ball tack testing, and probe tack tes-
5 minutes. Loosen the sample in holding the chucking
ting
allowance on the testing plate so that the testing plate will
The tests are conducted at 249 C ± 29C unless otherwise
not come into contact with the sample prior to adhesion. Ad-
specified. However, if the acceptable range of 249 C ± 29C
here the sample to the testing plate while crimping along the
temperature cannot be maintained, set a range as close to
long side direction with the roller. This will prevent air from
that as possible.
entering between the sample and the testing plate. If air en-
1. Preparation of samples ters, do not use the sample. Crimping should be done under
The following method will be followed to prepare samples constant load either at the rate of approximately 10 mm per
unless otherwise specified. Use a package, such as an alumi- second back and forth two times or at the rate of approxi-
num material that is not affected by humidity for the sam- mately 5 mm per second back and forth once. After crimp-
ple, and allow it to stand at 249C ± 29C for over 12 hours. ing the sample with the roller, perform a peel adhesion test
The sample can be cut to an appropriate size as needed. Fur- at the prescribed time (for example, 30 ± 10 minutes). Use a
thermore, visually confirm that dust is not adhered to the ad-
hesive side of the sample, take care not to touch the sample
with bare hands, and prevent foreign matter from adhering
to it.
2. Cleaning method for testing instruments
To clean the testing plates, balls, and probe for adhesion
testing, use cleaning solvents such as acetone, 2-butanone,
ethanol (99.5), ethyl acetate, heptane, water, and methanol.
Utilize soft cloth, such as gauze, absorbent cotton, or waste
cloth that does not generate lint or dust during use, is absor-
bent, and free from additives that dissolve in cleaning sol-
vents. Apply a cleaning solvent on a clean cloth, wipe the Fig. 6.12-1a Example of the automatic application device
surfaces of the instruments, and repeat wiping with a new
cloth until dry. Repeat this step until the instruments are de-
termined clean by visual observation. Lastly, apply acetone,
2-butanone, or another appropriate solvent on a cloth, wipe
the surfaces of the instruments, and repeat wiping with a
new cloth until dry. Use the cleaned instruments for testing
within 10 hours. Take care not to touch the surfaces with
fingers, and preserve them without damaging or contaminat-
ing. Do not use any instrument if it is dirty, discolored, or
has a number of scratches. With respect to new testing
plates, balls, and probe, wipe well using cloth soaked with a Fig. 6.12-1b Example of the manual application device

Fig. 6.12-2a Example of 180-degree peeling tester

Fig. 6.12-2b Example of 90-degree peeling tester Fig. 6.12-3 Example of inclined ball tack tester

Fig. 6.12-4 Example of rolling ball tack tester

(All dimensions are expressed in mm unless otherwise noted.)

3.3. Rolling ball tack testing

In the rolling ball tack test, a ball of specified size is rolled
down a ramp from the start position, and the distance to the
position at which the ball stops is measured.
3.3.1. Equipment
3.3.1.1. Ball-rolling device
The ball-rolling device has an inclination angle of 21.5
degrees, and Figure 6.12-4 shows an example.
3.3.1.2. Balls
Use No. 14 balls (a diameter of 7/16 inch) for adhesion
testing as noted in 3.2.1.2 unless otherwise specified.
3.3.2. Operation procedures
Secure the sample on the smooth, hard, and flat plate with
another adhesive tape. When fixing the sample, take care
that it will not be off the plate, wrinkled, or bent; if an edge
of the sample is curved and off the plate, stick the part to the Fig. 6.12-5 Example of probe tack tester
plate with another adhesive tape. Secure the ball rolling
device horizontally on the stand with the sample using a
level. Roll the ball down from the start position.
Measure the distance when the ball stopped on the adhe- Express the unit in N/cm2.
sive face. Measure the length between the end of the inclined
plane and the midpoint at which the adhesive is in contact
with the ball, which will be the value of the rolling ball tack
test. Express the unit in mm. 6.13 Release Test for Preparations
3.4. Probe tack testing for Cutaneous Application
The probe tack test measures the force required for peel-
ing a prescribed cylindrical probe after bringing the probe This chapter describes the method to measure release pro-
into contact with the adhesive side of a patch briefly. files of active ingredients from preparations for cutaneous
3.4.1. Equipment application and is provided to determine compliance of the
The equipment consists of a probe, a sample stage, and a preparations with drug-release requirements. Since the rela-
stress detector, and has a mechanism to give constant load tion between efficacy and release profile depends on each
for a certain period of time by a weight ring. As for the ma- characteristic of these preparations, this release test is an
terial of the probe for adhesion testing, use SUS304 with the effective method for a quality control of each preparation.
root-mean-square value (Rq) of 250 to 500 nm for surface Particularly, for transdermal absorption type pharmaceuti-
roughness, and a diameter of 5 mm unless otherwise speci- cal preparations, it is necessary to maintain an appropriate
fied. Furthermore, the equipment has a feature that can con- control on the release profiles of active ingredients.
trol the speed so that the probe's contact with the adhesive
1. Paddle over disk method
side of the patch and peeling will be done at a constant rate.
1.1. Apparatus
An example of device that applies a load by weight rings is
Use the assembly for the paddle method (Apparatus 2)
provided in Figure 6.12-5. A device without weight rings
described under Dissolution Test <6.10> and, addition to the
may also be used.
paddle and vessel, a stainless steel (SUS316) disk consisting
3.4.2. Operation procedures
of screen with wire opening of 125 mm is used to sink sam-
Adhere the sample to the weight ring ensuring that there is
ples on the bottom of the vessel. If necessary, other devices
no slack and place on the sample stage. Next, bring the
which is similar to Fig. 6.13-1 with a different size or shape
probe into contact with the adhesive side of the sample at the
may be used. Other appropriate devices instead of the disk
rate of 10 ± 0.01 mm per second, and maintain for 1.0 ±
may be used as long as they are chemically inert and do not
0.1 seconds with the contact load of 0.98 ± 0.01 N/cm2
interfere with the analysis. The disk attached with a sample
unless otherwise specified. Immediately after that, peel the
is installed parallel to the bottom of the paddle blade. The
probe vertically from the adhesive face at the rate of 10 ±
distance between the bottom of the paddle blade and the sur-
0.01 mm per second. Measure the maximum load required
face of the disk is 25 ± 2 mm, unless otherwise specified in
for peeling, which will be the value of the probe tack test.

Fig. 6.13-1 Paddle over disk

Fig. 6.13-3-1 Upper structure of cylinder stirring element

Fig. 6.13-2 Paddle and vessel

individual monograph (Fig. 6.13-2).

Additionally, apparatus suitability and handling of disso-
lution medium, etc., are in principle proceed as directed
under Dissolution Test <6.10>.
1.2. Procedure
Prior to set a disk, place the prescribed volume of the dis-
solution medium in the vessel and maintain the temperature
of the medium at 32 ± 0.59C. Fix the sample on a disk as
the release surface facing up in a suitable manner by using a Fig. 6.13-3-2 Cylinder stirring element
double-sided adhesive tape or the like. When a function of
the sample is not compromised by cutting, an appropriate
and exactly measured piece of the sample may be cut and
used for the test. If necessary, a porous membrane may be cal method.
attached to the release surface for suppressing a shape In the case where a different device in shape and material
change of the preparation. The characteristics of the mem- from that in Fig. 6.13-2 is used for sinking a sample with
brane used such as hydrophobicity or hydrophilicity and the almost the same procedure stated, the method can be consi-
pore size must be stated in the test method. When a mem- dered as the paddle over disk method, however, it needs to
brane is used, it is applied so that no air bubbles occur be- state the information about the used device.
tween the membrane and the release surface.
2. Cylinder method
Install the disk at the bottom of the vessel as that the
2.1. Apparatus
release surface of the sample is set upwards, and paralleled
Use the vessel of the apparatus for the Paddle Method
to the bottom of the paddle blade and to the surface of the
(Apparatus 2) described in Dissolution Test <6.10>, and use a
dissolution medium. Immediately after the installation,
stainless steel cylinder stirring element shown in Fig. 6.13-3-1
rotate the paddle at the specified rate, and then at specified
and Fig. 6.13-3-2 instead of a paddle. The cylinder is fabri-
interval or time, withdraw a specimen from the zone midway
cated of chemically inert material such as stainless steel
between the surface of the dissolution medium and the top
(SUS316), and the surface of the cylinder is electrolytically-
of the paddle blade, not less than 10 mm from the vessel
polished. The cylinder whose size is adjusted to the same size
wall. (Note: Where multiple sampling times are specified,
as Fig. 6.13-3-2 (B) by equipping with an additional cylindri-
replace the aliquots withdrawn for analysis with equal
cal device to the cylinder of Fig. 6.13-3-2 (A) can be used.
volumes of fresh dissolution medium of 329C or, where
The distance between the inside bottom of the vessel and
replacement of the medium is not necessary, correct for the
lower side of the cylinder is maintained at 25 ± 2 mm. Addi-
volume change in the calculation. Keep the vessel covered
tionally, apparatus suitability and handling of dissolution
for the duration of the test, and verify the temperature of the
medium, etc., are performed as directed under Dissolution
mixture under test at suitable intervals.) Perform the assay
Test <6.10>.
of the released active ingredients using the specified analyti-

2.2. Procedure rials such as glass or plastic, which do not interfere with the
Place the prescribed volume of the dissolution medium in analysis.
the vessel and maintain the temperature of the medium to 32 3.2. Procedure
± 0.59C. Remove the protective liner from the sample and Place the prescribed volume of the dissolution medium in
fix the sample on the cylinder as the release surface being the receptor chamber with a magnetic stirrer rotator and
outside by a suitable method using a double-sided adhesive maintain the medium temperature at 32 ± 1.09C. If neces-
tape or the like. If necessary, a porous membrane may be at- sary, a porous membrane may be attached to the release sur-
tached to the release surface. The characteristics of the mem-
brane used such as hydrophobicity or hydrophilicity and the
pore size must be stated in the test method.
Install the cylinder in the dissolution test apparatus, and Table 6.13-1 Acceptance criteria
immediately after the installation, rotate the cylinder at the
Number
specified rate. At specified interval or time, withdraw a Level Acceptance criteria
tested
specimen from a zone midway between the surface of the
dissolution medium and the bottom of the cylinder, not less L1 6 No individual value lies outside each of the
than 10 mm from the vessel wall. (Note: Where multiple stated ranges (including the limit values).
sampling times are specified, replace the aliquots withdrawn
for analysis with equal volumes of fresh dissolution medium L2 6 The average value of the 12 samples (L1 ＋
of 329C or, where replacement of the medium is not neces- L2) lies within each of the stated ranges (in-
sary, correct for the volume change in the calculation. Keep cluding the limit values) and no individual
the vessel covered for the duration of the test, and verify the value is more than 10z of labeled content
temperature of the mixture under test at appropriate inter- outside each of the stated ranges.
vals.) Perform the assay of the released active ingredients on
L3 12 The average value of the 24 samples (L1 ＋
each sample using the specified analytical method.
L2 ＋ L3) lies within each of the stated
3. Vertical diffusion cell method ranges (including the limit values), and not
3.1. Apparatus more than 2 of the 24 values are more than
The assembly is composed of a vertical diffusion cell sepa- 10z of labeled content outside each of the
rated to two chambers and the chambers are fixed by a stated ranges and no value is more than 20
clamp. An example of the vertical diffusion cell is shown in z of labeled content outside each of the
Fig. 6.13-4. These cells are made of chemically inert mate- stated ranges.

Fig. 6.13-4 Vertical diffusion cell

Injections Containers to be fused

Containers not to be fused (including injection
0.30 mL

The glass containers for injections do not interact physi- syringes used as containers) 2.00 mL
cally or chemically with the contained medicament to alter (ii) Method 2: This method is applied to containers not
any property or quality, can protect the contained medica- to be fused for aqueous infusions with a capacity exceeding
ment from the invasion of microbes by means of perfect 100 mL.
sealing or other suitable process, and meet the following re- Rinse thoroughly the inside and outside of the containers
quirements. The surface-treated container for aqueous infu- to be tested with water, and dry. Add a volume of water
sion is made from glass which meets the requirements for the equivalent to 90z of the overflow capacity of the container,
soluble alkali test for a container not to be fused under cover it with a small beaker of hard glass or close tightly with
method 1. a suitable stopper, heat in an autoclave at 1219 C for 1 hour,
(1) The containers are colorless or light brown and trans- and allow to stand until the temperature falls to room tem-
parent, and have no bubbles which interfere the test of the perature, measure exactly 100 mL of this solution, and trans-
Foreign Insoluble Matter Test for Injections <6.06>. fer to a 250-mL conical flask of hard glass. Add 5 drops of
(2) Multiple-dose containers are closed by rubber stop- bromocresol green-methyl red TS, and titrate <2.50> with
pers or any other suitable stoppers. The stoppers permit 0.01 mol/L sulfuric acid VS until the color of the solution
penetration of an injection needle without detachment of changes from green through slightly grayish blue to slightly
fragments, and upon withdrawal of the needle, they reclose grayish red-purple. Measure accurately 100 mL of water,
the containers immediately to prevent external contamina- transfer to a 250-mL conical flask of hard glass, perform a
tion, and also do not interact physically or chemically with blank determination in the same manner, and make any nec-
the contained medicaments. essary correction. The quantity of 0.01 mol/L sulfuric acid
Containers intended for aqueous infusions are closed by VS consumed does not exceed 0.10 mL.
rubber stoppers meeting the requirements of the test for (4) Soluble iron test for light-resistant containers—Rinse
Rubber Closure for Aqueous Infusions <7.03>. thoroughly five or more light-resistant containers to be

tested with water, and dry at 1059C for 30 minutes. Pour a 5). Filter through a glass filter (G3) if insoluble matter
volume of 0.01 mol/L hydrochloric acid VS corresponding remains. To the obtained filtrate add 10 mL of a solution of
to the labeled volume of the container into individual con- diammonium hydrogen citrate (1 in 4), 2 drops of bromo-
tainers, and fuse them. In the case of containers not to be thymol blue TS and ammonia TS until the color of the solu-
fused, cover them with small beakers of hard glass or watch tion changes from yellow to green. Then add 10 mL of a so-
glasses of hard glass. Heat them at 1059C for 1 hour. After lution of ammonium sulfate (2 in 5) and water to make 100
cooling, prepare the test solution with 40 mL of this solution mL. Add 20 mL of a solution of sodium N, N-diethyl-
according to Method 1 of the Iron Limit Test <1.10>, and dithiocarbamate trihydrate (1 in 20) to this solution, mix,
perform the test according to Method B. Prepare the control allow to stand for a few minutes, then add 20.0 mL of 4-
solution with 2.0 mL of the Standard Iron Solution. methyl-2-pentanone, and shake vigorously. Allow to stand
(5) Light transmission test for light-resistant contain- to separate the 4-methyl-2-pentanone layer, filter if neces-
ers—Cut five light-resistant containers to be tested, prepare sary, and use the layer as the sample solution. Separately, to
test pieces with surfaces as flat as possible, and clean the sur- 2.0 mL of Standard Lead Solution add water to make ex-
faces. Fix a test piece in a cell-holder of a spectrophotometer actly 10 mL. To 1.0 mL of this solution add 10 mL of a solu-
to allow the light pass through the center of the test piece tion of diammonium hydrogen citrate (1 in 4) and 2 drops of
perpendicularly to its surface. Measure the light transmit- bromothymol blue TS, then proceed in the same manner as
tance of the test piece with reference to air between 290 nm for the sample solution, and use the solution so obtained as
and 450 nm and also between 590 nm and 610 nm at intervals the standard solution. Perform the test with the sample solu-
of 20 nm each. The percent transmissions obtained between tion and standard solution according to Atomic Absorption
290 nm and 450 nm are not more than 50z and that between Spectrophotometry <2.23> under the following conditions,
590 nm and 610 nm are not less than 60z. In the case of and determine the concentration of lead in the sample solu-
containers not to be fused having a wall thickness not less tion.
than 1.0 mm, the percent transmissions between 590 nm and Gas: Combustible gas—Acetylene or hydrogen.
610 nm are not less than 45z. Supporting gas—Air.
Lamp: Lead hollow-cathode lamp.
Wavelength: 283.3 nm.
1.1.3.2. Method 2
7.02 Test Methods for Cut a container into pieces smaller than 5-mm square,
Plastic Containers take 2.0 g of the pieces into a glass beaker, add 50 mL of 2-
butanone and 0.1 mL of nitric acid, and warm to dissolve.
Test methods for plastic containers may be used for To this solution add 96 mL of methanol gradually to precipi-
designing and assuring quality of plastic containers. Not all tate a resinous substance, and filter by suction. Wash the
tests described here will be necessary in any phases for any beaker and the resinous substance with 12 mL of methanol
containers. On the other hand, the set does not include followed by 12 mL of water, combine the washings and the
sufficient numbers and kinds of tests needed for any design filtrate, and concentrate to about 10 mL under reduced pres-
verification and quality assurance of any containers. Addi- sure. Transfer into a separator, add 10 mL of ethyl acetate
tional tests may be considered if necessary. and 10 mL of water, shake vigorously, and allow to stand to
It is not allowable for plastic containers for the aqueous separate the water layer. Evaporate the water layer to dry-
injections to interact with the pharmaceutical contained ness, add 5 mL of hydrochloric acid to the residue, and
therein resulting in the deterioration of its efficacy, safety or warm to dissolve. Then add 1 mL of a mixture of a solution
stability, and to contaminate with microorganisms. They of citric acid monohydrate (1 in 2) and hydrochloric acid
should meet the requirements prescribed in 2. Requirements (1:1), and add 1 mL of a warmed solution of ammonium
for Plastic Containers for Aqueous Injections. acetate (2 in 5). Filter through a glass filter (G3) if insoluble
matter remains. To the solution so obtained add 10 mL of a
1. Test methods
solution of diammonium hydrogen citrate (1 in 4) and 2
1.1. Combustion tests
drops of bromothymol blue TS, and then add ammonia TS
1.1.1. Residue on ignition
until the color of the solution changes from yellow to green.
Weigh accurately about 5 g of cut pieces of the container
Further add 10 mL of a solution of ammonium sulfate (2 in
and perform the test according to Residue on Ignition
5) and water to make 100 mL. Add 20 mL of a solution of
<2.44>.
sodium N, N-diethyldithiocarbamate trihydrate (1 in 20) to
1.1.2. Heavy metals
this solution, mix, allow to stand for a few minutes, then
Place an appropriate amount of cut pieces of the container
add 20.0 mL of 4-methyl-2-pentanone, and shake vigor-
in a porcelain crucible, and perform the test according to
ously. Allow to stand to separate the 4-methyl-2-pentanone
Method 2 of Heavy Metals Limit Test <1.07>. Prepare the
layer, filter the layer if necessary, and use the layer as the
control solution with 2.0 mL of Standard Lead Solution.
sample solution. Separately, pipet 5 mL of Standard Lead
1.1.3. Lead
Solution, add water to make exactly 50 mL, and to 2.0 mL
1.1.3.1. Method 1
of this solution add 10 mL of a solution of diammonium
Place 2.0 g of cut pieces of a container in a crucible of
hydrogen citrate (1 in 4) and 2 drops of bromothymol blue
platinum or quartz, moisten with 2 mL of sulfuric acid, heat
TS, then proceed in the same manner as for the sample solu-
slowly to dryness, then heat to combustion at between 4509C
tion, and use the solution so obtained as the standard solu-
and 5009C. Repeat this procedure, if necessary. After cool-
tion. Perform the test with the sample solution and standard
ing, moisten the residue with water, add 2 to 4 mL of hydro-
solution according to Atomic Absorption Spectrophotome-
chloric acid, evaporate to dryness on a water bath, then add
try <2.23> under the conditions described in Method 1, and
1 to 5 mL of hydrochloric acid, and warm to dissolve. Then
determine the concentration of lead in the sample solution.
add 0.5 to 1 mL of a mixture of a solution of citric acid
1.1.4. Cadmium
monohydrate (1 in 2) and hydrochloric acid (1:1), and add
1.1.4.1. Method 1
0.5 to 1 mL of a warmed solution of ammonium acetate (2 in
To 2.0 mL of Standard Cadmium Solution add 10 mL of a

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
168 7.02 Test Methods for Plastic Containers / General Tests JP XVII
solution of diammonium hydrogen citrate (1 in 4) and 2 heating the vessel in an autoclave at 1219C for 1 hour, take
drops of bromothymol blue TS, and proceed in the same out the vessel, allow to stand until the temperature falls to
manner as for the sample solution in ``1.1.3.1. Method 1'', room temperature, and use the content as the test solution.
and use the solution so obtained as the standard solution. For containers made of composite plastics, the extraction
Perform the test with the sample solution obtained in may be performed by filling a labeled volume of water in the
``1.1.3.1. Method 1'' and the standard solution according to container. In this case, it is necessary to record the volume of
Atomic Absorption Spectrophotometry <2.23> under the fol- water used and the inside area of the container.
lowing conditions, and determine the concentration of cad- When containers are deformed at 1219C, the extraction
mium in the sample solution. may be performed at the highest temperature which does not
Gas: Combustible gas—Acetylene or hydrogen. cause deformation among the following conditions: at 100
Supporting gas—Air. ± 29 C for 2 ± 0.2 hours, at 70 ± 29C for 24 ± 2 hours, at
Lamp: Cadmium hollow-cathode lamp. 50 ± 29C for 72 ± 2 hours or at 37 ± 19C for 72 ± 2
Wavelength: 228.8 nm. hours.
1.1.4.2. Method 2 Prepare the blank solution with water in the same manner.
To 2.0 mL of Standard Cadmium Solution add 10 mL of a For containers made of composite plastics, water is used as
solution of diammonium hydrogen citrate (1 in 4) and 2 the blank solution.
drops of bromothymol blue TS, and proceed in the same Perform the following tests with the test solution and the
manner as for the sample solution in ``1.1.3.2. Method 2'', blank solution:
and use the solution so obtained as the standard solution. (i) Foaming test: Place 5 mL of the test solution in a
Perform the test with the sample solution obtained in glass-stoppered test tube about 15 mm in inside diameter and
``1.1.3.2. Method 2'' and the standard solution according to about 200 mm in length, shake vigorously for 3 minutes, and
Atomic Absorption Spectrophotometry <2.23> under the measure the time needed for almost complete disappearance
conditions described in ``1.1.4.1. Method 1'', and determine of the foam thus generated.
the concentration of cadmium in the sample solution. (ii) pH <2.54>: To 20 mL each of the test solution and the
1.1.5. Tin blank solution add 1.0 mL of a solution of potassium chlo-
Cut a container into pieces smaller than 5-mm square, ride (1 in 1000), and obtain the difference in the reading of
place 5.0 g of the pieces in a Kjeldahl flask, add 30 mL of a pH between these solutions.
mixture of sulfuric acid and nitric acid (1:1), and decompose (iii) Potassium permanganate-reducing substances: Place
by gentle heating in a muffle furnace, occasionally adding 20.0 mL of the test solution in a glass-stoppered, conical
dropwise a mixture of sulfuric acid and nitric acid (1:1) until flask, add 20.0 mL of 0.002 mol/L potassium permanganate
the content changes to a clear, light brown solution. Then VS and 1 mL of dilute sulfuric acid, and boil for 3 minutes.
heat until the color of the solution changes to a clear, light After cooling, add 0.10 g of potassium iodide, stopper
yellow, and heat to slowly concentrate to practical dryness. tightly, shake, then allow to stand for 10 minutes, and titrate
After cooling, dissolve the residue in 5 mL of hydrochloric <2.50> with 0.01 mol/L sodium thiosulfate VS (indicator: 5
acid by warming, and after cooling, add water to make ex- drops of starch TS). Perform the test in the same manner,
actly 10 mL. Pipet 5 mL of this solution into a 25-mL volu- using 20.0 mL of the blank solution, and obtain the differ-
metric flask (A). Transfer the remaining solution to a 25-mL ence of the consumption of 0.002 mol/L potassium perman-
beaker (B) by washing out with 10 mL of water, add 2 drops ganate VS between these solutions.
of bromocresol green TS, neutralize with diluted ammonia (iv) UV spectrum: Read the maximum absorbances be-
solution (28) (1 in 2), and measure the volume consumed for tween 220 nm and 240 nm and between 241 nm and 350 nm
neutralization as a mL. To the volumetric flask, A, add po- of the test solution against the blank solution as directed
tassium permanganate TS dropwise until a slight pale red under Ultraviolet-visible Spectrophotometry <2.24>.
color develops, and add a small amount of L-ascorbic acid to (v) Residue on evaporation: Evaporate 20 mL of the test
decolorize. Add 1.5 mL of 1 mol/L hydrochloric acid TS, 5 solution on a water bath to dryness, and weigh the residue
mL of a solution of citric acid monohydrate (1 in 10), a mL after drying at 1059 C for 1 hour.
of diluted ammonia solution (28) (1 in 2), 2.5 mL of poly- 1.3. Test for fine particles
vinyl alcohol TS, 5.0 mL of phenylfluorone-ethanol TS and 1.3.1. Test procedure
water to make 25 mL. Shake well, then allow to stand for Rinse thoroughly the inside and outside of containers to
about 20 minutes, and use this solution as the sample solu- be tested with water for particle matter test, fill the container
tion. Separately, pipet 1.0 mL of Standard Tin Solution, add with the labeled volume of water for particulate matter test
5 mL of water, add potassium permanganate TS dropwise or 0.9 w/vz sodium chloride solution, adjust the amount of
until a slight pale red color develops, proceed in the same air in the container to about 50 mL per 500 mL of the
manner as for the sample solution, and use this solution as labeled volume, put tight stopper to the container, and heat
the standard solution. Determine the absorbances of the it at 1219 C for 25 minutes in an autoclave. After allowing to
sample solution and the standard solution according to Ul- cool for 2 hours, take out the container from the autoclave,
traviolet-visible Spectrophotometry <2.24> at 510 nm, using and then allow to stand at ordinary temperature for about 24
water as the blank. hours. If the containers are deformed at 1219C, employ a
1.2. Extractable substances suitable temperature-time combination as directed under 1.2.
Cut the container at homogeneous regions of low curva- Extractable substances. Clean the outside of the container,
ture and preferably the same thickness, gather pieces to mix by turning upside-down 5 or 6 times, insert immediately
make a total surface area of about 1200 cm2 when the thick- a clean needle of filterless infusion set into the container
ness is 0.5 mm or less, or about 600 cm2 when the thickness through the rubber closure of the container, take the effluent
is greater than 0.5 mm, and subdivide in general into strips from the container while mixing gently in a clean container
approximately 0.5 cm in width and 5 cm in length. Wash for measurement, and use it as the test solution.
them with water, and dry at room temperature. Place these Counting of the fine particles must be performed in dust-
strips in a 300-mL hard glass vessel, add exactly 200 mL of less, clean facilities or apparatus, using a light-shielded auto-
water, and seal the opening with a suitable stopper. After matic fine particle counter. The sensor of the counter to be

used must be able to count fine particles of 1.5 mm or more 1.5.2. Method 2
in diameter. The volume to be measured is 10 mL. Adjust This test method is provided for evaluating moisture per-
the counter before measurement. For calibration of the di- meability of containers for hygroscopic drugs. Unless other-
ameter and number of particles, the standard particles for wise specified, perform the test according to the following
calibration of the light-shielded automatic fine particle coun- procedure.
ter should be used in suspension in water for particulate mat- 1.5.2.1. Desiccant
ter test or 0.9 w/vz sodium chloride solution. Place a quantity of calcium chloride for water determina-
Count five times the numbers of particles with diameters tion in a shallow container, taking care to exclude any fine
of 5 – 10 mm, 10 – 25 mm and more than 25 mm while stirring powder, then dry at 1109 C for 1 hour, and cool in a desicca-
the test solution, and calculate the average particle numbers tor.
of four counts, excluding the first, as the number of particles 1.5.2.2. Procedure
in 1.0 mL of the test solution. Select 12 containers, clean their surfaces with a dry cloth,
1.3.2. Reagent and close and open each container 30 times in the same
Water for particulate matter test and 0.9 w/vz sodium manner. Ten among the 12 containers are used as ``test
chloride solution to be used for the tests should not contain containers'' and the remaining two, as ``control containers''.
more than 0.5 particles of 5 – 10 mm in size per 1.0 mL. A torque for closing screw-capped containers is specified in
1.4. Transparency test Table 7.02-1. Add desiccant to 10 of the containers, desig-
1.4.1. Method 1 nated test containers, filling each to within 13 mm of the
This method can only be applied to containers which have closure if the container volume is 20 mL or more, or filling
a smooth and not embossed surface and rather low curva- each to two-thirds of capacity if the container volume is less
ture. Cut the container at homogeneous regions of low cur- than 20 mL. If the interior of the container is more than 63
vature and preferably the same thickness to make 5 pieces of mm in depth, an inert filler or spacer may be placed in the
about 0.9 × 4 cm in size, immerse each piece in water filled bottom to minimize the total mass of the container and
in a cell for determination of the ultraviolet spectrum, and desiccant; the layer of desiccant in such a container shall be
determine the transmittance at 450 nm as directed under Ul- not less than 5 cm in depth. Close each container immedi-
traviolet-visible Spectrophotomety <2.24> using a cell filled ately after adding desiccant, applying the torque designated
with water as a blank. in the table. To each of the control containers, add a
1.4.2. Method 2 sufficient number of glass beads to attain a mass approxi-
Sensory test—This method can be applied to containers mately equal to that of each of the test containers, and close,
which have a rough or embossed surface. It can also be ap- applying the torque designated in the table. Record the mass
plied to testing of the transparency of containers in case of the individual containers so prepared to the nearest 0.1
where the turbidity of their pharmaceutical contents must be mg if the container volume is less than 20 mL, to the nearest
checked. 1 mg if the container volume is 20 mL or more but less than
1.4.2.1. Test solutions 200 mL, or to the nearest 10 mg if the container volume is
(i) Formazin standard suspension: To 15 mL of the for- 200 mL or more, and store the containers at 75 ± 3z rela-
mazin stock suspension add water to make 1000 mL. Use tive humidity and a temperature of 20 ± 29C. After 14 days,
within 24 hours of preparation. Shake thoroughly before record the mass of the individual containers in the same
use.
(ii) Reference suspension: To 50 mL of Formazin stand-
ard suspension add water to make 100 mL.
1.4.2.2. Test procedures Table 7.02-1 Torque applicable to screw-type container
(i) Method 2A (with control): Take two of containers
Closure Diameter (mm) Torque (N・cm)
to be tested, and fill one of them with the labeled volume of
the reference suspension and the other with the same volume 8 59
of water. Show these two containers to five subjects, sepa- 10 60
rately, ask which one seems to be more turbid, and calculate 13 88
15 59 – 98
the rate of correct answers.
18 78 – 118
(ii) Method 2B (without control): Take six of contain-
20 88 – 137
ers to be tested, put number to each of them, and fill three of 22 98 – 157
them with the labeled volume of the reference suspension 24 118 – 206
and the others with the same volume of water. Show each 28 137 – 235
one of these containers at random order to five subjects, 30 147 – 265
separately, ask if it is turbid or not, and calculate the percen- 33 167 – 284
tage of the answer judged as ``turbid'' (100 X/15, X: num- 38 196 – 294
ber of containers judged as ``turbid'') for reference suspen- 43 196 – 304
sion-filled containers group and water-filled containers 48 216 – 343
53 235 – 402
group, respectively.
58 265 – 451
1.5. Water vapor permeability test
63 284 – 490
1.5.1. Method 1 66 294 – 510
This test method is applicable to containers for aqueous 70 314 – 569
injection. Fill the container with the labeled volume of 83 363 – 735
water. After closing it hermetically, accurately weigh the 86 451 – 735
container and record the value. Store the container at 65 ± 89 451 – 794
5z relative humidity and a temperature of 20 ± 29 C for 14 100 510 – 794
days, and then accurately weigh the container again and 110 510 – 794
record the value. Calculate the mass loss during storage. 120 618 – 1069
132 677 – 1069

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
170 7.02 Test Methods for Plastic Containers / General Tests JP XVII
manner. Completely fill 5 empty containers with water or a tle or a sterile disposable centrifuge tube. Cap the bottle or
non-compressible, free-flowing solid such as fine glass tube loosely and cover the cap with clean aluminum foil.
beads, to the level indicated by the closure surface when in Sterilize the bottle or tube by autoclaving at 1219C for 15
place. Transfer the contents of each to a graduated cylinder, minutes. When the material of the sample is not resistant to
and determine the average container volume, in mL. Calcu- heat during autoclaving, gas sterilization with ethylene oxide
late the rate of moisture permeability, in mg per day per (EO) may be used. In the case of EO sterilization, sufficient
liter, by use of the formula: aeration should be achieved to avoid an additional toxic ef-
fect of residual EO in the test results. To the bottle or tube
(1000/14V ) [(Tf － Ti) － (Cf － Ci)]
add the culture medium in a proportion of 1 mL per 2.5 cm2
V: Average volume (mL) (one side) or 10 mL per 1 g of the sample, loosely cap the
Tf － Ti: Difference between the final and initial masses of bottle or tube, and allow to stand in an incubator maintain-
each test container (mg) ing 5z carbon dioxide at 379C for 24 hours. Transfer the
Cf － Ci: Average of the differences between the final and culture medium extract, which is designated 100z sample
initial masses of the two controls (mg) solution, to a sterilized screw-capped glass bottle or a sterile
1.6. Leakage test disposable centrifuge tube. Dilute the 100z sample solution
Fill a container with a solution of fluorescein sodium (1 in with fresh culture medium using a dilution factor of two to
1000), stopper tightly, place filter papers on and under the prepare serial dilutions having extract concentrations of
container, and apply a pressure of 6.9 N (0.7 kg)/cm2 at 50z, 25z, 12.5z, 6.25z, 3.13z and so on.
209 C for 10 minutes. Judge the leakiness by observing the (iii) Preparation of cell suspension: Remove the culture
color of the paper. medium from the maintained cell culture vessel (flask or
1.7. Cytotoxicity test dish), and add gently a suitable volume of phosphate buffer
The following test methods are designed to detect cytotox- solution for cytotoxicity test. Rinse the cells by gentle rota-
ic substances in plastic materials by evaluating the cytotoxici- tion of the cell culture vessel two or three times, and discard
ty of the culture medium extracts from plastic containers for the phosphate buffer solution. Add a sufficient volume of
pharmaceutical products. Other appropriate standard trypsin solution to cover the cell layer. Cap the vessel and
methods of cytotoxicity testing may be used for the evalua- place in an incubator maintaining 5z carbon dioxide at
tion, if appropriate. However, the final decision shall be 379C for 1 to 2 minutes. After confirming detachment of the
made based upon the test methods given here, if the test cell layer from the bottom surface of the vessel by using a
results obtained according to the other methods are ques- microscope and by gently tapping of the vessel, add an
tionable. Other than those of the culture medium, reagents appropriate volume of the fresh culture medium and gently
and test solutions being specified for the test may be used if pipet the cells completely out of the vessel. Transfer the
they meet for the purpose of the test. pipetted cell suspension into a sterile disposable centrifuge
1.7.1. Cell lines tube and centrifuge. Discard the supernatant liquid,
The recommended cell lines are L929 cells (ATCC. CCL1) resuspend the cells in an appropriate volume of flesh phos-
and V79 cells (JCRB0603). In addition, other established cell phate buffer solution for cytotoxicity test by pipetting, and
lines may be used when it is confirmed that they form well- centrifuge the tube again. Discard the supernatant liquid,
defined colonies reproducibly, with characteristics compara- and add an appropriate volume of fresh culture medium to
ble to those of L929 cells and V79 cells. the tube. Resuspend the cells by gentle pipetting and make a
1.7.2. Culture medium homogeneous cell suspension. Determine the cell concentra-
(i) Medium for L929 cells: To Eagle's minimum essential tion using a hemocytometer.
medium add fetal calf serum (FCS) to make 10 volz FCS. (iv) Cytotoxicity test: Dilute the cell suspension prepared
(ii) Medium for V79 cells: M05 medium prepared by add- according to procedure (iii) with culture medium to adjust
ing 10 mL each of nonessential amino acid TS and 100 the cell concentration to 100 cells/mL. Place a 0.5 mL ali-
mmol/L sodium pyruvate TS to 1000 mL of Eagle's mini- quot of the diluted cell suspension on each well of a sterile
mum essential medium, then adding fetal calf serum (FCS) disposable multiple well plate (24 wells). Incubate the plate
to make 5 volz FCS. Medium for L929 cells may be used in the incubator maintaining 5z carbon dioxide at 379C for
instead if it gives equivalent sensitivity. 4 – 24 hours to attach the cells to the bottom surface of the
1.7.3. Reference materials and control substances well. Discard the medium from each well, and add a 0.5 mL
(i) Negative reference material: high-density polyethy- aliquot of the sample solution or fresh medium to at least 3
lene film wells each. Place the plate immediately in the incubator and
(ii) Positive reference material (A): polyurethane film incubate the plate for the appropriate period: 7 – 9 days for
containing 0.1z zinc diethyldithiocarbamate L929 cells; 6 – 7 days for V79 cells. After the incubation, dis-
(iii) Positive reference material (B): polyurethane film card the medium from the plate, add an appropriate volume
containing 0.25z zinc dibutyldithiocarbamate of methanol or dilute formaldehyde TS to each well and
(iv) Control substances: zinc diethyldithiocarbamate or allow the plate to stand for about 30 minutes to fix the cells.
zinc dibutyldithiocarbamate Discard the methanol or dilute formaldehyde TS from each
1.7.4. Test procedure well and add an appropriate volume of dilute Giemsa's TS to
(i) Sample preparation: When the material of the con- each well. After ensuring good staining of the colonies, dis-
tainer consists of a single homogeneous layer, subdivide the card the stain solution from the wells, wash with water, dry,
cut pieces of a container into pieces of the size of approxi- and count the number of colonies in each well. Calculate a
mately 2 × 15 mm and subject the pieces to the test. When mean number of colonies for each concentration of the sam-
the material of the container has multiple layers, such as ple solution, and divide the mean by the mean number of
laminated and coated materials, prepare cut pieces with a colonies for the fresh medium to obtain the relative plating
surface area of one side of 2.5 cm2 and subject the pieces to efficiency (z) for each extract concentration of the sample
the test without subdividing them into smaller pieces. solution. Plot the extract concentration (z) of the sample
(ii) Preparation of sample solutions: Transfer an ap- solution on a logarithmic scale and the relative plating
propriate amount of the sample to a screw-capped glass bot- efficiency on an ordinary scale on semilogarithmic graph

paper to obtain a colony formation inhibition curve of the tions''.

container. Read the 50z inhibition concentration, IC50 (z), (3) Appearance—Proceed as directed in (2) under ``2.1.
at which the colony number is half that in the control group, Polyethylene or polypropylene containers for aqueous injec-
from the inhibition curve. tions''.
It is recommended to check the sensitivity and the repro- (4) Leakage—Proceed with the test according to ``1.6.
ducibility of the test system by the use of suitable reference Leakage test''. The solution contained does not leak.
materials or control substances in the test system, if neces- (5) Flexibility—Insert the spike needle for infusion
sary. through a rubber closure of the container used in (4) Leak-
age. The contained solution is almost completely discharged
2. Requirements for plastic containers for aqueous injec-
without displacement by air.
tions
(6) Water vapor permeability—Proceed as directed in (3)
2.1. Polyethylene or polypropylene containers for aqueous
under ``2.1. Polyethylene or polypropylene containers for
injections
aqueous injections''.
The containers are made of polyethylene or polypropylene
(7) Heavy metals <1.07>—The turbidity of the test solu-
and free from any adhesive.
tion is not greater than that of the control solution when the
(1) Transparency—The containers have a transmittance
amount of the sample taken is 1.0 g.
of not less than 55z, when tested as directed in ``1.4.1.
(8) Lead—Perform the test as directed in ``1.1.3.2.
Method 1''. When ``1.4.1. Method 1'' can not be applied,
Method 2''. The absorbance of the sample solution is not
test according to ``1.4.2.2. (ii) Method 2B''. In this case, the
more than that of the standard solution.
rate that the water-containing container is judged as ``being
(9) Cadmium—Perform the test as directed in ``1.1.4.2.
turbid'' is not more than 20z, and the rate that the refer-
Method 2''. The absorbance of the sample solution is not
ence suspension-containing container is judged as ``being
more than that of the standard solution.
turbid'' is not less than 80z.
(10) Tin—The absorbance of the sample solution is not
(2) Appearance—The containers do not have strips,
more than that of the standard solution.
cracks, bubbles, or other faults which cause difficulties in
(11) Vinyl chloride—Wash cut pieces of a container with
practical use.
water, wipe them thoroughly with a filter paper, subdivide
(3) Water vapor permeability—Proceed as directed in
them into pieces smaller than 5 mm square, and put 0.5 g
``1.5.1. Method 1''. The loss of mass is not more than
of them into a 20-mL vial. Add 2.5 mL of N, N-
0.20z.
dimethylacetoamide to the vial to dissolve the sample pieces,
(4) Heavy metals <1.07>—The turbidity of the test solu-
put a tight stopper on the vial, and use the solution in the
tion is not greater than that of the control solution when the
vial as the sample solution. If the sample is hardly soluble,
amount of the sample taken is 1.0 g.
allow to stand the vial at room temperature for a night, put a
(5) Lead—Perform the test as directed in ``1.1.3.1.
tight stopper on the vial, and use the liquid part in the vial as
Method 1''. The absorbance of the sample solution is not
the sample solution. Separately, to a 20-mL vial add 2.5 mL
more than that of the standard solution.
of N, N-dimethylacetoamide, add 50 mL of Standard Vinyl
(6) Cadmium—Perform the test as directed in ``1.1.4.1.
Chloride Solution, previously cooled with dry ice-methanol,
Method 1''. The absorbance of the sample solution is not
put a tight stopper on the vial, and use the solution in the
more than that of the standard solution.
vial as the standard solution.
(7) Residue on ignition <2.44>—Not more than 0.1z
After heating the vials containing sample solution and
(5 g).
standard solution at 909 C for 1 hour, perform the test with
(8) Extractable substances—
0.5 mL each of vapor phases in these vials as directed under
(i) Foaming test: the foam formed almost disappears
Gas Chromatography <2.02> according to the following con-
within 3 minutes.
ditions: the peak area of vinyl chloride obtained from the
(ii) pH: the difference in the reading of pH between the
sample solution is not larger than that from the standard so-
test solution and the blank solution is not more than 1.5.
lution.
(iii) Potassium permanganate-reducing substances: The
Operating conditions—
difference in the consumption of 0.002 mol/L potassium
Detector: A hydrogen flame-ionization detector.
permanganate VS between the test solution and the blank so-
Column: A fused silicate column 0.25 mm in inside diame-
lution is not more than 1.0 mL.
ter and 25 m in length, coated the inside surface in 3 mm
(iv) UV spectrum: The maximum absorbance between
thickness with porous styrene-divinylbenzene copolymer for
220 nm and 240 nm is not more than 0.08, and that between
gas chromatography.
241 nm and 350 nm is not more than 0.05.
Column temperature: Maintain at 509C for 2 minutes
(v) Residue on evaporation: Not more than 1.0 mg.
after injection, then rise to 1209 C in the rate of 109C per
(9) Cytotoxicity—IC50 (z) is not less than 90z. The
minute, then rise to 2509C in the rate of 209C per minute,
result obtained by the other standard methods is negative.
and keep at 2509 C for 10 minutes.
2.2. Polyvinyl chloride containers for aqueous injections
Injection port temperature: A constant temperature of
The containers are composed of homopolymer of vinyl
about 2009C.
chloride, free from any adhesive, and the plasticizer added
Detector temperature: A constant temperature of about
to the material should be di(2-ethylhexyl)phthalate. The con-
2509C.
tainers may be covered with easily removable material to
Carrier gas: Nitrogen or helium.
prevent the permeation of water vapor. In this case, perform
Flow rate: Adjust so that the retention time of vinyl chlo-
the water vapor permeability test on the covered containers.
ride is about 7 minutes.
(1) Thickness—Measure the thickness of a container at
Split ratio: 1:5.
five different locations. The difference between the maxi-
System suitability—
mum and minimum values of thickness is 0.05 mm or less.
System performance: When the procedure is run under the
(2) Transparency—Proceed as directed in (1) under ``2.1.
above operating conditions with 0.5 mL of the vapor phase
Polyethylene or polypropylene containers for aqueous injec-
of the standard solution heated at 909C for 1 hour, vinyl

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
172 7.03 Test for Rubber Closure for Aqueous Infusions / General Tests JP XVII
chloride and ethanol are eluted in this order with the resolu- (1 in 2) and hydrochloric acid (1:1) and 0.5 to 1 mL of a
tion between these peaks being not less than 3.0. warmed solution of ammonium acetate (2 in 5). When any
System repeatability: When the test is repeated 6 times insoluble residue remains, filter through a glass filter. To the
under the above operating conditions with 0.5 mL of the solution thus obtained add 10 mL of a solution of diammo-
vapor phase of the standard solution heated at 909 C for 1 nium hydrogen citrate (1 in 4), 2 drops of bromothymol blue
hour, the relative standard deviation of the peak area of TS and ammonium TS until the color of the solution changes
vinyl chloride is not more than 5.0z. from yellow to green. Then add 10 mL of ammonium sulfate
(12) Fine particles—The number of fine particles in 1.0 solution (2 in 5) and water to make 100 mL. Next, add 20
mL of the test solution is counted as not more than 100 of 5 mL of a solution of sodium N,N-diethyldithiocarbamate tri-
to 10 mm, not more than 10 of 10 to 25 mm and not more hydrate (1 in 20), mix, allow to stand for a few minutes, add
than 1 of 25 mm or more. 20 mL of 4-methyl-2-pentanone, and mix by vigorous shak-
(13) Residue on ignition <2.44>—Not more than 0.1z ing. Allow to stand to separate the 4-methyl-2-pentanone
(5 g). layer from the solution, filter if necessary, and use as the
(14) Extractable substances—Proceed as directed in (8) sample solution. On the other hand, to exactly 10 mL of
under ``2.1. Polyethylene or polypropylene containers for Standard Cadmium Solution add 10 mL of a solution of di-
aqueous injections''. ammonium hydrogen citrate (1 in 4) and 2 drops of bromo-
(15) Cytotoxicity—Proceed as directed in (9) under ``2.1. thymol blue TS, proceed in the same manner as for the sam-
Polyethylene or polypropylene containers for aqueous injec- ple solution, and use this solution as the standard solution.
tions''. Perform the tests according to the Atomic Absorption Spec-
2.3. Plastic containers for aqueous injections being not trophotometry <2.23> under the following conditions, using
described above the sample solution and the standard solution. The absor-
The containers meet the following specifications and other bance of the sample solution is not more than that of the
necessary specifications for their materials with regard to standard solution.
heavy metals, residue on ignition and extractable substances, Gas: Combustible gas—Acetylene or hydrogen.
etc. Supporting gas—Air.
(1) Transparency—Proceed as directed in (1) under ``2.1. Lamp: Cadmium hollow-cathode lamp.
Polyethylene or polypropylene containers for aqueous injec- Wavelength: 228.8 nm.
tions''.
2. Lead
(2) Appearance—Proceed as directed in (2) under ``2.1.
To exactly 1 mL of the Standard Lead Solution add 10 mL
Polyethylene or polypropylene containers for aqueous injec-
of a solution of diammonium hydrogen citrate (1 in 4) and 2
tions''.
drops of bromothymol blue TS, proceed as directed for the
(3) Vapor permeability—Proceed as directed in (3) under
sample solution under 1, and use this solution as the stand-
``2.1. Polyethylene or polypropylene containers for aqueous
ard solution. Perform the tests according to the Atomic Ab-
injections''.
sorption Spectrophotometry <2.23> under the following con-
(4) Cytotoxicity—Proceed as directed in (9) under ``2.1.
ditions, using the sample solution and the standard solution
Polyethylene or polypropylene containers for aqueous injec-
obtained in 1. The absorbance of the sample solution is not
tions''.
more than that of the standard solution.
Gas: Combustible gas—Acetylene or hydrogen.
Supporting gas—Air.
7.03 Test for Rubber Closure for Lamp: Lead hollow-cathode lamp.
Wavelength: 283.3 nm.
Aqueous Infusions
3. Extractable substances
The rubber closure for aqueous infusions means a rubber Wash the rubber closures with water, and dry at room
closure (containing material coated or laminated with the temperature. Place an amount of them, equivalent to about
stuff like plastics) used for a container for aqueous infusion 150 cm2 in surface area, in a glass vessel, add 2 mL of water
having a capacity of 100 mL or more, and is in direct contact per cm2 of the sample, stopper adequately, heat at 1219C for
with the contained aqueous infusion. The rubber closure 1 hour in an autoclave, take out the glass vessel, allow to
when in use does not interact physically or chemically with cool to room temperature, then remove immediately the rub-
the contained medicament to alter any property or quality, ber closures, and use the remaining solution as the test solu-
does not permit the invasion of microbes, does not disturb tion. Prepare the blank solution with water in the same man-
the use of the contained infusion, and meets the following ner. Perform the following tests with the test solution and
requirements. the blank solution.
3.1. Description
1. Cadmium
The test solution is clear and colorless. Read the transmit-
Wash the rubber closures with water, dry at room temper-
tance of the test solution at 430 nm and 650 nm (10 mm),
ature, cut into minute pieces, mix well, place 2.0 g of them in
using the blank solution as the blank. Both of them are not
a crucible of platinum or quartz, moisten them with 2 mL of
less than 99.0z.
sulfuric acid, heat gradually to dryness, and ignite between
3.2. pH <2.54>
4509C and 5009C until the residue is incinerated. When in-
To 20 mL each of the test solution and the blank solution
cineration was insufficient, moisten the residue with 1 mL of
add 1 mL each of potassium chloride solution, prepared by
sulfuric acid, heat to dryness, and ignite again. Repeat the
dissolving 1.0 g of potassium chloride in water to make 1000
above-mentioned procedure if necessary. Cool the crucible,
mL. The difference of pH between the two solutions is not
moisten the residue with water, add 2 to 4 mL of hydrochlo-
more than 1.0.
ric acid, heat on a water bath to dryness, add 1 to 5 mL of
3.3. Zinc
hydrochloric acid, and dissolve by heating. Then add 0.5 to 1
To exactly 10 mL of the test solution add diluted dilute
mL of a mixture of a solution of citric acid monohydrate
nitric acid (1 in 3) to make exactly 20 mL, and use this solu-

tion as the sample solution. Further, to exactly 1 mL of (iv) Control substances: Zinc diethyldithiocarbamate
Standard Zinc Solution for atomic absorption spectropho- (reagent grade) or zinc dibutyldithiocarbamate
tometry add diluted nitric acid (1 in 3) to make exactly 20 4.4. Test procedure
mL, and use this solution as the standard solution. Perform (i) Sample preparation: Rubber closure is subjected to
the tests according to the Atomic Absorption Spectropho- the test without cutting into pieces. Reference material is
tometry <2.23>, using these solutions, under the following divided into pieces of approximately 2 × 15 mm and sub-
conditions. The absorbance of the sample solution is not jected to the test.
more than that of the standard solution. (ii) Preparation of sample solutions: Transfer an ap-
Gas: Combustible gas—Acetylene. propriate amount of the sample to a screw-capped glass bot-
Supporting gas—Air. tle or a sterile disposable centrifuge tube. Cap the bottle or
Lamp: Zinc hollow-cathode lamp. tube loosely and cover the cap with clean aluminum foil.
Wavelength: 213.9 nm. Sterilize the bottle or tube by autoclaving at 1219C for 15
3.4. Potassium Permanganate-reducing substances minutes. When the material of the sample is not resistant to
Measure 100 mL of the test solution in a glass-stoppered, heat during autoclaving, gas sterilization with ethylene oxide
Erlenmyer flask, add 10 mL of 0.002 mol/L potassium per- (EO) may be used. In the case of EO sterilization, sufficient
manganate VS, then add 5 mL of dilute sulfuric acid, and aeration should be achieved to avoid an additional toxic
boil for 3 minutes. After cooling, add 0.10 g of potassium effect of residual EO in the test results. To the bottle or tube
iodide, stopper, mix by shaking, then allow to stand for 10 add the culture medium in a proportion of 60 cm2 surface
minutes, and titrate <2.50> with 0.01 mol/L sodium thiosul- area or 10 mL per 1 g of the sample, loosely cap the bottle or
fate VS (indicator: 5 drops of starch TS). Perform the blank tube, and allow to stand in an incubator maintaining 5z
test in the same manner, using 100 mL of the blank solution. carbon dioxide at 379C for 24 hours. To the reference mate-
The difference in mL of 0.002 mol/L potassium permanga- rial add 10 mL of the culture medium per 1 g and extract in
nate VS required between the tests is not more than 2.0 mL. the same manner. Transfer the culture medium extract,
3.5. Residue on evaporation which is designated 100z sample solution, to a sterilized
Measure 100 mL of the test solution, evaporate on a water screw-capped glass bottle or a sterile disposable centrifuge
bath to dryness, and dry the residue at 1059C for 1 hour. tube. Dilute the 100z sample solution with fresh culture me-
The mass of the residue is not more than 2.0 mg. dium using a dilution factor of two to prepare serial dilu-
3.6. UV spectrum tions having extract concentrations of 50z, 25z, 12.5z,
Read the absorbance of the test solution between 220 nm 6.25z, 3.13z and so on.
and 350 nm against the blank solution as directed under Ul- (iii) Preparation of cell suspension: Remove the culture
traviolet-visible Spectrophotometry <2.24>: it is not more medium from the maintained cell culture vessel (flask or
than 0.20. dish), and add gently a suitable volume of phosphate buffer
solution for cytotoxicity test. Rinse the cells by gentle rota-
4. Cytotoxicity test
tion of the cell culture vessel two or three times, and discard
The following test methods are designed to detect cytotox-
the phosphate buffer solution. Add a sufficient volume of
ic substances in rubber materials by evaluating the cytotoxic-
trypsin solution to cover the cell layer. Cap the vessel and
ity of the culture medium extracts from rubber closure for
place in an incubator maintaining 5z carbon dioxide at
aqueous infusion. Other appropriate standard methods of
379C for 1 to 2 minutes. After confirming detachment of the
cytotoxicity testing may be used for the evaluation, if ap-
cell layer from the bottom surface of the vessel by using a
propriate. However, the final decision shall be made based
microscope and by gently tapping of the vessel, add an ap-
upon the test methods given here, if the test results obtained
propriate volume of the fresh culture medium and gently
according to the other methods are questionable. Other than
pipet the cells completely out of the vessel. Transfer the
those of the culture medium, reagents and test solutions
pipetted cell suspension into a sterile disposable centrifuge
being specified for the test may be used if they meet for the
tube and centrifuge. Discard the supernatant liquid,
purpose of the test.
resuspend the cells in an appropriate volume of flesh phos-
4.1. Cell lines
phate buffer solution for cytotoxicity test by pipetting, and
The recommended cell lines are L929 cells (ATCC. CCL1)
centrifuge the tube again. Discard the supernatant liquid,
and V79 cells (JCRB0603). In addition, other established cell
and add an appropriate volume of fresh culture medium to
lines may be used when it is confirmed that they form well-
the vessel. Resuspend the cells by gentle pipetting and make
defined colonies reproducibly, with characteristics compara-
a homogeneous cell suspension. Determine the cell concen-
ble to those of L929 cells and V79 cells.
tration using a hemocytometer.
4.2. Culture medium
(iv) Cytotoxicity test: Dilute the cell suspension prepared
(i) Medium for L929 cells: To Eagle's minimum essential
according to procedure (iii) with culture medium to adjust
medium add fetal calf serum (FCS) to make 10 volz FCS.
the cell concentration to 100 cells/mL. Place a 0.5 mL ali-
(ii) Medium for V79 cells: M05 medium prepared by
quot of the diluted cell suspension on each well of a sterile
adding 10 mL each of nonessential amino acid TS and 100
disposable multiple well plate (24 wells). Incubate the plate
mmol/L sodium pyruvate TS to 1000 mL of Eagle's mini-
in the incubator maintaining 5z carbon dioxide at 379 C for
mum essential medium, then adding fetal calf serum (FCS)
4 – 24 hours to attach the cells to the bottom surface of the
to make 5 volz FCS. Medium for L929 cells may be used in-
well. Discard the medium from each well, and add a 0.5 mL
stead if it gives equivalent sensitivity.
aliquot of the sample solution or fresh medium to at least 3
4.3. Reference materials and control substances
wells each. Place the plate immediately in the incubator and
(i) Negative reference material: Highdensity polyethylene
incubate the plate for the appropriate period: 7 – 9 days for
film
L929 cells; 6 – 7 days for V79 cells. After the incubation, dis-
(ii) Positive reference material (A): Polyurethane film
card the medium from the plate, add an appropriate volume
containing 0.1z zinc diethyldithiocarbamate
of methanol or dilute formaldehyde TS to each well and
(iii) Positive reference material (B): Polyurethane film
allow the plate to stand for about 30 minutes to fix the cells.
containing 0.25z zinc dibutyldithiocarbamate
Discard the methanol or dilute formaldehyde TS from each

Caffeine RS for Apparatus Suitability Fluocinolone Acetonide RS

Calcitonin Salmon RS Fluocinonide RS
Calcium Folinate RS Fluorometholone RS
Calcium Oxalate Monohydrate RS for Calibration of Ap- Fluoxymesterone RS
paratus Flutamide RS
Calcium Pantothenate RS Fluvoxamine Maleate RS
Camostat Mesilate RS Folic Acid RS
d-Camphor RS Furosemide RS
dl-Camphor RS Fursultiamine Hydrochloride RS
Carbidopa RS Gabexate Mesilate RS
Carboplatin RS Gefarnate RS
Cellacefate RS Ginsenoside Rb1 RS
Cetotiamine Hydrochloride RS Ginsenoside Rg1 RS
Chlordiazepoxide RS Gitoxin RS
Chlormadinone Acetate RS Glimepiride RS
Chlorpheniramine Maleate RS D-Glucuronolactone RS
Cholecalciferol RS Glycyrrhizic Acid RS
Ciclosporin RS Gonadorelin Acetate RS
Cilnidipine RS Guaifenesin RS
Cilostazol RS Heparin Sodium RS
Ciprofloxacin RS Heparin Sodium RS for Physicochemical Test
Cisplatin RS High-molecular Mass Urokinase RS
Citicoline RS Human Chorionic Gonadotrophin RS
Clobetasol Propionate RS Human Menopausal Gonadotrophin RS
Clofibrate RS Hydrochlorothiazide RS
Clomifene Citrate RS Hydrocortisone RS
Clopidogrel Sulfate RS Hydrocortisone Acetate RS
Cortisone Acetate RS Hydrocortisone Sodium Phosphate RS
Cyanocobalamin RS Hydrocortisone Succinate RS
Danazol RS Idoxuridine RS
Deferoxamine Mesilate RS Imipramine Hydrochloride RS
Deslanoside RS Indapamide RS
Dexamethasone RS Indometacin RS
Diclofenamide RS Insulin Glargine RS
Diethylcarbamazine Citrate RS Insulin Human RS
Diflorasone Diacetate RS Interleukin-2 RS
Diflucortolone Valerate RS Ipriflavone RS
Digitoxin RS Isoflurane RS
Digoxin RS Isomalt RS
Dihydroergotoxine Mesilate RS Kallidinogenase RS
Dobutamine Hydrochloride RS Lactose RS
Docetaxel RS Lactulose RS
Donepezil Hydrochloride RS Lanatoside C RS
Dorzolamide Hydrochloride RS Lansoprazole RS
Doxazosin Mesilate RS Lenograstim RS
Edrophonium Chloride RS Leuprorelin Acetate RS
Elcatonin RS Limaprost RS
Enalapril Maleate RS Losartan Potassium RS
Endotoxin RS Low-molecular Mass Heparin RS
Epalrestat RS Loxoprofen RS
Epitiostanol RS Lysozyme RS
Eplerenone RS Maltose RS
Epoetin Alfa RS Manidipine Hydrochloride RS
Epoetin Beta RS D-Mannitol RS
Ergocalciferol RS Mecobalamin RS
Ergometrine Maleate RS Medroxyprogesterone Acetate RS
Estradiol Benzoate RS Menatetrenone RS
Estriol RS Mestranol RS
Ethenzamide RS Methotrexate RS
Ethinylestradiol RS Methoxsalen RS
Ethyl Aminobenzoate RS Methyldopa RS
Ethyl Icosapentate RS Methylergometrine Maleate RS
Ethyl Parahydroxybenzoate RS Methyl Parahydroxybenzoate RS
Etoposide RS Methylprednisolone Succinate RS
Fexofenadine Hydrochloride RS Methyltestosterone RS
Filgrastim RS Metildigoxin RS
Fludrocortisone Acetate RS Mexiletine Hydrochloride RS

Cefazolin RS Meropenem RS
Cefbuperazone RS Micronomicin Sulfate RS
Cefcapene Pivoxil Hydrochloride RS Midecamycin RS
Cefdinir RS Midecamycin Acetate RS
Cefditoren Pivoxil RS Minocycline Hydrochloride RS
Cefepime Dihydrochloride RS Mitomycin C RS
Cefixime RS Mupirocin Lithium RS
Cefmenoxime Hydrochloride RS Nystatin RS
Cefmetazole RS Oxytetracycline Hydrochloride RS
Cefminox Sodium RS Panipenem RS
Cefodizime Sodium RS Peplomycin Sulfate RS
Cefoperazone RS Phenethicillin Potassium RS
Cefotaxime RS Pimaricin RS
Cefotetan RS Piperacillin RS
Cefotiam Hexetil Hydrochloride RS Pirarubicin RS
Cefotiam Hydrochloride RS Pivmecillinam Hydrochloride RS
Cefozopran Hydrochloride RS Polymixin B Sulfate RS
Cefpiramide RS Pyrrolnitrin RS
Cefpirome Sulfate RS Ribostamycin Sulfate RS
Cefpodoxime Proxetil RS Rifampicin RS
Cefroxadine RS Rokitamycin RS
Cefsulodin Sodium RS Roxithromycin RS
Ceftazidime RS Spectinomycin Hydrochloride RS
Cefteram Pivoxil Mesitylene Sulfonate RS Spiramycin II Acetate RS
Ceftibuten Hydrochloride RS Streptomycin Sulfate RS
Ceftizoxime RS Sulbactam RS
Ceftriaxone Sodium RS Sulbenicillin Sodium RS
Cefuroxime Axetil RS Sultamicillin Tosilate RS
Chloramphenicol RS Talampicillin Hydrochloride RS
Chloramphenicol Palmitate RS Tazobactam RS
Chloramphenicol Succinate RS Teicoplanin RS
Ciclacillin RS Tetracycline Hydrochloride RS
Clarithromycin RS Tobramycin RS
Clindamycin Hydrochloride RS Trichomycin RS
Clindamycin Phosphate RS Vancomycin Hydrochloride RS
Cloxacillin Sodium RS Zinostatin Stimalamer RS
Colistin Sodium Methanesulfonate RS
Colistin Sulfate RS
Cycloserine RS
Daunorubicin Hydrochloride RS Standard Solutions
Demethylchlortetracycline Hydrochloride RS
Dibekacin Sulfate RS
Dicloxacillin Sodium RS 9.21 Standard Solutions
Diethanolammonium Fusidate RS for Volumetric Analysis
Doxorubicin Hydrochloride RS
Doxycycline Hydrochloride RS Standard Solutions for Volumetric Analysis are the solu-
Enviomycin Sulfate RS tions of reagent with an accurately known concentration,
Epirubicin Hydrochloride RS mainly used for the volumetric analysis.
Erythromycin RS They are prepared to a specified molar concentration. A 1
Faropenem Sodium RS molar solution is a solution which contains exactly 1 mole of
Flomoxef Triethylammonium RS a specified substance in each 1000 mL of the solution and is
Fosfomycin Phenethylammonium RS designated as 1 mol/L.
Fradiomycin Sulfate RS If necessary, these solutions are diluted to other specified
Gentamicin Sulfate RS molar concentrations and the diluted solutions are also used
Gramicidin RS as standard solutions. For example, 0.1 mol/L solution is
Idarubicin Hydrochloride RS obtained by diluting 1 mol/L solution 10 times by volume.
Imipenem RS Unless otherwise directed, standard solutions for volumet-
Interferon Alfa RS ric analysis should be stored in colorless or light-resistant,
Isepamicin Sulfate RS glass-stoppered bottles.
Josamycin RS
Preparation and Standardization
Josamycin Propionate RS
A volumetric standard solution is prepared according to
Kanamycin Monosulfate RS
one of the following methods. The degree of difference from
Latamoxef Ammonium RS
a specified concentration n (mol/L) is expressed as a factor
Lenampicillin Hydrochloride RS
(molar concentration coefficient) f. Usually, standard solu-
Leucomycin A5 RS
tions are prepared so that the factor is in the range of 0.970 –
Lincomycin Hydrochloride RS
1.030. The determination procedure of the factor is called
Lithium Clavulanate RS

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
178 9.21 Standard Solutions for Volumetric Analysis / General Tests JP XVII
standardization of the standard solution. iron (II) sulfate hexahydrate [Fe(NH4)2(SO4)2.6H2O: 392.14].
(1) Weigh accurately a quantity equivalent to about 1 Preparation—Dissolve 40 g of ammonium iron (II) sulfate
mole or its multiple or a fractional mole number of the pure hexahydrate in a cooled mixture of 30 mL of sulfuric acid
substance, and dissolve it in the specified solvent to make ex- and 300 mL of water, dilute with water to make 1000 mL,
actly 1000 mL to prepare a standard solution having a con- and standardize the solution as follows:
centration close to the specified molarity n (mol/L). In this Standardization—Measure exactly 25 mL of the prepared
case, the factor f of the standard solution is obtained ammonium iron (II) sulfate solution, and add 25 mL of
by dividing the mass of the pure substance taken (g) by water and 5 mL of phosphoric acid. Titrate <2.50> the solu-
the molecular mass of the substance (g) and the specified tion with 0.02 mol/L potassium permanganate VS. Calculate
molarity number n. the molarity factor.
When a pure substance is not obtainable, it is permissible Note: Prepare before use.
to use a highly purified substance whose purity has been ex-
actly determined and certified. Ammonium Iron (II) Sulfate, 0.02 mol/L
(2) In the case where a pure substance or a highly puri- 1000 mL of this solution contains 7.843 g of ammonium
fied substance is not obtainable, weigh a quantity equivalent iron (II) sulfate hexahydrate [Fe(NH4)2(SO4)2.6H2O: 392.14].
to about 1 mole or its multiple or a fractional mole number Preparation—Before use, dilute 0.1 mol/L ammonium
of the substance specified for each standard solution and dis- Iron (II) sulfate VS with diluted sulfuric acid (3 in 100) to
solve it in the specified solvent to make about 1000 mL to make exactly 5 times the initial volume.
prepare a standard solution having a concentration close to
the specified molarity n (mol/L). The factor f of this solu- Ammonium Iron (III) Sulfate, 0.1 mol/L
tion is determined by applying the standardization procedure 1000 mL of this solution contains 48.22 g of ammonium
described for the respective standard solution. The proce- iron (III) sulfate dodecahydrate [FeNH4(SO4)2.12H2O:
dure is classified into direct and indirect methods, as 482.19].
follows: Preparation—Dissolve 49 g of ammonium iron (III) sul-
a) Direct method fate dodecahydrate in a cooled mixture of 6 mL of sulfuric
Weigh accurately a standard reagent or an indicated sub- acid and 300 mL of water, add water to make 1000 mL, and
stance specified for each standard solution, dissolve it in the standardize the solution as follows:
specified solvent, then titrate with the prepared standard so- Standardization—Measure exactly 25 mL of the prepared
lution to be standardized, and determine the factor f by ap- ammonium iron (III) sulfate solution into an iodine flask,
plying the following equation. add 5 mL of hydrochloric acid, and shake the mixture. Dis-
solve 2 g of potassium iodide, and stopper the flask. After
1000m
f＝ allowing the mixture to stand for 10 minutes, add 50 mL of
VMn
water, and titrate <2.50> the liberated iodine with 0.1 mol/L
M: Molecular mass equivalent to 1 mole of the standard sodium thiosulfate VS. When the solution assumes a pale
reagent or the specified substance (g) yellow color as the end point is approached, add 3 mL of
m: Mass of the standard reagent or the specified substance starch TS. Continue the titration, until the blue color dis-
taken (g) appears. Perform a blank determination. Calculate the
V: Volume of the prepared standard solution consumed molarity factor.
for the titration (mL) Note: Store protected from light. This solution, if stored
n: Arithmetical mole number of the specified molar con- for a long period of time, should be restandardized.
centration of the standard solution to be standardized
(e.g. n ＝ 0.02 for 0.02 mol/L standard solution) Ammonium Thiocyanate, 0.1 mol/L
1000 mL of this solution contains 7.612 g of ammonium
b) Indirect method
thiocyanate (NH4SCN: 76.12).
When an appropriate standard reagent is not available,
Preparation—Dissolve 8 g of ammonium thiocyanate in
titrate a defined volume V2 (mL) of a standard solution to be
water to make 1000 mL, and standardize the solution as
standardized with the specified standard solution having a
follows:
known factor ( f1), and calculate the factor ( f2) by applying
Standardization—Measure exactly 25 mL of the 0.1
the following equation.
mol/L silver nitrate VS, and add 50 mL of water, 2 mL of
V1 × f 1 nitric acid and 2 mL of ammonium iron (III) sulfate TS.
f2 ＝
V2 Titrate <2.50> the solution with the prepared ammonium
thiocyanate solution to the first appearance of a persistent
f1: Factor of the titrating standard solution having a
red-brown color with shaking. Calculate the molarity factor.
known factor
Note: Store protected from light.
f2: Factor of the prepared standard solution to be stand-
ardized
Ammonium Thiocyanate, 0.02 mol/L
V1: Volume of the titrating standard solution consumed
1000 mL of this solution contains 1.5224 g of ammonium
(mL)
thiocyanate (NH4SCN: 76.12).
V2: Volume of the prepared standard solution taken (mL)
Preparation—Before use, dilute 0.1 mol/L ammonium
(3) Standard solutions may be prepared by diluting ex- thiocyanate VS with water to make exactly 5 times the initial
actly an accurately measured volume of a standard solution volume.
having a known factor, according to the specified dilution
procedure. During this dilution procedure, the original fac- Barium chloride, 0.1 mol/L
tor of the standard solution is assumed to remain constant. 1000 mL of this solution contains 24.426 g of barium chlo-
ride dihydrate (BaCl2.2H2O: 244.26).
Ammonium Iron (II) Sulfate, 0.1 mol/L Preparation—Dissolve 24.5 g of barium chloride dihy-
1000 mL of this solution contains 39.214 g of ammonium drate in water to make 1000 mL, and standardize the solu-

tion as follows: mol/L disodium dihydrogen ethylenediamine tetraacetate

Standardization—Measure exactly 20 mL of the prepared VS until the red color changes to yellow. Calculate the
solution, add 3 mL of hydrochloric acid, and warm the molarity factor.
mixture. Add 40 mL of diluted sulfuric acid (1 in 130),
previously warmed, heat the mixture on a water bath for 30 Bromine, 0.05 mol/L
minutes, and allow it to stand overnight. Filter the mixture, 1000 mL of this solution contains 7.990 g of bromine (Br:
wash the precipitate on the filter paper with water until the 79.90).
last washing shows no turbidity with silver nitrate TS, trans- Preparation—Dissolve 2.8 g of potassium bromate and
fer the precipitate together with the filter paper to a tared 15 g of potassium bromide in water to make 1000 mL, and
crucible, and then heat strongly to ashes. After cooling, add standardize the solution as follows:
2 drops of sulfuric acid, and heat again at about 7009C for Standardization—Measure exactly 25 mL of the prepared
2 hours. After cooling, weigh accurately the mass of the solution into an iodine flask. Add 120 mL of water, quickly
residue, and calculate the molarity factor as barium sulfate add 5 mL of hydrochloric acid, stopper the flask immedi-
(BaSO4). ately, and shake it gently. Then add 5 mL of potassium
iodide TS, re-stopper immediately, shake the mixture gently,
Each mL of 0.1 mol/L barium chloride VS
and allow to stand for 5 minutes. Titrate <2.50> the liberated
＝ 23.34 mg of BaSO4
iodine with 0.1 mol/L sodium thiosulfate VS. When the so-
lution assumes a pale yellow color as the end point is ap-
Barium Chloride, 0.02 mol/L
proached, add 3 mL of starch TS. Continue the titration,
1000 mL of this solution contains 4.885 g of barium chlo-
until the blue color disappears. Perform a blank determina-
ride dihydrate (BaCl2.2H2O: 244.26).
tion. Calculate the molarity factor.
Preparation—Dissolve 4.9 g of barium chloride dihydrate
in water to make 1000 mL, and standardize the solution as
Ceric Ammonium Sulfate, 0.1 mol/L
follows:
See cerium (IV) tetraammonium sulfate, 0.1 mol/L.
Standardization—Measure exactly 100 mL of the prepared
barium chloride solution, add 3 mL of hydrochloric acid,
Ceric Ammonium Sulfate, 0.01 mol/L
and warm the mixture. Add 40 mL of diluted sulfuric acid (1
See cerium (IV) tetraammonium sulfate, 0.01 mol/L.
in 130), warmed previously, heat the mixture on a water bath
for 30 minutes, and allow to stand overnight. Filter the mix-
Cerium (IV) Tetraammonium Sulfate, 0.1 mol/L
ture, wash the collected precipitate of filter paper with water
1000 mL of this solution contains 63.26 g of cerium (IV)
until the last washing shows no turbidity with silver nitrate
tetraammonium sulfate dihydrate [Ce(NH4)4(SO4)4.2H2O:
TS, transfer the precipitate together with the filter paper to a
632.55].
tared crucible, and then heat strongly to ashes. After cool-
Preparation—Dissolve 64 g of cerium (VI) tetraammoni-
ing, add 2 drops of sulfuric acid, and heat strongly again at
um sulfate dihydrate in 0.5 mol/L sulfuric acid VS to make
about 7009C for 2 hours. After cooling, weigh accurately the
1000 mL, allow to stand for 24 hours, filter the solution
residue as barium sulfate (BaSO4), and calculate the molarity
through a glass filter (G3 or G4), if necessary, and stand-
factor.
ardize the solution as follows:
Each mL of 0.02 mol/L barium chloride VS Standardization—Measure exactly 25 mL of the prepared
＝ 4.668 mg of BaSO4 cerium (IV) tetraammonium sulfate solution into an iodine
flask. Add 20 mL of water and 20 mL of dilute sulfuric acid,
Barium Chloride, 0.01 mol/L then dissolve 1 g of potassium iodide in the mixture. Imme-
1000 mL of this solution contains 2.4426 g of barium chlo- diately titrate <2.50> the solution with 0.1 mol/L sodium
ride dihydrate (BaCl2.2H2O: 244.26). thiosulfate VS. When the solution assumes a pale yellow
Preparation—Before use, dilute 0.02 mol/L barium chlo- color as the end point is approached, add 3 mL of starch TS.
ride VS with water to make exactly twice the initial volume. Continue the titration, until the blue color disappears. Per-
form a blank determination. Calculate the molarity factor.
Barium Perchlorate, 0.005 mol/L Note: Store protected from light. This solution, if stored
1000 mL of this solution contains 1.6812 g of barium for a long period of time, should be restandardized.
perchlorate [Ba(ClO4)2: 336.23].
Preparation—Dissolve 1.7 g of barium perchlorate in 200 Cerium (IV) Tetraammonium Sulfate, 0.01 mol/L
mL of water, dilute with 2-propanol to make 1000 mL, and 1000 mL of this solution contains 6.326 g of cerium (IV)
standardize the solution as follows: tetraammonium sulfate dihydrate [Ce(NH4)4(SO4)4.2H2O:
Standardization—Measure exactly 20 mL of the prepared 632.55].
barium perchlorate solution, add 55 mL of methanol and Preparation—Before use, dilute 0.1 mol/L cerium (IV)
0.15 mL of arsenazo III TS. Titrate <2.50> the solution with tetraammonium sulfate VS with 0.5 mol/L sulfuric acid VS
0.005 mol/L sulfuric acid VS until its purple color changes to make exactly 10 times the initial volume.
through red-purple to red. Calculate the molarity factor.
Copper (II) Nitrate, 0.1 mol/L
Bismuth Nitrate, 0.01 mol/L 1000 mL of this solution contains 24.16 g of copper (II)
1000 mL of this solution contains 4.851 g of bismuth nitrate trihydrate [Cu(NO3)2.3H2O: 241.60].
nitrate pentahydrate [Bi(NO3)3.5H2O: 485.07]. Preparation—Dissolve 24.2 g of copper (II) nitrate trihy-
Preparation—Dissolve 4.86 g of bismuth nitrate pentahydrate in water to make 1000 mL, and standardize the solu-
drate in 60 mL of dilute nitric acid, add water to make 1000 tion as follows:
mL, and standardize the solution as follows: Standardization—Measure exactly 10 mL of the prepared
Standardization—Measure exactly 25 mL of the prepared 0.1 mol/L copper (II) nitrate, and add 1 mL of sodium ni-
bismuth nitrate solution, add 50 mL of water and 1 drop of trate solution (9 in 20), 20 mL of acetic acid-ammonium
xylenol orange TS, and titrate <2.50> the solution with 0.01 acetate buffer solution, pH 4.8, and 70 mL of water. Titrate

Disodium Dihydrogen Ethylenediamine Tetraacetate, Ferric Ammonium Sulfate, 0.1 mol/L

0.05 mol/L See Ammonium Iron (III) Sulfate, 0.1 mol/L.
1000 mL of this solution contains 18.612 g of disodium
dihydrogen ethylenediamine tetraacetate dehydrate Ferrous Ammonium Sulfate, 0.1 mol/L
(C10H14N2Na2O8.2H2O: 372.24). See Ammonium Iron (II) Sulfate, 0.1 mol/L.
Preparation—Dissolve 19 g of disodium dihydrogen ethyl-
enediamine tetraacetate dihydrate in water to make 1000 Ferrous Ammonium Sulfate, 0.02 mol/L
mL, and standardize the solution as follows: See Ammonium Iron (II) Sulfate, 0.02 mol/L.
Standardization—Wash zinc (standard reagent) with
dilute hydrochloric acid, water and then acetone, dry at Hydrochloric Acid, 2 mol/L
1109C for 5 minutes, and allow to cool in a desiccator (silica 1000 mL of this solution contains 72.92 g of hydrochloric
gel). Weigh accurately about 0.8 g of this zinc, add 12 mL of acid (HCl: 36.46).
dilute hydrochloric acid and 5 drops of bromine TS, and dis- Preparation—Dilute 180 mL of hydrochloric acid with
solve it by gentle warming. Expel any excess of bromine by water to make 1000 mL, and standardize the solution as
boiling, and add water to make exactly 200 mL. Measure follows:
exactly 20 mL of this solution, and neutralize with sodium Standardization—Proceed as directed for standardization
hydroxide solution (1 in 50). Add 5 mL of ammonia- under 1 mol/L hydrochloric acid VS, but weigh about 1.5 g
ammonium chloride buffer solution, pH 10.7, and 0.04 g of of sodium carbonate (standard reagent) accurately, and dis-
eriochrome black T-sodium chloride indicator. Titrate <2.50> solve in 100 mL of water.
the solution with the prepared disodium dihydrogen ethyl-
Each mL of 2 mol/L hydrochloric acid VS
enediamine tetraacetate solution until the red-purple color
＝ 106.0 mg of Na2CO3
changes to blue-purple. Calculate the molarity factor.
Each mL of 0.05 mol/L disodium dihydrogen Hydrochloric Acid, 1 mol/L
ethylenediamine tetraacetate VS 1000 mL of this solution contains 36.461 g of hydrochloric
＝ 3.269 mg of Zn acid (HCl: 36.46).
Preparation—Dilute 90 mL of hydrochloric acid with
Note: Store in polyethylene bottles.
water to make 1000 mL, and standardize the solution as
follows:
Disodium Dihydrogen Ethylenediamine Tetraacetate,
Standardization—Weigh accurately about 0.8 g of sodium
0.02 mol/L
carbonate (standard reagent), previously heated between
1000 mL of this solution contains 7.445 g of disodium
5009C and 6509 C for 40 to 50 minutes and allowed to cool in
dihydrogen ethylenediamine tetraacetate dihydrate
a desiccator (silica gel). Dissolve it in 50 mL of water, and
(C10H14N2Na2O8.2H2O: 372.24).

titrate <2.50> with the prepared hydrochloric acid to calcu- Hydrochloric Acid, 0.001 mol/L
late the molarity factor (Indicator method: 3 drops of methyl 1000 mL of this solution contains 0.036461 g of hydro-
red TS; or potentiometric titration). In the indicator method, chloric acid (HCl: 36.46).
when the end-point is approached, boil the content carefully, Preparation—Before use, dilute 0.2 mol/L hydrochloric
stopper the flask loosely, allow to cool, and continue the acid VS with water to make exactly 200 times the initial
titration until the color of the solution changes to persistent volume.
orange to orange-red. In the potentiometric titration, titrate
with vigorous stirring, without boiling. Iodine, 0.05 mol/L
1000 mL of this solution contains 12.690 g of iodine (I:
Each mL of 1 mol/L hydrochloric acid VS
126.90).
＝ 53.00 mg of Na2CO3
Preparation—Dissolve 13 g of iodine in 100 mL of a solu-
tion of potassium iodide (2 in 5), add 1 mL of dilute hydro-
Hydrochloric Acid, 0.5 mol/L
chloric acid and water to make 1000 mL, and standardize the
1000 mL of this solution contains 18.230 g of hydrochloric
solution as follows:
acid (HCl: 36.46).
Standardization—Measure exactly 15 mL of the iodine so-
Preparation—Dilute 45 mL of hydrochloric acid with
lution, and titrate <2.50> with 0.1 mol/L sodium thiosulfate
water to make 1000 mL, and standardize the solution as
VS (Indicator method: starch TS; or potentiometric titra-
follows:
tion: platinum electrode). In the indicator method, when the
Standardization—Proceed as directed for standardization
solution assumes a pale yellow color as the end point is ap-
under 1 mol/L hydrochloric acid VS, but weigh accurately
proached, add 3 mL of starch TS, and continue the titration
about 0.4 g of sodium carbonate (standard reagent), and dis-
until the blue color disappears. Calculate the molarity fac-
solve in 50 mL of water.
tor.
Each mL of 0.5 mol/L hydrochloric acid VS Note: Store protected from light. This solution, if stored
＝ 26.50 mg of Na2CO3 for a long period, should be restandardized before use.

Hydrochloric Acid, 0.2 mol/L Iodine, 0.01 mol/L

1000 mL of this solution contains 7.292 g of hydrochloric 1000 mL of this solution contains 2.5381 g of iodine (I:
acid (HCl: 36.46). 126.90).
Preparation—Dilute 18 mL of hydrochloric acid with Preparation—Before use, dilute 0.05 mol/L iodine VS
water to make 1000 mL, and standardize the solution as with water to make exactly 5 times the initial volume.
follows:
Standardization—Proceed as directed for standardization Iodine, 0.005 mol/L
under 1 mol/L hydrochloric acid VS, but weigh accurately 1000 mL of this solution contains 1.2690 g of iodine (I:
about 0.15 g of sodium carbonate (standard reagent), and 126.90).
dissolve in 30 mL of water. Preparation—Before use, dilute 0.05 mol/L iodine VS
with water to make exactly 10 times the initial volume.
Each mL of 0.2 mol/L hydrochloric acid VS
＝ 10.60 mg of Na2CO3
Iodine, 0.002 mol/L
1000 mL of this solution contains 0.5076 g of iodine (I:
Hydrochloric Acid, 0.1 mol/L
126.90).
1000 mL of this solution contains 3.6461 g of hydrochloric
Preparation—Before use, dilute 0.05 mol/L iodine VS
acid (HCl: 36.46).
with water to make exactly 25 times the initial volume.
Preparation—Before use, dilute 0.2 mol/L hydrochloric
acid VS with water to make exactly twice the initial volume.
Magnesium Chloride, 0.05 mol/L
Each mL of 0.1 mol/L hydrochloric acid VS 1000 mL of this solution contains 10.165 g of magnesium
＝ 5.300 mg of Na2CO3 chloride hexahydrate (MgCl2.6H2O: 203.30).
Preparation—Dissolve 10.2 g of magnesium chloride
Hydrochloric Acid, 0.05 mol/L hexahydrate in freshly boiled and cooled water to make
1000 mL of this solution contains 1.8230 g of hydrochloric 1000 mL, and standardize the solution as follows:
acid (HCl: 36.46). Standardization—Measure exactly 25 mL of the prepared
Preparation—Before use, dilute 0.2 mol/L hydrochloric magnesium chloride solution. Add 50 mL of water, 3 mL of
acid VS with water to make exactly 4 times the initial pH 10.7 ammonia-ammonium chloride buffer solution and
volume. 0.04 g of eriochrome black T-sodium chloride indicator, and
titrate <2.50> with 0.05 mol/L disodium dihydrogen ethyl-
Hydrochloric Acid, 0.02 mol/L enediamine tetraacetate VS until the red-purple color of the
1000 mL of this solution contains 0.7292 g of hydrochloric solution changes to blue-purple. Calculate the molarity fac-
acid (HCl: 36.46). tor.
Preparation—Before use, dilute 0.2 mol/L hydrochloric
acid VS with water to make exactly 10 times the initial Magnesium Chloride, 0.01 mol/L
volume. 1000 mL of this solution contains 2.0330 g of magnesium
chloride hexahydrate (MgCl2.6H2O: 203.30).
Hydrochloric Acid, 0.01 mol/L Preparation—Before use, dilute 0.05 mol/L magnesium
1000 mL of this solution contains 0.36461 g of hydrochlo- chloride VS with water to make exactly 5 times the initial
ric acid (HCl: 36.46). volume.
Preparation—Before use, dilute 0.2 mol/L hydrochloric
acid VS with water to make exactly 20 times the initial Oxalic Acid, 0.05 mol/L
volume. 1000 mL of this solution contains 6.303 g of oxalic acid

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
182 9.21 Standard Solutions for Volumetric Analysis / General Tests JP XVII
(C2H2O4.2H2O: 126.07). acid (HClO4: 100.46).
Preparation—Dissolve 6.3 g of oxalic acid dihydrate in Preparation—Before use, dilute 0.1 mol/L perchloric acid
water to make 1000 mL, and standardize the solution as VS with acetic acid for nonaqueous titration to make exactly
follows: 5 times the initial volume. Perform quickly the test as di-
Standardization—Measure exactly 25 mL of the prepared rected under Water Determination with 8.0 mL of acetic acid
oxalic acid solution in a 500-mL conical flask, and add 200 for nonaqueous titration, and designate the water content as
mL of diluted sulfuric acid (1 in 20), previously boiled for A (g/dL). If A is not less than 0.03, add [(A － 0.03) × 52.2]
10 to 15 minutes and then cooled to 27 ± 39C. Transfer mL of acetic anhydride to 1000 mL of acetic acid for nona-
freshly standardized 0.02 mol/L potassium permanganate queous titration, and use it for the preparation.
VS to a burette. Add quickly 22 mL of the 0.02 mol/L potas-
sium permanganate VS to the oxalic acid solution from the Perchloric Acid-1,4-Dioxane, 0.1 mol/L
burette under gentle stirring, and allow to stand until the red 1000 mL of this solution contains 10.046 g of perchloric
color of the mixture disappears. Heat the solution up to be- acid (HClO4: 100.46).
tween 559C and 609C, and complete the titration <2.50> by Preparation—Dilute 8.5 mL of perchloric acid with 1,4-
adding 0.02 mol/L potassium permanganate VS until a faint dioxane to make 1000 mL, and standardize the solution as
red color persists for 30 seconds. Add the last 0.5 to 1 mL follows:
dropwise, being particularly careful to allow the solution to Standardization—Weigh accurately about 0.5 g of potas-
become decolorized before the next drop is added. Calculate sium hydrogen phthalate (standard reagent), previously
the molarity factor. dried at 1059C for 4 hours and allowed to cool in a desicca-
Note: Store protected from light. tor (silica gel). Dissolve it in 80 mL of acetic acid for nona-
queous titration, and add 3 drops of crystal violet TS.
Oxalic Acid, 0.005 mol/L Titrate <2.50> the solution with the prepared perchloric acid-
1000 mL of this solution contains 0.6303 g of oxalic acid 1,4-dioxane solution until it acquires a blue color. Perform a
dihydrate (C2H2O4.2H2O: 126.07). blank determination. Calculate the molarity factor.
Preparation—Before use, dilute 0.05 mol/L oxalic acid
Each mL of 0.1 mol/L perchloric acid-1,4-dioxane VS
VS with water to make exactly 10 times the initial volume.
＝ 20.42 mg of KHC6H4(COO)2
Perchloric Acid, 0.1 mol/L Note: Store in a cold place, protected from moisture.
1000 mL of this solution contains 10.046 g of perchloric
acid (HClO4: 100.46). Perchloric Acid-1,4-Dioxane, 0.05 mol/L
Preparation—Add slowly 8.7 mL of perchloric acid to 1000 mL of this solution contains 5.023 g of perchloric
1000 mL of acetic acid (100) while keeping the temperature acid (HClO4: 100.46).
at about 209C. Allow the mixture to stand for about 1 hour. Preparation—Before use, dilute 0.1 mol/L perchloric
Perform quickly the test as directed under Water Determina- acid-1,4-dioxane VS with 1,4-dioxane to make exactly twice
tion with 3.0 mL of the mixture, and designate the water the initial volume.
content as A (g/dL). To the rest mixture add slowly [(A －
0.03) × 52.2] mL of acetic anhydride with shaking at about Perchloric Acid-1,4-Dioxane, 0.004 mol/L
209 C. Allow the solution to stand for 24 hours, and stand- 1000 mL of this solution contains 0.4018 g of perchloric
ardize it as follows: acid (HClO4: 100.46).
Standardization—Weigh accurately about 0.3 g of potas- Preparation—Before use, dilute 0.1 mol/L perchloric
sium hydrogen phthalate (standard reagent), previously acid-1,4-dioxane VS with 1,4-dioxane to make exactly 25
dried at 1059 C for 4 hours and allowed to cool in a desicca- times the initial volume.
tor (silica gel). Dissolve it in 50 mL of acetic acid (100), and
titrate <2.50> the solution with the prepared perchloric acid Potassium Bichromate, 1/60 mol/L
solution (Indicator method: 3 drops of crystal violet TS; or See Potassium Dichromate, 1/60 mol/L.
potentiometric titration). In the indicator method, titrate
until the solution acquires a blue color. Perform a blank de- Potassium Bromate, 1/60 mol/L
termination. Calculate the molarity factor. 1000 mL of this solution contains 2.7833 g of potassium
bromate (KBrO3: 167.00).
Each mL of 0.1 mol/L perchloric acid VS
Preparation—Dissolve 2.8 g of potassium bromate in
＝ 20.42 mg of KHC6H4(COO)2
water to make 1000 mL, and standardize the solution as
Note: Store protected from moisture. follows:
Standardization—Measure exactly 25 mL of the prepared
Perchloric Acid, 0.05 mol/L potassium bromate solution into an iodine flask. Add 2 g of
1000 mL of this solution contains 5.023 g of perchloric potassium iodide and 5 mL of dilute sulfuric acid, stopper
acid (HClO4: 100.46). the flask, and allow the solution to stand for 5 minutes. Add
Preparation—Before use, dilute 0.1 mol/L perchloric acid 100 mL of water, and titrate <2.50> the liberated iodine with
VS with acetic acid for nonaqueous titration to make exactly 0.1 mol/L sodium thiosulfate VS. When the solution as-
twice the initial volume. Perform quickly the test as directed sumes a pale yellow color as the end point is approached,
under Water Determination with 8.0 mL of acetic acid for add 3 mL of starch TS. Continue the titration until the blue
nonaqueous titration, and designate the water content as A color disappears. Perform a blank determination. Calculate
(g/dL). If A is not less than 0.03, add [(A － 0.03) × 52.2] the molarity factor.
mL of acetic anhydride to 1000 mL of acetic acid for nona-
queous titration, and use it for the preparation. Potassium Dichromate, 1/60 mol/L
1000 mL of this solution contains 4.903 g of potassium
Perchloric Acid, 0.02 mol/L dichromate (K2Cr2O7: 294.18).
1000 mL of this solution contains 2.0092 g of perchloric Preparation—Weigh accurately about 4.903 g of potas-

sium dichromate (standard reagent), previously powdered, sium hydroxide VS, and standardize the solution as follows:
dried between 1009C and 1109 C for 3 to 4 hours and allowed Standardization—Proceed as directed for standardization
to cool in a desiccator (silica gel), dissolve it in water to make under 1 mol/L potassium hydroxide VS, but weigh accu-
exactly 1000 mL, and calculate the molarity factor. rately about 1.3 g of amidosulfuric acid (standard reagent).
Each mL of 0.5 mol/L potassium hydroxide VS
Potassium Ferricyanide, 0.1 mol/L
＝ 48.55 mg of HOSO2NH2
See Potassium Hexacyanoferrate (III), 0.1 mol/L.
Note: Store as directed under 1 mol/L potassium hydrox-
Potassium Ferricyanide, 0.05 mol/L ide VS. This solution, if stored for a long period, should be
See Potassium Hexacyanoferrate (III), 0.05 mol/L. restandardized.

Potassium Hexacyanoferrate (III), 0.1 mol/L Potassium Hydroxide, 0.1 mol/L

1000 mL of this solution contains 32.924 g of potassium 1000 mL of this solution contains 5.611 g of potassium hy-
hexacyanoferrate (III) [K3Fe(CN)6: 329.24]. droxide (KOH: 56.11).
Preparation—Dissolve 33 g of potassium hexacyano- Preparation—Weigh 6.5 g of potassium hydroxide,
ferrate (III) in water to make 1000 mL, and standardize the proceed as directed for preparation under 1 mol/L potas-
solution as follows: sium hydroxide VS, and standardize the solution as follows:
Standardization—Measure exactly 25 mL of the prepared Standardization—Proceed as directed for standardization
potassium hexacyanoferrate (III) solution into an iodine under 1 mol/L potassium hydroxide VS, but weigh accu-
flask. Add 2 g of potassium iodide and 10 mL of dilute hy- rately about 0.25 g of amidosulfuric acid (standard reagent).
drochloric acid, stopper the flask, and allow to stand for 15
Each mL of 0.1 mol/L potassium hydroxide VS
minutes. Add 15 mL of zinc sulfate TS, and titrate <2.50> the
＝ 9.709 mg of HOSO2NH2
liberated iodine with 0.1 mol/L sodium thiosulfate VS.
When the solution assumes a pale yellow color as the end Note: Store as directed under 1 mol/L potassium hydrox-
point is approached, add 3 mL of starch TS. Continue the ide VS. This solution, if stored for a long period, should be
titration, until the blue color disappears. Perform a blank restandardized.
determination. Calculate the molarity factor.
Note: Store protected from light. This solution, if stored Potassium Hydroxide-Ethanol, 0.5 mol/L
for a long period, should be restandardized. 1000 mL of this solution contains 28.053 g of potassium
hydroxide (KOH: 56.11).
Potassium Hexacyanoferrate (III), 0.05 mol/L Preparation—Dissolve 35 g of potassium hydroxide in 20
1000 mL of this solution contains 16.462 g of potassium mL of water, and add aldehyde-free ethanol to make 1000
hexacyanoferrate (III) [K3Fe(CN)6: 329.24]. mL. Allow the solution to stand for 24 hours in a tightly
Preparation—Before use, dilute 0.1 mol/L potassium stoppered bottle. Then quickly decant the supernatant liq-
hexacyanoferrate (III) VS with water to make exactly twice uid, and standardize the solution as follows:
the initial volume. Standardization—Measure exactly 15 mL of 0.25 mol/L
sulfuric acid VS, add 50 mL of water, and titrate with the
Potassium Hydroxide, 1 mol/L prepared potassium hydroxide-ethanol solution to calculate
1000 mL of this solution contains 56.11 g of potassium hy- the molarity factor (Indicator method: 2 drops of phenol-
droxide (KOH: 56.11). phthalein TS; or potentiometric titration). In the indicator
Preparation—Dissolve 65 g of potassium hydroxide in 950 method, titrate <2.50> until the solution acquires a pale red
mL of water. Add a freshly prepared, saturated solution of color.
barium hydroxide octahydrate until no more precipitate is Note: Store in tightly stoppered bottles, protected from
produced. Shake the mixture thoroughly, and allow it to light. Standardize before use.
stand for 24 hours in a tightly stoppered bottle. Decant the
supernatant liquid or filter the solution through a glass filter Potassium Hydroxide-Ethanol, 0.1 mol/L
(G3 or G4), and standardize the solution as follows: 1000 mL of this solution contains 5.611 g of potassium hy-
Standardization—Weigh accurately about 2.5 g of amido- droxide (KOH: 56.11).
sulfuric acid (standard reagent), previously dried in a desic- Preparation—Weigh 7 g of potassium hydroxide, proceed
cator (in vacuum, silica gel) for about 48 hours. Dissolve it as directed for preparation under 0.5 mol/L potassium hy-
in 25 mL of freshly boiled and cooled water, and add 2 drops droxide-ethanol VS, and standardize the solution as follows:
of bromothymol blue TS. Titrate <2.50> the solution with the Standardization—Proceed as directed for standardization
prepared potassium hydroxide solution until it acquires a under 0.5 mol/L potassium hydroxide-ethanol VS, but
green color. Calculate the molarity factor. measure exactly 15 mL of 0.05 mol/L sulfuric acid VS.
Note: Store as directed under 0.5 mol/L potassium
Each mL of 1 mol/L potassium hydroxide VS
hydroxide-ethanol VS. Standardize before use.
＝ 97.09 mg of HOSO2NH2
Note: Store in tightly stoppered bottles or in containers Potassium Iodate, 0.05 mol/L
provided with a carbon dioxide-absorbing tube (soda-lime). 1000 mL of this solution contains 10.700 g of potassium
This solution, if stored for a long period, should be restand- iodate (KIO3: 214.00).
ardized. Preparation—Weigh accurately about 10.700 g of potas-
sium iodate (standard reagent), previously dried between
Potassium Hydroxide, 0.5 mol/L 1209C and 1409 C for 1.5 to 2 hours and allowed to cool
1000 mL of this solution contains 28.053 g of potassium in a desiccator (silica gel), and dissolve it in water to make
hydroxide (KOH: 56.11). exactly 1000 mL. Calculate the molarity factor.
Preparation—Weigh 32 g of potassium hydroxide,
proceed as directed for preparation under 1 mol/L potas-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
184 9.21 Standard Solutions for Volumetric Analysis / General Tests JP XVII
Potassium Iodate, 1/60 mol/L titration: silver electrode). In the indicator method, titrate
1000 mL of this solution contains 3.567 g of potassium until the color of the solution changes from yellow-green to
iodate (KIO3: 214.00). orange through yellow.
Preparation—Weigh accurately about 3.567 g of potas-
Each mL of 0.1 mol/L silver nitrate VS
sium iodate (standard reagent), previously dried between
＝ 5.844 mg of NaCl
1209C and 1409 C for 2 hours and allowed to cool in a desic-
cator (silica gel), and dissolve it in water to make exactly Note: Store protected from light.
1000 mL. Calculate the molarity factor.
Silver Nitrate, 0.02 mol/L
Potassium Iodate, 1/1200 mol/L 1000 mL of this solution contains 3.3974 g of silver nitrate
1000 mL of this solution contains 0.17833 g of potassium (AgNO3: 169.87).
iodate (KIO3: 214.00). Preparation—Before use, dilute 0.1 mol/L silver nitrate
Preparation—Weigh accurately about 0.17833 g of potas- VS with water to make exactly 5 times the initial volume.
sium iodate, previously dried between 1209C and 1409 C for
1.5 to 2 hours and allowed to cool in a desiccator (silica gel), Silver Nitrate, 0.01 mol/L
and dissolve it in water to make exactly 1000 mL. Calculate 1000 mL of this solution contains 1.6987 g of silver nitrate
the molarity factor. (AgNO3: 169.87).
Preparation—Before use, dilute 0.1 mol/L silver nitrate
Potassium Permanganate, 0.02 mol/L VS with water to make exactly 10 times the initial volume.
1000 mL of this solution contains 3.1607 g of potassium
permanganate (KMnO4: 158.03). Silver Nitrate, 0.005 mol/L
Preparation—Dissolve 3.2 g of potassium permanganate 1000 mL of this solution contains 0.8494 g of silver nitrate
in water to make 1000 mL, and boil the solution for 15 (AgNO3: 169.87).
minutes. Allow the solution to stand for at least 48 hours in Preparation—Before use, dilute 0.1 mol/L silver nitrate
a tightly stoppered flask, and filter it through a glass filter VS with water to make exactly 20 times the initial volume.
(G3 or G4). Standardize the solution as follows:
Standardization—Weigh accurately about 0.3 g of sodium Silver Nitrate, 0.001 mol/L
oxalate (standard reagent), previously dried between 1509C 1000 mL of this solution contains 0.16987 g of silver ni-
and 2009C for 1 to 1.5 hours and allowed to cool in a desic- trate (AgNO3: 169.87).
cator (silica gel), transfer it to a 500 mL conical flask, dis- Preparation—Dilute 0.1 mol/L silver nitrate VS with
solve in 30 mL of water, add 250 mL of diluted sulfuric acid water to make exactly 100 times of the initial volume before
(1 in 20), and warm the mixture between 309C and 359C. use.
Transfer the prepared potassium permanganate solution to a
buret, add quickly 40 mL of the solution under gentle stir- Sodium Acetate, 0.1 mol/L
ring from the buret, and allow to stand until the red color of 1000 mL of this solution contains 8.203 g of sodium ace-
the mixture disappears. Warm the solution between 559 C tate (CH3COONa: 82.03).
and 609C, and complete the titration <2.50> with the potas- Preparation—Dissolve 8.20 g of anhydrous sodium ace-
sium permanganate solution until a faint red color persists tate in acetic acid (100) to make 1000 mL, and standardize
for 30 seconds. Add the last 0.5 to 1 mL dropwise before the the solution as follows:
end point, being particularly careful to allow the solution to Standardization—Pipet 25 mL of the prepared sodium
be decolorized before the next drop is added. Calculate the acetate solution, add 50 mL of acetic acid (100) and 1 mL of
molarity factor. p-naphtholbenzeine TS, and titrate <2.50> with 0.1 mol/L
perchloric acid VS until a yellow-brown color changes
Each mL of 0.02 mol/L potassium permanganate VS
through yellow to green. Perform a blank determination.
＝ 6.700 mg of Na2C2O4
Calculate the molarity factor.
Note: Store protected from light. This solution, if stored
for a long period, should be restandardized. Sodium Hydroxide, 1 mol/L
1000 mL of this solution contains 39.997 g of sodium hy-
Potassium Permanganate, 0.002 mol/L droxide (NaOH: 40.00).
1000 mL of this solution contains 0.31607 g of potassium Preparation—Dissolve 42 g of sodium hydroxide in 950
permanganate (KMnO4: 158.03). mL of water. Add a freshly prepared, saturated solution of
Preparation—Before use, dilute 0.02 mol/L potassium barium hydroxide octahydrate until no more precipitate is
permanganate VS with water to make exactly 10 times the produced. Mix well the mixture, and allow to stand for 24
initial volume. hours in a tightly stoppered bottle. Decant the supernatant
liquid or filter the solution through a glass filter (G3 or G4),
Silver Nitrate, 0.1 mol/L and standardize the solution as follows:
1000 mL of this solution contains 16.987 g of silver Standardization—Weigh accurately about 1.5 g of amido-
nitrate (AgNO3: 169.87). sulfuric acid (standard reagent), previously dried in a desic-
Preparation—Dissolve 17.0 g of silver nitrate in water to cator (in vacuum, silica gel) for about 48 hours. Dissolve it
make 1000 mL, and standardize the solution as follows: in 25 mL of freshly boiled and cooled water, and titrate
Standardization—Weigh accurately about 80 mg of so- <2.50> the solution with the prepared sodium hydroxide solu-
dium chloride (standard reagent), previously dried between tion to calculate the molarity factor (Indicator method: 2
5009C and 6509C for 40 to 50 minutes and allowed to cool in drops of bromothymol blue TS; or potentiometric titration).
a desiccator (silica gel), dissolve it in 50 mL of water, and In the indicator method, titrate until the solution acquires a
titrate <2.50> under vigorous stirring with the prepared silver green color.
nitrate solution to calculate the molarity factor (Indicator
method: 3 drops of fluorescein sodium TS; or potentiometric

Each mL of 1 mol/L sodium hydroxide VS droxide VS with freshly boiled and cooled water to make ex-
＝ 97.09 mg of HOSO2NH2 actly 5 times the initial volume.
Note: Store in tightly stoppered bottles or in containers
Sodium Hydroxide, 0.01 mol/L
provided with a carbon dioxide-absorbing tube (soda lime).
1000 mL of this solution contains 0.39997 g of sodium hy-
This solution, if stored for a long period, should be restand-
droxide (NaOH: 40.00).
ardized.
Preparation—Before use, dilute 0.1 mol/L sodium hy-
droxide VS with freshly boiled and cooled water to make ex-
Sodium Hydroxide, 0.5 mol/L
actly 10 times the initial volume.
1000 mL of this solution contains 19.999 g of sodium hy-
droxide (NaOH: 40.00).
Sodium Hydroxide-Ethanol, 0.025 mol/L
Preparation—Weigh 22 g of sodium hydroxide, proceed
1000 mL of this solution contains 1.000 g of sodium hy-
as directed for preparation under 1 mol/L sodium hydroxide
droxide (NaOH: 40.00).
VS, and standardize the solution as follows:
Preparation—Dissolve 2.1 g of sodium hydroxide in 100
Standardization—Proceed as directed for standardization
mL of ethanol (99.5), stopper tightly, and allow to stand for
under 1 mol/L sodium hydroxide VS, but weigh accurately
a night. To 50 mL of the supernatant liquid add 650 mL of
about 0.7 g of amidosulfuric acid (standard reagent).
ethanol (99.5) and freshly boiled and cooled water to make
Each mL of 0.5 mol/L sodium hydroxide VS 1000 mL, and standardize the solution as follows:
＝ 48.55 mg of HOSO2NH2 Standardization—Weigh accurately about 25 mg of ami-
dosulfuric acid (standard reagent), previously dried in a
Note: Store as directed under 1 mol/L sodium hydroxide
desiccator (in vacuum, silica gel) for 48 hours. Dissolve in 30
VS. This solution, if stored for a long period, should be
mL of diluted ethanol (99.5) with fleshly boiled and cooled
restandardized.
water (7 in 10), and titrate <2.50> with the prepared sodium
hydroxide-ethanol to calculate the molarity factor (potentio-
Sodium Hydroxide, 0.2 mol/L
metric titration).
1000 mL of this solution contains 7.999 g of sodium hy-
droxide (NaOH: 40.00). Each mL of 0.025 mol/L sodium hydroxide-ethanol VS
Preparation—Weigh 9 g of sodium hydroxide, proceed as ＝ 2.427 mg of HOSO2NH2
directed for preparation under 1 mol/L sodium hydroxide
Note: Store in light-resistant, well-stoppered bottles. The
VS, and standardize the solution as follows:
standardization should be performed before using.
Standardization—Proceed as directed for standardization
under 1 mol/L sodium hydroxide VS, but weigh accurately
Sodium Lauryl Sulfate, 0.01 mol/L
about 0.3 g of amidosulfuric acid (standard reagent).
1000 mL of this solution contains 2.8838 g of sodium
Each mL of 0.2 mol/L sodium hydroxide VS lauryl sulfate (C12H25NaO4S: 288.38).
＝ 19.42 mg of HOSO2NH2 Preparation—Dissolve 2.9 g of sodium lauryl sulfate in
water to make 1000 mL, and standardize the solution as
Note: Store as directed under 1 mol/L sodium hydroxide
follows:
VS. This solution, if stored for a long period, should be
Standardization—Weigh accurately about 0.3 g of papa-
restandardized.
verine hydrochloride for assay, previously dried, and dis-
solve in water to make exactly 100 mL. Pipet 10 mL of this
Sodium Hydroxide, 0.1 mol/L
solution into a glass-stoppered conical flask, add 5 mL each
1000 mL of this solution contains 3.9997 g of sodium hy-
of water and dilute sulfuric acid and 60 mL of dichlorometh-
droxide (NaOH: 40.00).
ane, then add 5 to 6 drops of a solution of methyl yellow in
Preparation—Weigh 4.5 g of sodium hydroxide, proceed
dichloromethane (1 in 500) as indicator, and titrate <2.50>,
as directed for preparation under 1 mol/L sodium hydroxide
while vigorous shaking, with the sodium lauryl sulfate solu-
VS, and standardize the solution as follows.
tion prepereed above, using a buret with a minimum gradua-
Standardization—Proceed as directed for standardization
tion of 0.02 mL. End point is reached when the color of the
under 1 mol/L sodium hydroxide VS, but weigh accurately
dichloromethane layer changes from yellow to orange-red
about 0.15 g of amidosulfuric acid (standard reagent).
after dropwise addition of the sodium lauryl sulfate solution,
Each mL of 0.1 mol/L sodium hydroxide VS vigorous shaking and standing for a while.
＝ 9.709 mg of HOSO2NH2
Each mL of 0.01 mol/L sodium lauryl sulfate VS
Note: Store as directed under 1 mol/L sodium hydroxide ＝ 3.759 mg of C20H21NO4.HCl
VS. This solution, if stored for a long period, should be
restandardized. Sodium Methoxide, 0.1 mol/L
1000 mL of this solution contains 5.402 g of sodium
Sodium Hydroxide, 0.05 mol/L methoxide (CH3ONa: 54.02).
1000 mL of this solution contains 1.9999 g of sodium hy- Preparation—Add little by little 2.5 g of freshly cut so-
droxide (NaOH: 40.00). dium pieces to 150 mL of methanol cooled in ice-water.
Preparation—Before use, dilute 0.1 mol/L sodium hy- After the sodium has dissolved, add benzene to make 1000
droxide VS with freshly boiled and cooled water to make ex- mL, and standardize the solution as follows:
actly twice the initial volume. Standardization—Weigh accurately about 0.3 g of benzoic
acid, previously dried for 24 hours in a desiccator (silica gel),
Sodium Hydroxide, 0.02 mol/L dissolve it in 80 mL of N, N-dimethylformamide, and add 3
1000 mL of this solution contains 0.7999 g of sodium hy- drops of thymol blue-N, N-dimethylformamide TS. Titrate
droxide (NaOH: 40.00). <2.50> the solution with the prepared sodium methoxide so-
Preparation—Before use, dilute 0.1 mol/L sodium hy- lution until a blue color appears. Perform a blank determi-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
186 9.21 Standard Solutions for Volumetric Analysis / General Tests JP XVII
nation. Calculate the molarity factor. add 2 mL of acetic acid (31), and warm to 509C in a water
bath. Add slowly 50 mL of the prepared sodium tetraphenyl-
Each mL of 0.1 mol/L sodium methoxide VS
borate solution under stirring from a buret, then cool the
＝ 12.21 mg of C6H5COOH
mixture quickly, and allow to stand for 1 hour at room tem-
Note: Store in a cold place, protected from moisture. perature. Transfer the precipitate to a tared glass filter (G4),
Standardize before use. wash with three 5 mL portions of potassium tetraphenyl-
borate TS, dry at 1059C for 1 hour, and weigh accurately the
Sodium Methoxide-Dioxane, 0.1 mol/L glass filter. Calculate the molarity factor from the mass of
See Sodium Methoxide-1,4-Dioxane, 0.1 mol/L. potassium tetraphenylborate [KB(C6H5)4: 358.32].
Each mL of 0.02 mol/L sodium tetraphenylborate VS
Sodium Methoxide-1,4-Dioxane, 0.1 mol/L
＝ 7.166 mg of KB(C6H5)4
1000 mL of this solution contains 5.402 g of sodium
methoxide (CH3ONa: 54.02). Note: Prepare before use.
Preparation—Add little by little 2.5 g of freshly cut so-
dium pieces to 150 mL of methanol cooled in ice-water. Sodium Tetraphenylboron, 0.02 mol/L
After the sodium has dissolved, add 1,4-dioxane to make See Sodium Tetraphenylborate, 0.02 mol/L.
1000 mL, and standardize the solution as follows:
Standardization—Weigh accurately about 0.3 g of benzoic Sodium Thiosulfate, 0.1 mol/L
acid, previously dried in a desiccator (silica gel) for 24 hours, 1000 mL of this solution contains 24.818 g of sodium thio-
dissove it in 80 mL of N, N-dimethylformamide, and add 3 sulfate pentahydrate (Na2S2O3.5H2O: 248.18).
drops of thymol blue-N, N-dimethylformamide TS. Titrate Preparation—Dissolve 25 g of sodium thiosulfate and
<2.50> the solution with the prepared sodium methoxide-1,4- 0.2 g of anhydrous sodium carbonate in freshly boiled and
dioxane solution until a blue color appears. Perform a blank cooled water to make 1000 mL, allow to stand for 24 hours,
determination. Calculate the molarity factor. and standardize the solution as follows:
Standardization—Weigh accurately about 50 mg of potas-
Each mL of 0.1 mol/L sodium methoxide-1,4-dioxane VS
sium iodate (standard reagent), previously dried between
＝ 12.21 mg of C6H5COOH
1209C and 1409 C for 1.5 to 2 hours and allowed to cool in a
Note: Store in a cold place, protected from moisture. desiccator (silica gel), and transfer to an iodine flask. Dis-
Standardize before use. solve it in 25 mL of water, add 2 g of potassium iodide and
10 mL of dilute sulfuric acid, and stopper the flask. After
Sodium Nitrite, 0.1 mol/L allowing the mixture to stand for 10 minutes, add 100 mL of
1000 mL of this solution contains 6.900 g of sodium nitrite water, and titrate <2.50> the liberated iodine with the pre-
(NaNO2: 69.00). pared sodium thiosulfate solution (Indicator method; or
Preparation—Dissolve 7.2 g of sodium nitrite in water to potentiometric titration: platinum electrode). In the indica-
make 1000 mL, and standardize the solution as follows: tor method, when the solution assumes a pale yellow color as
Standardization—Weigh accurately about 0.44 g of sul- the end point is approached, add 3 mL of starch TS. Con-
fanilamide for titration of diazotization, previously dried at tinue the titration, until the blue color disappears. Perform a
1059C for 3 hours and allowed to cool in a desiccator (silica blank determination. Calculate the molarity factor.
gel), dissolve in 10 mL of hydrochloric acid, 40 mL of water
Each mL of 0.1 mol/L sodium thiosulfate VS
and 10 mL of a solution of potassium bromide (3 in 10), cool
＝ 3.567 mg of KIO3
below 159 C, and titrate with the prepared sodium nitrite so-
lution as directed in the potentiometric titration or ampero- Note: This solution, if stored for a long period, should be
metric titration under Endpoint Detection Methods in Titri- restandardized.
metry <2.50>. Calculate the molarity factor.
Sodium Thiosulfate, 0.05 mol/L
Each mL of 0.1 mol/L sodium nitrite VS
1000 mL of this solution contains 12.409 g of sodium thio-
＝ 17.22 mg of H2NC6H4SO2NH2
sulfate pentahydrate (Na2S2O3.5H2O: 248.18).
Note: Store protected from light. This solution, if stored Preparation—Before use, dilute 0.1 mol/L sodium thio-
for a long period, should be restandadized. sulfate VS with freshly boiled and cooled water to make ex-
actly 2 times the initial volume.
Sodium Oxalate, 0.005 mol/L
1000 mL of this solution contains 0.6700 g of sodium oxa- Sodium Thiosulfate, 0.02 mol/L
late (Na2C2O4: 134.00). 1000 mL of this solution contains 4.964 g of sodium thio-
Preparation—Weigh accurately about 0.6700 g of sodium sulfate pentahydrate (Na2S2O3.5H2O: 248.18).
oxalate (standard reagent), previously dried between 1509C Preparation—Before use, dilute 0.1 mol/L sodium thio-
and 2009C for 2 hours and allowed to cool in a desiccator sulfate VS with freshly boiled and cooled water to make ex-
(silica gel), dissolve it in water to make exactly 1000 mL, and actly 5 times the initial volume.
calculate the molarity factor.
Sodium Thiosulfate, 0.01 mol/L
Sodium Tetraphenylborate, 0.02 mol/L 1000 mL of this solution contains 2.4818 g of sodium thio-
1000 mL of this solution contains 6.844 g of sodium sulfate pentahydrate (Na2S2O3.5H2O: 248.18).
tetraphenylborate [NaB(C6H5)4: 342.22]. Preparation—Before use, dilute 0.1 mol/L sodium thio-
Preparation—Dissolve 7.0 g of sodium tetraphenylborate sulfate VS with freshly boiled and cooled water to make ex-
in water to make 1000 mL, and standardize the solution as actly 10 times the initial volume.
follows:
Standardization—Weigh 0.5 g of potassium hydrogen Sodium Thiosulfate, 0.005 mol/L
phthalate (standard reagent), dissolve it in 100 mL of water, 1000 mL of this solution contains 1.2409 g of sodium thio-

sulfate pentahydrate (Na2S2O3.5H2O: 248.18). Standardization—Proceed as directed for standardization

Preparation—Before use, dilute 0.1 mol/L sodium thio- under 0.5 mol/L sulfuric acid VS, but weigh accuretely
sulfate VS with freshly boiled and cooled water to make ex- about 80 mg of sodium carbonate (standard reagent), and
actly 20 times the initial volume. dissolve in 30 mL of water.
Each mL of 0.05 mol/L sulfuric acid VS
Sodium Thiosulfate, 0.002 mol/L
＝ 5.300 mg of Na2CO3
1000 mL of this solution contains 0.4964 g of sodium thio-
sulfate pentahydrate (Na2S2O3.5H2O: 248.18).
Sulfuric Acid, 0.025 mol/L
Preparation—Before use, dilute 0.1 mol/L sodium thio-
1000 mL of this solution contains 2.4520 g of sulfuric acid
sulfate VS with freshly boiled and cooled water to make ex-
(H2SO4: 98.08).
actly 50 times the initial volume.
Preparation—Before use, dilute 0.05 mol/L sulfuric acid
VS with water to make exactly twice the initial volume.
Sulfuric Acid, 0.5 mol/L
1000 mL of this solution contains 49.04 g of sulfuric acid
Sulfuric Acid, 0.02 mol/L
(H2SO4: 98.08).
1000 mL of this solution contains 1.9616 g of sulfuric acid
Preparation—Add slowly, under stirring, 30 mL of sulfu-
(H2SO4: 98.08).
ric acid to 1000 mL of water, allow to cool, and standardize
Preparation—Before use, dilute 0.05 mol/L sulfuric acid
the solution as follows:
VS with water to make exactly 2.5 times the initial volume.
Standardization—Weigh accurately about 0.8 g of sodium
carbonate (standard reagent), previously heated between
Sulfuric Acid, 0.01 mol/L
5009C and 6509C for 40 to 50 minutes and allowed to cool in
1000 mL of this solution contains 0.9808 g of sulfuric acid
a desiccator (silica gel). Dissolve it in 50 mL of water, and
(H2SO4: 98.08).
titrate <2.50> the solution with the prepared sulfuric acid (In-
Preparation—Before use, dilute 0.05 mol/L sulfuric acid
dicator method: 3 drops of methyl red TS; or potentiometric
VS with water to make exactly 5 times the initial volume.
titration). In the indicator method, when the end point is ap-
proached, boil the solution carefully, stopper the flask loose-
Sulfuric Acid, 0.005 mol/L
ly, allow to cool, and continue the titration, until the color
1000 mL of this solution contains 0.4904 g of sulfuric acid
of the solution changes to persistent orange to orange-red.
(H2SO4: 98.08).
Calculate the molarity factor. In the potentiometric titra-
Preparation—Before use, dilute 0.05 mol/L sulfuric acid
tion, titrate with vigorous stirring without boiling.
VS with water to make exactly 10 times the initial volume.
Each mL of 0.5 mol/L sulfuric acid VS
＝ 53.00 mg of Na2CO3 Surfuric Acid, 0.0005 mol/L
1000 mL of this solution contains 0.04904 g of sulfuric
Sulfuric Acid, 0.25 mol/L acid (H2SO4: 98.08).
1000 mL of this solution contains 24.520 g of sulfuric acid Preparation—Before use, dilute 0.05 mol/L sulfuric acid
(H2SO4: 98.08). VS with water to make exactly 100 times the initial volume.
Preparation—Add slowly, under stirring, 15 mL of sulfu-
ric acid to 1000 mL of water, allow to cool, and standardize Tetrabutylammonium Hydroxide, 0.1 mol/L
the solution as follows: 1000 mL of this solution contains 25.947 g of tetrabutyl
Standardization—Proceed as directed for standardization ammonium hydroxide [(C4H9)4NOH: 259.47].
under 0.5 mol/L sulfuric acid VS, but weigh accurately Preparation—Before use, dilute a volume of 10z
about 0.4 g of sodium carbonate (standard reagent), and dis- tetrabutylammonium hydroxide-methanol TS, equivalent to
solve in 50 mL of water. 26.0 g of tetrabutylammonium hydroxide, with 2-propanol
to make 1000 mL, and standardize the solution as follows:
Each mL of 0.25 mol/L sulfuric acid VS
Standardization—Weigh accurately about 0.3 g of benzoic
＝ 26.50 mg of Na2CO3
acid, previously dried in a desiccator (silica gel) for 24 hours,
dissolve it in 50 mL of acetone, and titrate <2.50> the solu-
Sulfuric Acid, 0.1 mol/L
tion with the prepared tetrabutylammonium hydroxide solu-
1000 mL of this solution contains 9.808 g of sulfuric acid
tion (potentiometric titration). Perform a blank determina-
(H2SO4: 98.08).
tion in the same manner.
Preparation—Add slowly, under stirring, 6 mL of sulfuric
acid to 1000 mL of water, allow to cool, and standardize the Each mL of 0.1 mol/L tetrabutylammonium
solution as follows: hydroxide VS
Standardization—Proceed as directed for standardization ＝ 12.21 mg of C6H5COOH
under 0.5 mol/L sulfuric acid VS, but weigh accurately
Note: Store in tightly stoppered bottles. This solution, if
about 0.15 g of sodium carbonate (standard reagent), and
stored for a long period, should be restandardized.
dissolve in 50 mL of water.
Each mL of 0.1 mol/L sulfuric acid VS Tetramethylammonium Hydroxide, 0.2 mol/L
＝ 10.60 mg of Na2CO3 1000 mL of this solution contains 18.231 g of tetramethy-
lammonium hydroxide [(CH3)4NOH: 91.15].
Sulfuric Acid, 0.05 mol/L Preparation—Before use, dilute a volume of tetramethy-
1000 mL of this solution contains 4.904 g of sulfuric acid lammonium hydroxide-methanol TS, equivalent to 18.4 g of
(H2SO4: 98.08). tetramethylammonium hydroxide, with water to make 1000
Preparation—Add slowly, under stirring, 3 mL of sulfuric mL, and standardize the solution as follows:
acid to 1000 mL of water, allow to cool, and standardize the Standardization—Weigh accurately about 0.4 g of benzoic
solution as follows: acid, previously dried in a desiccator (silica gel) for 24 hours,

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
188 9.21 Standard Solutions for Volumetric Analysis / General Tests JP XVII
dissolve it in 60 mL of N, N-dimethylformamide, and titrate <2.50> with the prepared titanium (III) chloride solution until
<2.50> the solution with the prepared 0.2 mol/L tetramethyl the calculated end point is approached, then add 5 g of am-
ammonium hydroxide solution (Indicator method: 3 drops monium thiocyanate immediately, and continue the titration
of thymol blue-N, N-dimethylformamide TS; or potentio- with the prepared titanium (III) chloride solution until the
metric titration). In the indicator method, titrate until a blue color of the solution disappears. Perform a blank determina-
color is produced. Perform a blank determination in the tion. Calculate the molarity factor.
same manner. Calculate the molarity factor. Note: Store after the air has been displaced with hydro-
gen.
Each mL of 0.2 mol/L tetramethylammonium
hydroxide VS
Titanium Trichloride, 0.1 mol/L
＝ 24.42 mg of C6H5COOH
See Titanium (III) Chloride, 0.1 mol/L.
Note: Store in tightly stoppered bottles. This solution, if
stored for a long period, should be restandardized. Zinc, 0.1 mol/L
1000 mL of this solution contains 6.538 g of zinc (Zn:
Tetramethylammonium Hydroxide, 0.1 mol/L 65.38).
1000 mL of this solution contains 9.115 g of tetramethy- Preparation—To 6.538 g of zinc (standard reagent), previ-
lammonium hydroxide [(CH3)4NOH: 91.15]. ously washed with dilute hydrochloric acid, with water and
Preparation—Before use, dilute a volume of tetramethy- then acetone, and cooled in a desiccator (silica gel) after dry-
lammonium hydroxide-methanol TS, equivalent to 9.2 g of ing at 1109C for 5 minutes, add 80 mL of dilute hydrochloric
tetramethylammonium hydroxide, with water to make 1000 acid and 2.5 mL of bromine TS, dissolve by gentle warming,
mL, and standardize the solution as follows: evaporate excess bromine by boiling, and add water to make
Standardization—Proceed as directed for standardization exactly 1000 mL.
under 0.2 mol/L tetramethylammonium hydroxide VS.
Weigh accurately about 0.2 g of benzoic acid and titrate Zinc Acetate, 0.05 mol/L
<2.50>. 1000 mL of this solution contains 10.975 g of zinc acetate
dihydrate [Zn(CH3COO)2.2H2O: 219.50].
Each mL of 0.1 mol/L tetramethylammonium
Preparation—Dissolve 11.1 g of zinc acetate dihydrate in
hydroxide VS
40 mL of water and 4 mL of dilute acetic acid, add water to
＝ 12.21 mg of C6H5COOH
make 1000 mL, and standardize the solution as follows:
Note: Store in tightly stoppered bottles. This solution, if Standardization—Measure exactly 20 mL of 0.05 mol/L
stored for a long period, should be restandardized. disodium dihydrogen ethylenediamine tetraacetate VS, and
add 50 mL of water, 3 mL of ammonia-ammonium chloride
Tetramethylammonium Hydroxide, 0.02 mol/L buffer solution, pH 10.7, and 0.04 g of eriochrome black T-
1000 mL of this solution contains 1.8231 g of tetramethy- sodium chloride reagent. Titrate <2.50> the solution with the
lammonium hydroxide [(CH3)4NOH: 91.15]. prepared zinc acetate solution, until the blue color changes
Preparation—Before use, dilute 0.1 mol/L tetramethy- to blue-purple. Calculate the molarity factor.
lammonium hydroxide VS with freshly boiled and cooled
water to make exactly 5 times the initial volume. Zinc Acetate, 0.02 mol/L
1000 mL of this solution contains 4.390 g of zinc acetate
Tetramethylammonium Hydroxide-Methanol, 0.1 mol/L dihydrate [Zn(CH3COO)2.2H2O: 219.50].
1000 mL of this solution contains 9.115 g of tetramethy- Preparation—Dissolve 4.43 g of zinc acetate dihydrate in
lammonium hydroxide [(CH3)4NOH: 91.15]. 20 mL of water and 2 mL of dilute acetic acid, add water to
Preparation—Before use, dilute a volume of tetramethy- make 1000 mL, and standardize the solution as follows:
lammonium hydroxide-methanol TS, equivalent to 9.2 g of Standardization—Proceed as directed for standardization
tetramethylammonium hydroxide, with methanol to make under 0.05 mol/L zinc acetate VS, but measure exactly 20
1000 mL, and standardize the solution as follows: mL of 0.02 mol/L disodium dihydrogen ethylenediamine
Standardization—Proceed as directed for standardization tetraacetate VS.
under 0.1 mol/L tetramethylammonium hydroxide VS.
Note: Store in tightly stoppered bottles. This solution, if Zinc Sulfate, 0.1 mol/L
stored for a long period, should be restandardized. 1000 mL of this solution contains 28.755 g of zinc sulfate
heptahydrate (ZnSO4.7H2O: 287.55).
Titanium (III) Chloride, 0.1 mol/L Preparation—Dissolve 28.8 g of zinc sulfate heptahydrate
1000 mL of this solution contains 15.423 g of titanium in water to make 1000 mL, and standardize the solution as
(III) chloride (TiCl3: 154.23). follows:
Preparation—Add 75 mL of hydrochloric acid to 75 mL Standardization—Pipet 25 mL of the prepared zinc sulfate
of titanium (III) chloride (20), and dilute with freshly boiled solution, add 5 mL of ammonia-ammonium chloride buffer
and cooled water to make 1000 mL. Transfer the solution solution, pH 10.7, and 0.04 g of eriochrome black T-sodium
into a buret provided with a reservoir protected from light, chloride indicator, and titrate <2.50> with 0.1 mol/L disodi-
replace the air with hydrogen, and allow to stand for 48 um dihydrogen ethylenediamine tetraacetate VS until the
hours. Before use, standardize the solution as follows: color of the solution changes from red-purple to blue-purple.
Standardization—Weigh 3 g of ammonium iron (II) sul- Calculate the molarity factor.
fate hexahydrate in a wide-mouthed, 500 mL conical flask.
Passing carbon dioxide through the flask, dissolve it in 50 Zinc sulfate, 0.02 mol/L
mL of freshly boiled and cooled water, and add 25 mL of 1000 mL of this solution contains 5.7510 g of zinc sulfate
diluted sulfuric acid (27 in 100). Rapidly add exactly 40 mL heptahydrate (ZnSO4.7H2O: 287.55).
of 0.02 mol/L potassium permanganate VS to the mixture, Preparation—Before use, dilute 0.1 mol/L zinc sulfate VS
while passing carbon dioxide through the flask. Titrate with water to make exactly 5 times the initial volume.

limit test to make exactly 1000 mL. Measure exactly 10 mL

9.22 Standard Solutions of this solution, and add water for ammonium limit test to
make exactly 1000 mL. Each mL of this solution contains
Standard Solutions are used as the standard for the com- 0.01 mg of ammonium (NH4＋).
parison in a text of the Pharmacopoeia.
Standard Arsenic Solution See Arsenic Limit Test
Borate pH Standard Solution See pH Determination <1.11>.
<2.54>.
Standard Arsenic Stock Solution See Arsenic Limit Test
Calcium Hydroxide pH Standard Solution See pH De- <1.11>.
termination <2.54>.
Standard Boron Solution Weigh exactly 0.286 g of boric
Carbonate pH Standard Solution See pH Determination acid, previously dried in a desiccator (silica gel) to constant
<2.54>. mass, and dissolve in water to make exactly 1000 mL. Pipet
10 mL of this solution, and add water to make exactly 1000
Certified Standard Arsenic Solution See Arsenic Limit
mL. Each mL of this solution contains 0.5 mg of boron (B).
Test <1.11>.
Standard Cadmium Solution Measure exactly 10 mL of
Formazin stock suspension To 25 mL of hexamethyl-
Standard Cadmium Stock Solution, and add diluted nitric
enetetramine TS add 25 mL of hydrazinium sulfate TS, mix,
acid (1 in 3) to make exactly 1000 mL. Pipet 10 mL of this
and use after allowing to stand at room temperature for 24
solution, and add diluted nitric acid (1 in 3) to make 100 mL.
hours. Store in a glass container free from surface defects.
Each mL of this solution contains 0.001 mg of cadmium
Use within 2 months. Shake thoroughly before use. The tur-
(Cd). Prepare before use.
bidity of this suspension is equivalent to 4000 NTU.
Standard Cadmium Stock Solution Dissolve 1.000 g of
Oxalate pH Standard Solution See pH Determination
cadmium ground metal, exactly weighed, in 100 mL of dilute
<2.54>.
nitric acid by gentle heating, cool, and add dilute nitric acid
pH Standard Solution, Borate See pH Determination to make exactly 1000 mL.
<2.54>.
Standard Calcium Solution Weigh exactly 0.250 g of cal-
pH Standard Solution, Calcium Hydroxide See pH De- cium carbonate, add 5 mL of dilute hydrochloric acid and 25
termination <2.54>. mL of water, and dissolve by heating. After cooling, add
water to make exactly 1000 mL. Each mL of this solution
pH Standard Solution, Carbonate See pH Determina-
contains 0.1 mg of calcium (Ca).
tion <2.54>.
Standard Calcium Solution for Atomic Absorption Spec-
pH Standard Solution, Oxalate See pH Determination
trophotometry Weigh accurately 0.250 g of calcium
<2.54>.
carbonate, and add 1 mol/L hydrochloric acid TS to make
pH Standard Solution, Phosphate See pH Determina- exactly 100 mL. Each mL of this solution contains 1.00 mg
tion <2.54>. of calcium (Ca).
pH Standard Solution, Phthalate See pH Determination Standard Chromium Solution for Atomic Absorption
<2.54>. Spectrophotometry Weigh exactly 0.283 g of potassium
dichromate (standard reagent), dissolve in water to make
Phosphate pH Standard Solution See pH Determination
exactly 1000 mL. Each mL contains 0.10 mg of chromium
<2.54>.
(Cr).
Phthalate pH Standard Solution See pH Determination
Standard Copper Solution Pipet 10 mL of Standard
<2.54>.
Copper Stock Solution, and dilute with water to make ex-
Standard Aluminum Solution for Atomic Absorption actly 1000 mL. Each mL of this solution contains 0.01 mg of
Spectrophotometry To exactly 10 mL of Standard Alumi- copper (Cu). Prepare before use.
num Stock Solution add water to make exactly 100 mL. Pre-
Standard Copper Stock Solution Weigh exactly 1.000 g
pare before use. Each mL of this solution contains 0.100 mg
of copper (standard reagent), add 100 mL of dilute nitric
of aluminum (Al).
acid, and dissolve by heating. After cooling, add water to
Standard Aluminum Stock Solution Weigh 1.0 g of alu- make exactly 1000 mL.
minum, add 60 mL of diluted hydrochloric acid (1 in 2), dis-
Standard Cyanide Solution Measure exactly a volume of
solve by heating, cool, add water to make 1000 mL. Pipet 10
Standard Cyanide Stock Solution, equivalent to 10 mg of
mL of this solution, add 30 mL of water and 5 mL of acetic
cyanide (CN), add 100 mL of sodium hydroxide TS and
acid-ammonium acetate buffer solution, pH 3.0, and adjust
water to make exactly 1000 mL. Each mL of this solution
the pH to about 3 with ammonia TS added dropwise. Then,
contains 0.01 mg of cyanide (CN). Prepare before use.
add 0.5 mL of Cu-PAN TS, and titrate <2.50> with 0.01
mol/L disodium dihydrogen ethylenediamine tetraacetate Standard Cyanide Stock Solution Dissolve 2.5 g of po-
VS while boiling until the color of the solution changes from tassium cyanide in water to make exactly 1000 mL. Measure
red to yellow lasting for more than 1 minute. Perform a exactly 100 mL of this solution, add 0.5 mL of 4-
brank determination. dimethylaminobenzylidene rhodanine TS, and titrate <2.50>
with 0.1 mol/L silver nitrate VS until the solution shows a
Each mL of 0.01 mol/L disodium dihydrogen
red color.
ethylenediamine tetraacetate VS
＝ 0.2698 mg of Al Each mL of 0.1 mol/L silver nitrate VS
＝ 5.204 mg of CN
Standard Ammonium Solution Dissolve 2.97 g of am-
monium chloride, exactly weighed, in water for ammonium Standard Fluorine Solution See Oxygen Flask Combus-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
190 General Tests / 9.22 Standard Solutions JP XVII
tion Method <1.06>. Standard Magnesium Stock Solution Dissolve exactly
8.365 g of magnesium chloride hexahydrate in 2 mol/L hy-
Standard Gold Solution for Atomic Absorption Spectro-
drochloric acid TS to make exactly 1000 mL.
photometry To 25 mL of Standard Gold Stock Solution,
exactly measured, add water to make exactly 1000 mL. Each Standard Mercury Solution Weigh exactly 13.5 mg of
mL of this solution contains 0.025 mg of gold (Au). mercury (II) chloride, previously dried for 6 hours in a desic-
cator (silica gel), dissolve in 10 mL of dilute nitric acid, and
Standard Gold Stock Solution Dissolve 0.209 g of
add water to make exactly 1000 mL. Pipet 10 mL of this so-
hydrogen tetrachloroaurate (III) tetrahydrate, exactly
lution, and add 10 mL of dilute nitric acid and water to
weighed, in 2 mL of aqua regia, heat on a water bath for 10
make exactly 1000 mL. Each mL of this solution contains
minutes, and add 1 mol/L hydrochloric acid TS to make ex-
0.1 mg of mercury (Hg). Prepare before use.
actly 100 mL. Each mL of this solution contains 1.00 mg of
gold (Au). Standard Methanol Solution See Methanol Test <1.12>.
Standard Hydrogen Peroxide Stock Solution To an Standard Nickel Solution Dissolve 6.73 g of ammonium
amount of hydrogen peroxide (30) add water to make a solu- nickel (II) sulfate hexahydrate, exactly weighed, in water to
tion so that each mL contains 0.30 g of hydrogen peroxide make exactly 1000 mL. Pipet 5 mL of this solution, add
(H2O2:34.01). Pipet 1 mL of this solution, add water to water to make exactly 1000 mL. Each mL of this solution
make exactly 10 mL, pipet 1 mL of this solution, transfer it contains 0.005 mg of nickel (Ni).
to a flask containing 10 mL of water and 10 mL of dilute sul-
Standard Nickel Solution for Atomic Absorption Spectro-
furic acid, and titrate <2.50> with 0.02 mol/L potassium per-
photometry To exactly 10 mL of Standard Nickel Stock
manganate VS until the color of the solution changes to
Solution add water to make exactly 1000 mL. Prepare before
slightly red. Perform a blank determination in the same
use. Each mL of this solution contains 0.01 mg of nickel
manner, and make any necessary correction.
(Ni).
Each mL of 0.02 mol/L potassium permanganate VS
Standard Nickel Stock Solution Dissolve exactly 4.48 g
＝ 1.701 mg of H2O2
of nickel (II) sulfate hexahydrate in water to make exactly
Standard Hydrogen Peroxide Solution To exactly 10 mL 1000 mL.
of Standard Hydrogen Peroxide Stock Solution add water to
Standard Nitric Acid Solution Weigh exactly 72.2 mg of
make exactly 100 mL. Prepare before use. Each mL contains
potassium nitrate, dissolve in water to make exactly 1000
30 mg of hydrogen peroxide (H2O2:34.01).
mL. Each mL of this solution contains 0.01 mg of nitrogen
Standard Iron Solution Weigh exactly 86.3 mg of ammo- (N).
nium iron (III) sulfate dodecahydrate, dissolve in 100 mL of
Standard Palladium Solution for ICP Analysis Standard
water, and add 5 mL of dilute hydrochloric acid and water
solution specified by the Measurement Law. Each mL of this
to make exactly 1000 mL. Each mL of this solution contains
solution contains 1 mg of palladium (Pd).
0.01 mg of iron (Fe).
Standard Phosphoric Acid Solution Weigh exactly
Standard Iron Solution for Atomic Absorption Spectro-
0.358 g of potassium dihydrogen phosphate, previously
photometry To exactly 5 mL of Standard Iron Stock Solu-
dried to constant mass in a desiccator (silica gel), and add 10
tion add water to make exactly 200 mL. Prepare before use.
mL of diluted sulfuric acid (3 in 10) and water to make ex-
Each mL of this solution contains 0.250 mg of iron (Fe).
actly 1000 mL. Pipet 10 mL of this solution, and add water
Standard Iron Solution (2) for Atomic Absorption Spec- to make exactly 100 mL. Each mL of this solution contains
trophotometry To exactly 2 mL of Standard Iron Stock So- 0.025 mg of phosphoric acid (as PO4).
lution add water to make exactly 250 mL. Pipet 10 mL of
Standard Potassium Stock Solution Weigh exactly
this solution, add water to make exactly 100 mL. Prepare be-
9.534 g of potassium chloride, previously dried at 1309C for
fore use. Each mL contains 8 mg of iron (Fe).
2 hours, and dissolve in water to make exactly 1000 mL.
Standard Iron Stock Solution Dissolve exactly 4.840 g of Each mL of this solution contains 5.00 mg of potassium (K).
iron (III) chloride hexahydrate in diluted hydrochloric acid
Standard Selenium Solution To exactly 1 mL of Stand-
(9 in 25) to make exactly 100 mL.
ard Selenium Stock Solution add water to make exactly 1000
Standard Lead Solution Measure exactly 10 mL of mL. Prepare before use. It contains 1.0 mg of selenium (Se)
Standard Lead Stock Solution, and add water to make ex- per mL.
actly 100 mL. Each mL of this solution contains 0.01 mg of
Standard Selenium Stock Solution Dissolve exactly
lead (Pb). Prepare before use.
1.405 g of selenium dioxide in 0.1 mol/L nitric acid to make
Standard Lead Stock Solution Weigh exactly 159.8 mg exactly 1000 mL.
of lead (II) nitrate, dissolve in 10 mL of dilute nitric acid,
Standard Silver Solution for Atomic Absorption Spectro-
and add water to make exactly 1000 mL. Prepare and store
photometry Measure exactly 10 mL of Standard Silver
this solution using glass containers, free from soluble lead
Stock Solution, and add water to make exactly 1000 mL.
salts.
Each mL of this solution contains 0.01 mg of silver (Ag).
Standard Liquids for Calibrating Viscosimeters [JIS, Prepare before use.
Standard Liquids for Calibrating Viscosimeters (Z 8809)]
Standard Silver Stock Solution Dissolve 1.575 g of silver
Standard Magnesium Solution for Atomic Absorption nitrate, exactly weighed, in water to make exactly 1000 mL.
Spectrophotometry To exactly 1 mL of Standard Magnesi- Each mL of this solution contains 1.00 mg of silver (Ag).
um Stock Solution add water to make exactly 100 mL. Pre-
Standard Sodium Dodecylbenzene Sulfonate Solution
pare before use. Each mL of this solution contains 0.0100
Weigh exactly 1.000 g of sodium dodecylbenzene sulfonate,
mg of magnesium (Mg).
and dissolve in water to make exactly 1000 mL. Pipet 10 mL

of this solution, and add water to make exactly 1000 mL.

Each mL of this solution contains 0.01 mg of sodium Reagents, Test Solutions, etc.
dodecylbenzene sulfonate [CH3(CH2)11C6H4SO3Na].
Standard Sodium Stock Solution Weigh exactly 2.542 g
of sodium chloride (standard reagent), previously dried at 9.41 Reagents, Test Solutions
1309C for 2 hours, and dissolve in water to make exactly
Reagents are the substances used in the tests of the Phar-
1000 mL. Each mL of this solution contains 1.00 mg of so-
macopoeia. The reagents that are described as ``Standard
dium (Na).
reagent for volumetric analysis'', ``Special class'', ``First
Standard Sulfite Solution Dissolve exactly 3.150 g of an- class'', ``For water determination'', etc. in square brackets
hydrous sodium sulfite in freshly prepared distilled water to meet the corresponding requirements of the Japan Industrial
make exactly 100 mL. Pipet 0.5 mL of this solution, add Standards (JIS). The tests for them are performed according
freshly prepared distilled water to make exactly 100 mL. to the test methods of JIS. The reagents that are described as
Each mL of this solution contains 80 mg of sulfur dioxide ``Certified reference material'' are those noted a certificate
(SO2). Prepare before use. on the basis of JIS Q 0030 and guaranteed the traceability of
the international system of units. These reference materials
Standard Tin Solution Weigh exactly 0.250 g of tin, and
are provided by the Metrology Management Center,
dissolve in 10 mL of sulfuric acid by heating. After cooling,
National Institute of Advanced Industrial Science and Tech-
transfer this solution with 400 mL of diluted hydrochloric
nology (AIST) and manufacturers of the certified reference
acid (1 in 5) to a 500-mL volumetric flask, and add diluted
materials. In the case where the reagent name in the Pharma-
hydrochloric acid (1 in 5) to make 500 mL. Pipet 10 mL of
copoeia differs from that of JIS, the JIS name is given in the
this solution, and add diluted hydrochloric acid (1 in 5) to
brackets. The reagents for which a monograph's title is given
make exactly 1000 mL. Each mL of this solution contains
in the brackets meet the requirements of the corresponding
0.005 mg of tin (Sn). Prepare before use.
monograph. In the case of the reagents that are described
Standard Vinyl Chloride Solution Transfer about 190 merely as test items, the corresponding test method of the
mL of ethanol for gas chromotography into a 200-mL volu- Pharmacopoeia is applied.
metric flask, and stopper with a silicone rubber stopper. Test Solutions are the solutions prepared for use in the
Cooling this volumetric flask in a methanol-dry ice bath, in- tests of the Pharmacopoeia.
ject 0.20 g of vinyl chloride, previously liquidized, through
Acemetacin C21H18ClNO6 [Same as the namesake
the silicone rubber stopper, and then inject ethanol for gas
monograph]
chromatography, previously cooled in a methanol-dry ice
bath, through the silicone rubber stopper to make 200 mL. Acemetacin for assay C21H18ClNO6 [Same as the
Pipet 1 mL of this solution, add ethanol for gas chromatog- monograph Acemetacin. When dried, it contains not less
raphy, cooled previously in a methanol-dry ice bath to make than 99.5z of acemetacin (C21H18ClNO6) meeting the fol-
exactly 100 mL. Preserve in a hermetic container, at a tem- lowing additional specifications.]
perature not exceeding －209C. This solution contains 10 mg Purity Related substances—Dissolve 40 mg of acemeta-
of vinyl chloride per mL. cin for assay in 10 mL of methanol, and use this solution as
the sample solution. Pipet 1 mL of this solution, and add
Standard Water-Methanol Solution See Water Determi-
methanol to make exactly 10 mL. Pipet 1 mL of this solu-
nation <2.48>.
tion, add methanol to make exactly 20 mL, and use this solu-
Standard Zinc Solution Measure exactly 25 mL of tion as the standard solution. Perform the test with exactly
Standard Zinc Stock Solution, and add water to make ex- 10 mL each of the sample solution and standard solution as
actly 1000 mL. Prepare before use. Each mL of this solution directed under Liquid Chromatography <2.01> according to
contains 0.025 mg of zinc (Zn). the following conditions. Determine each peak area of both
solutions by the automatic integration method: the area of
Standard Zinc Solution for Atomic Absorption Spectro-
each peak other than acemetacin obtained from the sample
photometry To exactly 10 mL of Standard Zinc Stock So-
solution is not larger than 1/2 times the peak area of
lution add water to make exactly 1000 mL. Prepare before
acemetacin obtained from the standard solution, and the
use. Each mL of this solution contains 0.01 mg of zinc (Zn).
total area of the peaks other than acemetacin is not larger
Standard Zinc Stock Solution Dissolve exactly 1.000 g of than the peak area of acemetacin from the standard solu-
zinc (standard reagent), in 100 mL of water and 5 mL of hy- tion.
drochloric acid with the aid of gentle heating, cool, and add Operating conditions
water to make exactly 1000 mL. Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
the Assay under Acemetacin Tablets.
Time span of measurement: About 4 times as long as the
9.23 Matching Fluids for Color retention time of Acemetacin.
System Suitability
Refer to Color Comparison Tests <2.65>.
Test for required detectability: Pipet 1 mL of the standard
solution, and add methanol to make exactly 20 mL. Confirm
that the peak area of acemetacin obtained from 10 mL of this
solution is equivalent to 3 to 7z of that of acemetacin ob-
tained from 10 mL of the standard solution.
System performance: Dissolve 75 mg of acemetacin and 75
mg of indometacin in 50 mL of methanol. To 4 mL of this
solution add 1 mL of a solution of hexyl parahydroxybenzo-
ate in methanol (1 in 250), and add methanol to make 50

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
192 General Tests / 9.41 Reagents, Test Solutions JP XVII
mL. When the procedure is run with 10 mL of this solution acetaldehyde under reduced pressure, discard the first 20 mL
under the above operating conditions, acemetacin, indo- of the distillate, and use the subsequent distillate. Prepare
metacin and hexyl parahydroxybenzoate are eluted in this before use.
order with the resolutions between the peaks of acemetacin
Acetaldehyde for gas chromatography CH3CHO A
and indometacin and between the peaks of indometacin and
clear and colorless, flammable liquid. Miscible with water
hexyl parahydroxybenzoate being not less than 3, respec-
and with ethanol (95).
tively.
Refractive index <2.45> n 20
D : about 1.332
System repeatability: When the test is repeated 6 times
Specific gravity <2.56> d 20
20: about 0.788
with 10 mL of the standard solution under the above operat-
Boiling point <2.57>: about 219C
ing conditions, the relative standard deviation of the peak
area of acemetacin is not more than 1.5z. 2-Acetamidoglutarimide C7H10N2O3: 170.17
Identification—Determine the infrared absorption spec-
Acenaphthene C12H10 White to pale yellowish white,
trum of 2-acetamidoglutarimide as directed in the potassium
crystals or crystalline powder, having a characteristic aroma.
bromide disk method under Infrared Spectrophotometry
Freely soluble in diethyl ether and in chloroform, soluble in
<2.25>: it exhibits absorption at the wave numbers of about
acetonitrile, sparingly soluble in methanol, and practically
3350 cm－1, 1707 cm－1, 1639 cm－1 and 1545 cm－1.
insoluble in water.
Purity Related substances—Dissolve 10 mg of 2-ace-
Identification—Determine the infrared absorption spec-
tamidoglutarimide in 100 mL of the mobile phase, and use
trum of acenaphthene according to the paste method under
this solution as the sample solution. Pipet 1 mL of the sam-
Infrared Spectrophotometry <2.25>: it exhibits absorption at
ple solution, add the mobile phase to make exactly 100 mL,
the wave numbers of about 1605 cm－1, 840 cm－1, 785 cm－1
and use this solution as the standard solution. Proceed with
and 750 cm－1.
exactly 20 mL each of the sample solution and standard solu-
Melting point <2.60>: 93 – 969C
tion as directed in the Purity (3) under Aceglutamide Alumi-
Purity—Dissolve 0.1 g of acenaphthene in 5 mL of chlo-
num: the total of the peak areas other than 2-acetamido-
roform, and use this solution as the sample solution. Per-
glutarimide from the sample solution is not larger than the
form the test with 2 mL of the sample solution as directed
peak area from the standard solution.
under Gas Chromatography <2.02> according to the follow-
Content: not less than 98.0z. Assay—Weigh accurately
ing conditions. Measure each peak area by the automatic in-
about 20 mg of 2-acetamidoglutarimide, and perform the
tegration method, and calculate the amount of acenaphthene
test as directed under Nitrogen Determination <1.08>.
by the area percentage method: it shows a purity of not less
than 98.0z. Each mL of 0.005 mol/L sulfuric acid VS
Operating conditions ＝ 0.8509 mg of C7H10N2O3
Detector: A hydrogen flame-ionization detector.
Acetaminophen C8H9NO2 [Same as the namesake
Column: A glass column about 3 mm in inside diameter
monograph]
and about 2 m in length, packed with 150 to 180 mm siliceous
earth for gas chromatography coated with 10z of polyethy- Acetanilide C8H9NO White, crystals or crystalline
lene glycol 20 M. powder.
Column temperature: A constant temperature of about Melting point <2.60>: 114 – 1179C
2109C.
o-Acetanisidide C9H11NO2 White to light brownish,
Carrier gas: Nitrogen.
crystals or crystalline powder. Freely soluble in ethanol
Flow rate: Adjust so that the retention time of acenaph-
(99.5) and in acetonitrile, and slightly soluble in water.
thene is about 8 minutes.
Melting point: 86 – 899C
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of acenaphthene obtained from 2 mL of p-Acetanisidide C9H11NO2 White to purplish white,
the solution prepared by adding chloroform to 1.0 mL of the crystals or crystalline powder, having a characteristic odor.
sample solution to make 100 mL is 5 to 15z of the full scale. It is freely soluble in ethanol (95) and in acetonitrile, and
Time span of measurement: About 3 times as long as the very slightly soluble in water.
retention time of acenaphthene, beginning after the solvent Melting point <2.60>: 126 – 1329C
peak. Content: not less than 98.0z. Assay—Dissolve 0.1 g of
Residue on ignition <2.44>—Not more than 0.1z (1 g). p-acetanisidide in 5 mL of ethanol (95). Perform the test
with 2 mL of this solution as directed under Gas Chromatog-
Acetal C6H14O2 A clear and colorless, volatile liquid.
raphy <2.02> according to the following conditions, and de-
Miscible with water and with ethanol (95).
termine the area of each peak by the automatic integration
Refractive index <2.45> n 20
D : about 1.382
method.
Specific gravity <2.56> d 20
20: about 0.824
Boiling point <2.57>: about 1039C peak area of p-acetanisidide
Content (z) ＝ × 100
total of all peak areas
Acetaldehyde CH3CHO [K 8030, First class]
Operating conditions
Acetaldehyde ammonia trimer trihydrate (C2H5N)3.3H2O
Detector: A hydrogen flame-ionization detector.
Colorless or white to pale yellow, crystals or powder.
Column: A glass tube 3 mm in inside diameter and 2 m in
Content: not less than 95.0z. Assay—Weigh accurately
length, packed with acid-treated and silanized siliceous earth
about 0.9 g of acetaldehyde ammonia trimer trihydrate,
for gas chromatography coated with alkylene glycol phtha-
dissolve in 50 mL of water, and titrate <2.50> with 1 mol/L
late ester for gas chromatography in 1z (177–250 mm in par-
hydrochloric acid VS (potentiometric titration).
ticle diameter).
Each mL of 1 mol/L hydrochloric acid VS Column temperature: A constant temperature of about
＝ 61.08 mg of (C2H5N)3.3H2O 2109C.
Carrier gas: Nitrogen.
Acetaldehyde for assay CH3CHO Distil 100 mL of

Flow rate: Adjust to a constant flow rate of between 30 acetic acid for nonaqueous titration to make 100 mL, and
mL and 50 mL per minute and so that the retention time of use this solution as the sample solution. Pipet 25 mL of the
p-acetanisidide is between 11 minutes and 14 minutes. sample solution, titrate <2.50> with 0.1 mol/L perchloric
Time span of measurement: About 3 times as long as the acid VS, and designate the consumed volume as A (mL). A is
retention time of p-acetanisidide, beginning after the solvent not less than 26 mL. Pipet 25 mL of the sample solution, add
peak. 75 mL of acetic acid for nonaqueous titration, and titrate
<2.50> with 0.1 mol/L perchloric acid VS, and designate the
Acetate buffer solution (pH 3.5) Dissolve 50 g of ammo-
consumed volume as B (mL) (potentiometric titration). A －
nium acetate in 100 mL of 6 mol/L hydrochloric acid TS,
B is not more than 0.1 (mL) (not more than 0.001 g/dL).
adjust to pH 3.5 with ammonia TS or 6 mol/L hydrochloric
acid TS, if necessary, and add water to make 200 mL. Acetic acid, glacial See acetic acid (100).
0.05 mol/L Acetate buffer solution (pH 4.0) To 3.0 mL Acetic acid-potassium acetate buffer solution (pH 4.3)
of acetic acid (100) add 900 mL of water, adjust to pH 4.0 Dissolve 14 g of potassium acetate in 20.5 mL of acetic acid
with sodium hydroxide TS, and add water to make 1000 mL. (100), and add water to make 1000 mL.
Acetate buffer solution (pH 4.5) Dissolve 63 g of anhy- Acetic acid-sodium acetate buffer solution (pH 4.0) Dis-
drous sodium acetate in a suitable amount of water, add 90 solve 5.44 g of sodium acetate trihydrate in 900 mL of water,
mL of acetic acid (100) and water to make 1000 mL. adjust the pH 4.0 with acetic acid (100), and add water to
make 1000 mL.
0.01 mol/L Acetate buffer solution (pH 5.0) Dissolve
385 g of ammonium acetate in 900 mL of water, add acetic 0.05 mol/L Acetic acid-sodium acetate buffer solution
acid (31) to adjust the pH to 5.0, and then add water to (pH 4.0) To 3.0 g of acetic acid (100) add water to make
make 1000 mL. 1000 mL. Adjust to pH 4.0 with a solution prepared by dis-
solving 3.4 g of sodium acetate trihydrate in water to make
Acetate buffer solution (pH 5.4) To 5.78 mL of acetic
500 mL.
acid (100) add water to make 1000 mL (solution A). Dissolve
8.2 g of anhydrous sodium acetate in water to make 1000 mL 0.1 mol/L Acetic acid-sodium acetate buffer solution (pH
(solution B). Mix 176 mL of the solution A and 824 mL of 4.0) Dissolve 13.61 g of soduim acetate trihydrate in 750
the solution B, and adjust, if necessary, the pH to 5.4 with mL of water, adjust the pH to 4.0 with acetic acid (100), and
the solution A or the solution B. add water to make 1000 mL.
Acetate buffer solution (pH 5.5) Dissolve 2.72 g of sodi- Acetic acid-sodium acetate buffer solution (pH 4.5) To
um acetate trihydrate in water to make 1000 mL, and adjust 80 mL of sodium acetate TS add 120 mL of dilute acetic acid
the pH to 5.5 with diluted acetic acid (100) (3 in 2500). and water to make 1000 mL.
0.02 mL/L Acetate buffer solution (pH 6.0) Dissolve Acetic acid-sodium acetate buffer solution (pH 4.5), for
1.76 g of sodium chloride in 4 mL of 1 mol/L acetic acid- iron limit test Dissolve 75.4 mL of acetic acid (100) and
sodium acetate buffer solution (pH 6.0), and add water to 111 g of sodium acetate trihydrate in 1000 mL of water.
make 200 mL.
0.05 mol/L Acetic acid-sodium acetate buffer solution
Acetic acid See acetic acid (31). (pH 4.6) Dissolve 6.6 g of sodium acetate trihydrate in
900 mL of water, and add 3 mL of acetic acid and water to
Acetic acid (31) Dilute 31.0 g of acetic acide (100) with
make 1000 mL.
water to make 100 mL (5 mol/L).
Acetic acid-sodium acetate buffer solution (pH 4.7) Dis-
Acetic acid (100) CH3COOH [K 8355, Acetic Acid,
solve 27.2 g of sodium acetate trihydrate in 900 mL of water,
Special class]
adjust the pH to 4.7 by adding acetic acid (100) dropwise,
Acetic acid-ammonium acetate buffer solution (pH 3.0) and add water to make 1000 mL.
Add acetic acid (31) to ammonium acetate TS, and adjust
Acetic acid-sodium acetate buffer solution (pH 5.0) To
the pH to 3.0.
140 mL of sodium acetate TS add 60 mL of dilute acetic acid
Acetic acid-ammonium acetate buffer solution (pH 4.5) and water to make 1000 mL.
Dissolve 77 g of ammonium acetate in 200 mL of water,
1 mol/L Acetic acid-sodium acetate buffer solution (pH
adjust the pH to 4.5 by adding acetic acid (100), and add
5.0) To sodium acetate TS add dilute acetic acid, and
water to make 1000 mL.
adjust the pH to 5.0.
Acetic acid-ammonium acetate buffer solution (pH 4.8)
Acetic acid-sodium acetate buffer solution (pH 5.5) Dis-
Dissolve 77 g of ammonium acetate in about 200 mL of
solve 20 g of sodium acetate trihydrate in 80 mL of water,
water, and add 57 mL of acetic acid (100) and water to make
adjust the pH to 5.5 by adding acetic acid (100) dropwise,
1000 mL.
and add water to make 100 mL.
Acetic acid buffer solution containing 0.1z bovine serum
Acetic acid-sodium acetate buffer solution (pH 5.6) Dis-
albumin Dissolve 0.1 g of bovine serum albumin in sodium
solve 12 g of sodium acetate trihydrate in 0.66 mL of acetic
acetate trihydrate solution (1 in 100) to make exactly 100
acid (100) and water to make 100 mL.
mL, and adjust the pH to 4.0 with 1 mol/L hydrochloric
acid TS. 1 mol/L Acetic acid-sodium acetate buffer solution (pH
6.0) Adjust the pH of sodium acetate TS to 6.0 with dilute
Acetic acid, dilute Dilute 6 g of acetic acid (100) with
acetic acid.
water to make 100 mL (1 mol/L).
Acetic acid-sodium acetate TS Mix 17 mL of 1 mol/L
Acetic acid for nonaqueous titration CH3COOH
sodium hydroxide VS with 40 mL of dilute acetic acid, and
[K 8355, Special class. meeting with following requirement.]
add water to make 100 mL.
Purity Acetic anhydride—Dissolve 1.0 g of aniline in

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
194 General Tests / 9.41 Reagents, Test Solutions JP XVII
Acetic acid-sodium acetate TS (pH 7.0) Dissolve 4.53 g peak.
of sodium acetate trihydrate in water to make 100 mL, and
Acetonitrile CH3CN [K 8032, Special class]
adjust the pH to 7.0 with diluted acetic acid (100) (1 in 50).
Acetonitrile for liquid chromatography CH3CN Color-
0.02 mol/L Acetic acid-sodium acetate TS Dissolve
less and clear liquid. Miscible with water.
2.74 g of sodium acetate trihydrate in a suitable amount of
Purity Ultraviolet light absorbing substances—Deter-
water, and add 2 mL of acetic acid (100) and water to make
mine the absorbances of acetonitrile for liquid chromato-
1000 mL.
graphy at the following wavelengths as directed under Ultra-
0.25 mol/L Acetic acid TS To 3 g of acetic acid (100) violet-visible Spectrophotometry <2.24>, using water as the
add water to make 200 mL. control: not more than 0.07 at 200 nm, not more than 0.046
at 210 nm, not more than 0.027 at 220 nm, not more than
2 mol/L Acetic acid TS To 12 g of acetic acid (100) add
0.014 at 230 nm and not more than 0.009 at 240 nm.
water to make 100 mL.
Acetrizoic acid C9H6I3NO3 White powder.
6 mol/L Acetic acid TS Dilute 36 g of acetic acid (100)
Purity Related substances—Dissolve 60 mg of acetrizoic
with water to make 100 mL.
acid in a solution of meglumine (3 in 1000) to make 100 mL.
Acetic acid-sulfuric acid TS To 5 mL of acetic acid (100) To 10 mL of this solution add water to make 100 mL, and
add cautiously 5 mL of sulfuric acid while cooling in an ice use this solution as the sample solution. Proceed the test with
bath, and mix. 5 mL of the sample solution as directed in the Assay under
Meglumine Sodium Amidotrizoate Injection: any peaks
Acetic anhydride (CH3CO)2O [K 8886, Special class]
other than the principal peak are not observed.
Acetic anhydride-pyridine TS Place 25 g of acetic anhy-
Acetylacetone CH3COCH2COCH3 [K 8027, Special
dride in a 100 mL volumetric flask, add pyridine to make
class]
100 mL, and mix well. Preserve in light-resistant containers,
protected from air. This solution may be used even if it Acetylacetone TS Dissolve 150 g of ammonium acetate
becomes colored during storage. in a sufficient quantity of water, and add 3 mL of acetic acid
(100), 2 mL of acetylacetone and water to make 1000 mL.
Acetone CH3COCH3 [K 8034, Special class]
Prepare before use.
Acetone for nonaqueous titration Add potassium per-
Acetyl chloride CH3COCl A clear and colorless liquid.
manganate to acetone in small portions, and shake. When
the mixture keeps its purple color after standing for 2 to 3 Acetylene See dissolved acetylene.
days, distil, and dehydrate with freshly ignited anhydrous
N-Acetylgalactosamine C8H15NO6 White, crystals or
potassium carbonate. Distil by using a fractionating column
crystalline powder.
under protection from moisture, and collect the fraction dis-
Content: not less than 98.0z. Assay－Dissolve 36 mg of
tilling at 569C.
N-acetylgalactosamine in 1 mL of water. Perform the test
Acetone for purity of crude drug CH3COCH3 [K 8034, with 15 mL of this solution as directed under Liquid Chroma-
Acetone, Special class] Use acetone meeting the following tography <2.01> according to the following conditions. De-
additional specification. Evaporate 300.0 mL of acetone to termine each peak area by the automatic integration method,
be tested in vacuum at a temperature not higher than 409C, and calculate their amounts by the area percentage method.
add the acetone to make exactly 1 mL, and use this solution Operating conditions
as the sample solution. Separately, dissolve 2.0 mg of g-BHC Detector: A differential refractometer (Detector tempera-
in hexane for purity of crude drug to make exactly 100 mL. ture: a constant temperature of about 409 C).
Pipet 1 mL of this solution, and add hexane for purity of Column: A stainless steel column 8 mm in inside diameter
crude drug to make exactly 100 mL. Further pipet 2 mL of and 30 cm in length, packed with styrene-divinylbenzene
this solution, add hexane for purity of crude drug to make copolymer for liquid chromatography (7 mm in particle
exactly 100 mL, and use this solution as the standard solu- diameter).
tion (1). Perform the test with exactly 1 mL each of the sam- Column temperature: A constant temperature of about
ple solution and standard solution (1) as directed under Gas 809C.
Chromatography <2.02> according to the following operat- Mobile phase: Water.
ing conditions, and determine each peak area by the auto- Flow rate: 0.5 mL per minute.
matic integration method: the total area of peaks other than Time span of measurement: About 3 times as long as the
the solvent peak from the sample solution is not larger than retention time of N-acetylgalactosamine.
the peak area of g-BHC from the standard solution (1).
N-Acetylneuraminic acid C11H19NO9 White, crystals or
Operating conditions
crystalline powder.
Proceed the operating conditions in 4.3. under Crude
Content: not less than 98.0z. Assay—Dissolve 30 mg of
Drugs Test <5.01> except detection sensitivity and time span
N-acetylneuraminic acid in 1 mL of the mobile phase. Per-
of measurement.
form the test with 15 mL of this solution as directed under
Detection sensitivity: Pipet 1 mL of the standard solution
Liquid Chromatography <2.01> according to the following
(1), add hexane for purity of crude drug to make exactly 20
conditions. Determine each peak area by the automatic in-
mL, and use this solution as the standard solution (2).
tegration method, and calculate their amounts by the area
Adjust the detection sensitivity so that the peak area of g-
percentage method.
BHC obtained from 1 mL of the standard solution (2) can be
Operating conditions
measured by the automatic integration method, and the peak
Detector: A differential refractometer (detector tempera-
height of g-BHC from 1 mL of the standard solution (1) is
ture: a constant temperature of about 409 C).
about 20z of the full scale.
Column: A stainless steel column 8 mm in inside diameter
Time span of measurement: About three times as long as
and 30 cm in length, packed with styrene-divinylbenzene co-
the retention time of g-BHC, beginning after the solvent

polymer for liquid chromatography (6 mm in particle

diameter). R f value Color and shape of the spot
Column temperature: A constant temperature of about
Around 0.25 A weak spot, strongly purplish red
509 C.
Around 0.25 – 0.3 A leading spot or two strong spots,
Mobile phase: 10 mmol/L perchloric acid.
strongly purplish red
Flow rate: 0.5 mL per minute.
Around 0.35 A deep purplish red spot
Time span of measurement: About 3 times as long as the
Around 0.4 A weak spot, dull red
retention time of N-acetylneuraminic acid.
Around 0.42 A dark red spot
N-Acetylneuraminic acid for epoetin alfa C11H19NO9 Around 0.45 A weak spot, grayish red
White needle crystalline powder. Around 0.65 A weak spot, dull greenish yellow
Around 0.7 A weak spot, grayish red
0.4 mmol/L N-Acetylneuraminic acid TS Weigh accu-
Around 0.85 A weak spot, grayish red
rately about 15.5 mg of N-acetylneuraminic acid for epoetin
Around 0.95 A weak spot, dull yellow-red
alfa, dissolve in water to make exactly 50 mL. To exactly
V mL of this solution add water to make exactly 100 mL.
V (mL) ＝ 309.3 × 2/amount (mg) Acidic ferric chloride TS See iron (III) chloride TS,
of N-acetylneuraminic acid acidic.

Achyranthes root for thin-layer chromatography A heat- Acidic potassium chloride TS See potassium chloride
dried, pulverized root of Achyranthes fauriei Leveill áe et TS, acidic.
Vaniot (Amaranthaceae) meeting the following additional Acidic potassium permanganate TS See potassium per-
specifications. manganate TS, acidic.
Identification (1) To 2 g of pulverized achyranthes root
for thin-layer chromatography add 10 mL of water, shake Acidic stannous chloride TS See tin (II) chloride TS,
for 10 minutes, add 5 mL of 1-butanol, shake for 10 acidic.
minutes, centrifuge, and use the supernatant liquid as the Acid-treated gelatin See gelatin, acid-treated.
sample solution. Separately, dissolve 1 mg of chikuset-
susaponin IV for thin-layer chromatography in 1 mL of Aconitine for purity C34H47NO11 White, crystals or
methanol, and use this solution as the standard solution. crystalline powder. Sparingly soluble in acetonitrile and in
Perform the test with these solutions as directed under Thin- ethanol (99.5), slightly soluble in diethyl ether, and practi-
layer Chromatography <2.03>. Spot 10 mL each of the sample cally insoluble in water. Melting point: about 1859 C (with
solution and standard solution on a plate of silica gel for decomposition).
thin-layer chromatography. Develop the plate with a mixture Identification—Determine the infrared absorption spec-
of ethyl acetate, water and formic acid (5:1:1) to a distance trum of aconitine for purity as directed in the potassium
of about 10 cm, and air-dry the plate. Spray evenly dilute bromide disk method under Infrared Spectrophotometry
sulfuric acid on the plate, and heat the plate at 1059C for 10 <2.25>: it exhibits absorption at the wave numbers of about
minutes: the standard solution shows a deep purplish red 3500 cm－1, 1718 cm－1, 1278 cm－1, 1111 cm－1, 1097 cm－1
spot at around R f value of 0.35, and the sample solution and 717 cm－1.
shows spots equivalent to those described below: Absorbance <2.24> E 11zcm (230 nm): 211 – 243 [5 mg,
ethanol (99.5), 200 mL].
Purity Related substances—(1) Dissolve 5.0 mg of
R f value Color and shape of the spot
aconitine for purity in 2 mL of acetonitrile, and use this so-
Around 0 A weak spot, black lution as the sample solution. Pipet 1 mL of the sample solu-
Around 0.1 A weak spot, strong purplish red tion, add acetonitrile to make exactly 50 mL, and use this so-
Around 0.2 A weak, tailing spot, strong purplish lution as the standard solution. Perform the test with these
red solutions as directed under Thin-layer Chromatography
Around 0.25 A strong spot, deep purplish red <2.03>. Spot 20 mL each of the sample solution and standard
Around 0.35 A leading spot, deep purplish red solution on a plate of silica gel for thin-layer chromatogra-
Around 0.45 A weak spot, dull yellow phy, and proceed the test as directed in the Identification in
Around 0.5 A weak spot, grayish purplish red Processed Aconite Root: the spot other than the principal
Around 0.75 A weak spot, grayish red spot obtained with the sample solution is not more intense
Around 0.9 A weak spot, dull red than the spot obtained with the standard solution.
(2) Dissolve 5.0 mg of aconitine for purity in 5 mL of
acetonitrile, and use this solution as the sample solution.
(2) Perform the test as directed in the operating condi-
Pipet 1 mL of the sample solution, add acetonitrile to make
tions under (1), except using a mixture of 1-propanol, ethyl
exactly 50 mL, and use this solution as the standard solution.
acetate and water (4:4:3) as the developing solvent: the
Perform the test with exactly 10 mL each of the sample solu-
standard solution shows a deep purplish red spot at around
tion and standard solution as directed under Liquid Chroma-
R f value of 0.45, and the sample solution shows spots
tography <2.01> according to the following conditions, and
equivalent to those described below:
determine each peak area by the automatic integration
method: the total area of the peaks other than the peak of
aconitine with the sample solution is not larger than the peak
area of aconitine with the standard solution.
Operating conditions
Detector, column, and column temperature: Proceed as
directed in the operating conditions in the Purity (3) under
Processed Aconitine Root.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
196 General Tests / 9.41 Reagents, Test Solutions JP XVII
Mobile phase: A mixture of phosphate buffer solution for Acteoside for thin-layer chromatography See Verbasco-
processed aconite root and tetrahydrofuran (9:1). side for thin-layer chromatography.
Flow rate: Adjust so that the retention time of aconitine is
Activated alumina Aluminum oxide with specially strong
about 26 minutes.
adsorptive activity.
Time span of measurement: About 3 times as long as the
retention time of aconitine, beginning after the solvent peak. Activated charcoal [Same as the monograph Medicinal
System suitability Carbon]
Test for required detectability: Pipet 1 mL of the standard
Activated thromboplastin-time assay reagent It is pre-
solution, and add acetonitrile to make exactly 20 mL. Con-
pared by lyophilization of phospholipid (0.4 mg/mL) which
firm that the peak area of aconitine obtained from 10 mL of
is suspended in 1 mL of a solution of N-2-Hydroxyethyl-
this solution is equivalent to 3.5 to 6.5z of that obtained
piperazine-N?-2-ethanesulfonic acid (61 in 5000), mixed with
from 10 mL of the standard solution.
both silica-gel (4.3 mg/mL) and dextran after the extraction
System performance: Dissolve 1 mg each of aconitine for
and purificaton from rabbit brain. Activated throm-
purity, hypaconitine for purity and mesaconitine for purity,
boplastin-time: 25 – 45 seconds (as assayed with human nor-
and 8 mg of jesaconitine for purity in 200 mL of acetonitrile.
mal plasma).
When the procedure is run with 10 mL of this solution under
the above operating conditions, mesaconitine, hypaconitine, Activated thromboplastin-time assay TS Dissolve an
aconitine and jesaconitine are eluted in this order, and each aliquot of activated thromboplastin-time assay reagent
resolution between these peaks is not less than 1.5, respec- equivalent to 0.4 mg of phospholipid in 1 mL of water.
tively.
Adipic acid C4H8(COOH)2 White, crystals or crystal-
System repeatability: When the test is repeated 6 times
line powder. Freely soluble in ethanol (95), and sparingly
with 10 mL of the standard solution under the above operat-
soluble in water.
ing conditions, the relative standard deviation of the peak
Melting point <2.60>: 151 – 1549C
area of aconitine is not more than 1.5z.
Content: not less than 98.0z. Assay—Weigh accurately
Water <2.48>: not more than 1.0z (5 mg, coulometric
about 1 g of adipic acid, and dissolve in 100 mL of water by
titration).
warming, cool, and titrate <2.50> with 1 mol/L sodium hy-
Aconitum diester alkaloids standard TS for purity It is a droxide VS (indicator: 2 drops of phenolphthalein TS).
solution containing 10 mg of aconitine for purity, 10 mg of
Each mL of 1 mol/L sodium hydroxide VS
jesaconitine for purity, 30 mg of hypaconitine for purity and
＝ 73.07 mg of C6H10O4
20 mg of mesaconitine for purity in 1000 mL of a mixture of
phosphate buffer solution for processed aconite root and Agar [K 8263, Special class, or same as the monograph
acetonitrile (1:1). When proceed the test with 20 mL of this Agar or Agar Powder. Loss on drying is not more than
solution as directed in the Purity (3) under Processed 15z.]
Aconite Root at the detection wavelength 231 nm, the peaks
Agar medium, ordinary See ordinary agar medium.
of aconitine, jesaconitine, hypaconitine and mesaconitine
are observed, and the ratio of their peak heights is about Agar slant Dispense portions of about 10 mL of ordina-
10:1:35:30. When proceed the test at the detection wave- ry agar medium into test tubes, and sterilize by autoclaving.
length 254 nm, the peaks of aconitine, jesaconitine, Before the medium congeals, allow to stand in a slanting
hypaconitine and mesaconitine are observed, and the ratio of position, and solidify. When the coagulating water is lost,
their peak heights is about 2:8:7:6. reprepare by dissolving with the aid of heat.
Aconitum monoester alkaloids standard TS for assay Ajmaline for assay C20H26N2O2 [Same as the mono-
Weigh accurately about 20 mg of benzoylmesaconine hydro- graph Ajmaline. When dried, it contains not less than 99.0z
chloride for assay (separately, determine the water), about of ajmaline (C20H26N2O2).]
10 mg of benzoylhypaconine hydrochloride for assay (sepa-
Alacepril C20H26N2O5S [Same as the namesake mono-
rately, determine the water) and about 20 mg of 14-anisoy-
graph]
laconine hydrochloride for assay (separately, determine the
water), dissolve in a mixture of phosphate buffer solution Alacepril for assay C20H26N2O5S [Same as the mono-
for processed aconite root and tetrahydrofuran (183:17) to graph Alacepril. When dried, it contains not less than 99.0z
make exactly 1000 mL. Perform the test with 20 mL of this of alacepril (C20H26N2O5S).]
solution as directed in the Purity under benzoylmesaconine
b-Alanine C3H7NO2 Colorless crystals or a white crys-
hydrochloride for assay: the peaks of benzoylmesaconine,
talline powder. Freely soluble in water, very slightly soluble
benzoylhypaconine and 14-anisoylaconine appear with a
in methanol, and practically insoluble in ethanol (99.5) and
peak area ratio of about 2:1:2.
in diethyl ether.
Aconitum monoester alkaloids standard TS for compo- Purity Related substances—Dissolve 5.0 mg of b-Ala-
nent determination See aconitum monoester alkaloids nine in 10 mL of diluted methanol (4 in 5), and use this solu-
standard TS for assay. tion as the sample solution. Pipet 1 mL of the sample solu-
tion, add diluted methanol (4 in 5) to make exactly 100 mL,
Acrinol See acrinol hydrate.
and use this solution as the standard solution. Perform the
Acrinol hydrate C15H15N3O.C3H6O3.H2O [Same as the test with these solutions as directed under Thin-layer Chro-
namesake monograph] matography <2.03>. Spot 2 mL each of the sample solution
and standard solution on a plate of silica gel for thin-layer
Acrylamide CH2CHCONH2 White or pale yellow crys-
chromatography. Develop the plate with a mixture of 1-
talline powder.
butanol, water and acetic acid (100) (5:2:2) to a distance of
Melting point <2.60>: 83 – 879C
about 8 cm, and air-dry the plate. Spray evenly a solution of
Content: not less than 97.0z.
ninhydrin in acetone (1 in 50) on the plate, and heat at 809C

for 5 minutes: the spot other than the principal spot obtained Aldehyde-free ethanol See ethanol, aldehyde-free.
from the sample solution is not more intense than the spot
Aldioxa for assay C4H7AlN4O5 [Same as the mono-
obtained from the standard solution.
graph Aldioxa. When dried, it contains not less than 67.3z
L-Alanine C3H7NO2 [K 9101, Special class] and not more than 71.0z of allantoin (C4H6N4O3) and not
less than 11.6z and not more than 12.5z of aluminum
Albiflorin C23H28O11 White powder having no odor.
(Al).]
Freely soluble in water, in methanol and in ethanol (99.5).
Identification—Determine the absorption spectrum of a Alendronate sodium hydrate C4H12NNaO7P2.3H2O
solution of albiflorin in diluted methanol (1 in 2) (1 in [Same as the namesake monograph]
100,000) as directed under Ultraviolet-visible Spectropho-
Alisma tuber triterpenes TS for identification Dissolve
tometry <2.24>: it exhibits a maximum between 230 nm and
1 mg of alisol A for thin-layer chromatography, 1 mg of
234 nm.
alisol B and 1 mg of alisol B monoacetate in 5 mL of metha-
Purity (1) Related substances 1—Dissolve 1 mg of al-
nol.
biflorin in 1 mL of methanol, and perform the test with 10
mL of this solution as directed in the Identification (2) under Alisol A for thin-layer chromatography C30H50O5 A
Peony Root: any spot other than the principal spot with an white to pale yellow powder. Very soluble in methanol,
R f value of about 0.2 does not appear. freely soluble in ethanol (99.5), and practically insoluble in
(2) Related substances 2—Dissolve 1 mg of albiflorin in water.
10 mL of diluted methanol (1 in 2), and use this solution as Optical rotation <2.49> [a]20D : ＋86 – ＋1069(5 mg previ-
the sample solution. Perform the test with 10 mL of the sam- ously dried on silica gel for 24 hours, methanol, 1 mL, 50
ple solution as directed in the Assay under Peony Root: mm).
when measure the peak areas for 2 times the retention time Purity Related substances—Dissolve 1 mg of alisol A for
of paeoniflorin, the total area of the peaks other than al- thin-layer chromatography in 1 mL of methanol. Proceed
biflorin obtained from the sample solution is not larger than the test with 5 mL of this solution as directed in the Identifi-
1/10 times the total area of the peaks other than the solvent cation (6) under Saireito Extract: no spot appears other than
peak. the principal spot of around R f value of 0.3.
Albumin TS Carefully separate the white from the yolk Alisol B C30H48O4 White, crystals or crystalline pow-
of a fresh hen's egg. Shake the white with 100 mL of water der. Freely soluble in methanol and in ethanol (99.5), and
until the mixture is thoroughly mixed, and filter. Prepare practically insoluble in water.
before use. Identification Determine the infrared absorption spec-
trum of alisol B as directed in the potassium bromide disk
Alcian blue 8 GX C56H68Cl14CuN16S4 Dark blue-purple
method under Infrared Spectrophotometry <2.25>: it exhibits
powder.
absorption at the wave numbers of about 1704 cm－1, 1458
Alcian blue staining solution Dissolve 0.5 g of alcian cm－1 and 1244 cm－1.
blue 8 GX in 100 mL of diluted acetic acid (100) (3 in 100). Purity Related substances—Dissolve 1 mg of alisol B in
1 mL of methanol, and use this solution as the sample solu-
Aldehyde dehydrogenase Each mg contains not less than
tion. To exactly 0.5 mL of the sample solution add methanol
2 enzyme activity units. White powder.
to make exactly 25 mL, and use this solution as the standard
Assay—Dissolve about 20 mg of aldehyde dehydrogenase,
solution. Perform the test with these solutions as directed
accurately weighed, in 1 mL of water, add ice-cold solution
under Thin-layer Chromatography <2.03>. Spot 2 mL each of
of bovine serum albumin (1 in 100) to make exactly 200 mL,
the sample solution and standard solution on a plate of silica
and use this solution as the sample solution. In a spec-
gel for thin-layer chromatography. Develop the plate with a
trophotometric cell, place 2.50 mL of pyrophosphate buffer
mixture of ethyl acetate, hexane and acetic acid (100)
solution (pH 9.0), 0.20 mL of a solution prepared by dissolv-
(10:10:3) to a distance of about 7 cm, and air-dry the plate.
ing 20.0 mg of b-nicotinamide adenine dinucleotide (b-NAD)
Spray evenly vanillin-sulfuric acid-ethanol TS for spraying
in water to make exactly 1 mL, 0.10 mL of a pyrazole solu-
on the plate, and heat at 1059C for 5 minutes: the spot other
tion (17 in 2500) and 0.10 mL of the sample solution, stir,
than the principal spot which appeares at an R f value of
stopper tightly, and allow to stand at 25 ± 19C for 2
about 0.4 obtained from the sample solution is not more in-
minutes. To this solution add 0.01 mL of an acetaldehyde
tense than the spot obtained from the standard solution.
solution (3 in 1000), stir, stopper tightly, determine every 30
seconds the absorbance at 340 nm as directed under Ultravi- Alisol B monoacetate C32H50O5 White, crystals or crys-
olet-visible Spectrophotometry <2.24>, and calculate a talline powder. Freely soluble in methanol and in ethanol
change (DA) in absorbance per minute starting from the spot (99.5), and practically insoluble in water.
where the relation of time and absorbance is shown with a Identification Determine the infrared absorption spec-
straight line. One enzyme activity unit means an amount of trum of alisol B monoacetate as directed in the potassium
enzyme which oxidizes 1 mmol of acetaldehyde per minute bromide disk method under Infrared Spectrophotometry
when the test is conducted under the conditions of the Proce- <2.25>: it exhibits absorption at the wave numbers of about
dure. 3480 cm－1, 1743 cm－1, 1704 cm－1 and 1232 cm－1.
Purity Related substances—Dissolve 1 mg of alisol B
Enzyme activity unit (unit/mg) of aldehyde dehydrogenase
monoacetate in 1mL of methanol, and use this solution as
2.91 × DA × 200 the sample solution. To exactly 0.5 mL of the sample solu-
＝
6.3 × M × 0.10 × 1000 tion add methanol to make exactly 25 mL, and use this solu-
tion as the standard solution. Perform the test with these so-
M: Amount (g) of aldehyde dehydrogenase taken
lutions as directed under Thin-layer Chromatography <2.03>.
Aldehyde dehydrogenase TS Dissolve an amount equiva- Spot 2 mL each of the sample solution and standard solution
lent to 70 aldehyde dehydrogenase units in 10 mL of water. on a plate of silica gel for thin-layer chromatography. De-
Prepare before use. velop the plate with a mixture of ethyl acetate, hexane and

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
198 General Tests / 9.41 Reagents, Test Solutions JP XVII
acetic acid (100) (10:10:3) to a distance of about 7 cm, and Alkaline copper TS for protein content determination
air-dry the plate. Spray evenly vanillin-sulfuric acid-ethanol Dissolve 0.8 g of sodium hydroxide in water to make 100
TS for spraying on the plate, and heat at 1059C for 5 mL. Dissolve 4 g of anhydrous sodium carbonate in this so-
minutes: the spot other than the principal spot which is ap- lution to make solution A. Combine 1 mL of copper (II) sul-
peares at an Rf value of about 0.5 obtained from the sample fate pentahydrate solution (1 in 50) and 1 mL of sodium tar-
solution is not more intense than the spot obtained from the trate dihydrate solution (1 in 25) to make solution B. Mix 50
standard solution. mL of solution A and 1 mL of solution B. Prepare at the
time of use.
Alizarin complexone C19H15NO8 (1,2-Dihydroxyan-
thraquino-3-ylmethylamine-N, N-diacetate) A yellow- Alkaline copper TS (2) Dissolve 20 g of anhydrous so-
brown powder. Soluble in ammonia TS, and practically dium carbonate in dilute sodium hydroxide TS to make 1000
insoluble in water, in ethanol (95) and in diethl ether. mL, and use this solution as solution A. Dissolve 0.5 g of
Sensitivity—Dissolve 0.1 g of alizarin complexone by add- copper (II) sulfate pentahydrate in potassium sodium tar-
ing 2 drops of ammonia solution (28), 2 drops of ammonium tarate tetrahydrate solution (1 in 100) to make 100 mL, and
acetate TS and 20 mL of water. To 10 mL of this solution use this solution as solution B. To 50 mL of solution A add 1
add acetic acid-potassium acetate buffer solution (pH 4.3) mL of solution B. Prepare before use.
to make 100 mL. Place 1 drop of this solution on a white
Alkaline 1,3-dinitrobenzen TS See 1,3-dinitrobenzene
spot plate, add 1 drop of a solution of sodium fluoride (1 in
TS, alkaline.
100,000) and 1 drop of cerium (III) nitrate TS, stir, and ob-
serve under scattered light after 1 minute: a blue-purple Alkaline m-dinitrobenzene TS See 1,3-dinitrobenzene
color is produced, and the color of the control solution is TS, alkaline.
red-purple. Use a solution prepared in the same manner, to
Alkaline glycerin TS To 200 g of glycerin add water to
which 1 drop of water is added in place of a solution of so-
make 235 g, and add 142.5 mL of sodium hydroxide TS and
dium fluoride, as the control solution.
47.5 mL of water.
Alizarin complexone TS Dissolve 0.390 g of alizarin
Alkaline hydroxylamine TS See hydroxylamine TS, alka-
complexone in 20 mL of a freshly prepared solution of so-
line.
dium hydroxide (1 in 50), then add 800 mL of water and
0.2 g of sodium acetate trihydrate, and dissolve. Adjust the Alkaline phenolphthalein TS See Alcohol Number De-
pH to 4 to 5 with 1 mol/L hydrochloric acid VS, and add termination <1.01>.
water to make 1000 mL.
Alkaline phosphatase See phosphatase, alkaline.
Alizarin red S C14H7NaO7S [K 8057, Special class]
Alkaline phosphatase TS See phosphatase TS, alkaline.
Alizarin red S TS Dissolve 0.1 g of alizarin red S in water
Alkaline picric acid TS See 2,4,6-trinitrophenol TS,
to make 100 mL, and filter if necessary.
alkaline.
Alizarin S See alizarin red S.
Alkaline 2,4,6-trinitrophenol TS See 2,4,6-trinitrophenol
Alizarin S TS See alizarin red S TS. TS, alkaline.
Alizarin yellow GG C13H8N3NaO5 [K 8056, Special Alkaline potassium ferricyanide TS See potassium hexa-
class] cyanoferrate (III) TS, alkaline.
Alizarin yellow GG-thymolphthalein TS Mix 10 mL of Alkylene glycol phthalate ester for gas chromatography
alizarin GG TS with 20 mL of thymolphthalein TS. Prepared for gas chromatography.
Alizarin yellow GG TS Dissolve 0.1 g of alizarin yellow Allantoin for thin-layer chromatography C4H6N4O3 A
GG in 100 mL of ethanol (95), and filter if necessary. white, crystalline powder or powder. Slightly soluble in
water, and practically insoluble in methanol and in ethanol
Alkali copper TS Dissolve 70.6 g of disodium hydrogen
(99.5).
phosphate dodecahydrate, 40.0 g of potassium sodium tar-
Identification Determine the infrared absorption spec-
trate tetrahydrate and 180.0 g of anhydrous sodium sulfate
trum of the substance to be examined as directed in the po-
in 600 mL of water, and add 20 mL of a solution of sodium
tassium bromide disk method under Infrared Spectropho-
hydroxide (1 in 5). To this mixture add, with stirring, 100
tometry <2.25>: it exhibits absorption at the wave numbers of
mL of a solution of copper (II) sulfate pentahydrate (2 in
about 3440 cm－1, 3340 cm－1, 1721 cm－1, 1532 cm－1 and
25), 33.3 mL of 0.05 mol/L potassium iodate VS and water
1061 cm－1.
to make 1000 mL.
Purity Related substances—Dissolve 2 mg of the sub-
Alkaline blue tetrazolium TS See blue tetrazolium TS, stance to be examined in 1 mL of water by warming, and add
alkaline. 2 mL of methanol. Perform the test with 5 mL of this solu-
tion as directed in the Identification (3) under Dioscorea
Alkaline copper solution See alkaline copper TS for pro-
Rhizome: no spot is observed except the principal spot with
tein content determination.
an R f value of about 0.5.
Alkaline copper sulfate TS See copper (II) sulfate TS,
Allopurinol C5H 4N 4O [Same as the namesake mono-
alkaline.
graph]
Alkaline copper TS Dissolve 2 g of anhydrous sodium
Allopurinol for assay C5H4N4O [Same as the mono-
carbonate in 100 ml of dilute sodium hydroxide TS. To 50
graph Allopurinol. When dried, it contains not less than
mL of this solution add 1 mL of a mixture of a solution of
99.0z of allopurinol (C5H4N4O).]
copper (II) sulfate pentahydrate (1 in 100) and a solution of
potassium tartrate (1 in 50) (1:1), and mix. Alminoprofen for assay C13H17NO2 [Same as the
monograph Alminoprofen. When dried, it contains not less

than 99.5z of alminoprofen (C13H17NO2).] Each mL of 0.1 mol/L silver nitrate VS

＝ 23.97 mg of C11H13N3O.HCl
Alternative thioglycolate medium See Sterility Test
<4.06>. 4-Aminoantipyrine hydrochloride TS Dissolve 1 g of 4-
aminoantipyrine hydrochloride in water to make 50 mL.
Aluminon C22H23N3O9 [K 8011, Special class]
4-Aminoantipyrine TS Dissolve 0.1 g of 4-aminoantipy-
Aluminon TS Dissolve 0.1 g of aluminon in water to
rine in 30 mL of water, add 10 mL of a solution of sodium
make 100 mL, and allow this solution to stand for 24 hours.
carbonate decahydrate (1 in 5), 2 mL of sodium hydroxide
Aluminum Al [K 8069, Special class] TS and water to make 100 mL. Prepare before use.
Aluminum chloride See aluminum (III) chloride hexahy- 2-Aminobenzimidazole C7H7N3 White to light yellow,
drate. C
crystals or crystalline powder. Melting point: about 2319
(with decomposition).
Aluminum chloride TS See Aluminum (III) chloride TS.
Aminobenzoate derivatization TS To 0.28 g of ethyl
Aluminum (III) chloride TS Dissolve 64.7 g of alumi-
aminobenzoate add 600 mL of methanol, warm at about
num (III) chloride hexahydrate in 71 mL of water, add 0.5 g
509C to dissolve, and add 170 mL of acetic acid and 145 mL
of activated charcoal, then shake for 10 minutes, and filter.
of borane-pyridine complex.
Adjust the pH of the filtrate to 1.5 with a solution of sodium
hydroxide (1 in 100) with stirring, and filter if necessary. p-Aminobenzoic acid See 4-aminobenzoic acid.
Aluminum (III) chloride hexahydrate AlCl3.6H2O 4-Aminobenzoic acid H2NC6H4COOH White to very
[K 8114, Special class] pale yellow crystalline powder.
Purity Clarity of solution—Dissolve 0.1 g of 4-amino-
Aluminum oxide Al2O3 White crystals, crystalline pow-
benzoic acid in 10 mL of ethanol (95): the solution is clear.
C. Melting point:
der, or powder. Boiling point: about 30009
about 20009C. 2-Amino-1-butanol CH3CH2CH(NH2)CH2OH
Clear, colorless to light yellow liquid. Miscible with water
Aluminum potassium sulfate dodecahydrate
and with methanol.
AlK(SO4)2.12H2O [K 8255, Special class]
Refractive index <2.45> n 2020: 1.450 – 1.455
6-Amidino-2-naphthol methanesulfonate Specific gravity <2.56> d 20
20: 0.944 – 0.950
C11H10N2O.CH4O3S A white to pale yellow crystalline Purity Related substances—Weigh 50 mg of 2-amino-1-
powder. Melting point: about 2339C (with decomposition). butanol, and mix with exactly 10 mL of methanol. Perform
Purity—A solution obtained by dissolving 0.5 g of 6- the test with 2 mL of this solution as directed in the Purity (4)
amidino-2-naphthol methanesulfonate in 10 mL of methanol under Ethambutol Hydrochloride: any spot other than the
is clear. principal spot at the R f value of about 0.3 does not appear.
Amidosulfuric acid (standard reagent) HOSO2NH2 In 4-Aminobutylic acid H2NCH2CH2CH2COOH White,
addition to JIS K 8005 standard reagent for volumetric crystals or crystalline powder. Melting point: about 2009
C
analysis, certified reference material which can be used for (with decomposition).
volumetric analysis may be used.
e-Aminocaproic acid C6H13NO2 White, crystals or
Amidotrizoic acid for assay C11H9I3N2O4 [Same as the crystalline powder, having no odor or slightly a characteris-
monograph Amidotrizoic Acid. It contains not less than tic odor. Freely soluble in water and in acetic acid (100),
99.0z of amidotrizoic acid (C11H9I3N2O4), calculated on the slightly soluble in methanol, and practically insoluble in
dried basis.] ethanol. Melting point: about 2009 C (with decomposition).
Identification—Determine the infrared absorption spec-
p-Aminoacetophenone See 4-aminoacetophenone.
trum of e-aminocaproic acid as directed in the potassium
p-Aminoacetophenone TS See 4-aminoacetophenone bromide disk method under Infrared Spectrophotometry
TS. <2.25>: it exhibits absorption at the wave numbers of about
1564 cm－1, 1541 cm－1, 1391 cm－1 and 1269 cm－1.
4-Aminoacetophenone H2NC6H4COCH3 Light yellow,
crystals or crystalline powder, having a characteristic odor. 2-Amino-5-chlorobenzophenone for thin-layer chroma-
Melting point <2.60>: 105 – 1089 C tography C13H10ClNO Yellow crystalline powder.
Melting point <2.60>: 97 – 1019C
4-Aminoacetophenone TS Dissolve 0.100 g of 4-aminoa-
Purity Related substances—Dissolve 10 mg of 2-amino-
cetophenone in methanol to make exactly 100 mL.
5-chlorobenzophenone for thin-layer chromatography in
4-Aminoantipyrine C11H13N3O [K 8048, Special class] methanol to make exactly 200 mL, and perform the test with
this solution as directed in the purity (2) under Chlordia-
4-Aminoantipyrine hydrochloride C11H13N3O.HCl
zepoxide: any spot other than the principal spot at the R f
Light yellow crystalline powder. It dissolves in water. Melt-
value about 0.7 does not appear.
ing point: 232 – 2389 C (decomposition).
Purity Clarity of solution—Dissolve 1 g of 4-aminoan- 4-Amino-N, N-diethylaniline sulfate monohydrate
tipyrine hydrochloride in 25 mL of water: the solution is H2NC6H4N(C2H5)2.H2SO4.H2O White to slightly colored
almost clear. powder. It dissolves in water.
Content: 100.6 – 108.5z. Assay—Weigh accurately about Melting point <2.60>: 173 – 1769C
0.5 g of 4-aminoantipyrine hydrochloride, dissolve in 50 mL Residue on ignition <2.44>: not more than 0.1z (1 g).
of water, and, if necessary, neutralize with 0.1 mol/L so-
4-Amino-N, N-diethylaniline sulfate TS Dissolve 0.2 g of
dium hydroxide VS (indicator: red litmus paper). Add 4
4-amino-N, N-diethylaniline sulfate monohydrate in water to
drops of dichlorofluorescein TS, and titrate <2.50> with 0.1
make 100 mL. Prepare before use, protected from light.
mol/L silver nitrate VS.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
200 General Tests / 9.41 Reagents, Test Solutions JP XVII
2-Aminoethanethiol hydrochloride H2NCH2CH2SH.HCl [K 8050, Special class]
White, crystal or granule.
1-Amino-2-naphthol-4-sulfonic acid TS Mix thoroughly
Melting point <2.60>: 65 – 719C
5 g of anhydrous sodium sulfite, 94.3 g of sodium bisulfite
2-Aminoethanol NH2CH2CH2OH [K 8109, Special and 0.7 g of 1-amino-2-naphthol-4-sulfonic acid. Before use,
class] dissolve 1.5 g of this mixture in water to make 10 mL.
3-(2-Aminoethyl)indole C10H12N2 Yellowish-brown m-Aminophenol See 3-aminophenol.
crystals.
3-Aminophenol H2NC6H4OH White, crystals or crys-
Melting point <2.60>: about 1189C.
talline powder.
N-Aminohexamethyleneimine (CH2)6NNH2 Clear, col- Melting point <2.60>: 121 – 1259C
orless to pale yellow liquid. Content: not less than 97.0z. Assay—Weigh accurately
Refraction index <2.45> n 20D : 1.482 – 1.487 about 0.2 g, dissolve in 50 mL of acetic acid for nonaqueous
Specific gravity <2.56> d 20
20: 0.936 – 0.942 titration, and titrate <2.50> with 0.1 mol/L perchloric acid
VS (potentiometric titration). Perform a blank determina-
2-Amino-2-hydroxymethyl-1,3-propanediol C4H11NO3
tion in the same manner, and make any necessary correction.
[K 9704, Special class]
Each mL of 0.1 mol/L perchloric acid VS
2-Amino-2-hydroxymethyl-1,3-propanediol hydrochloride
＝ 10.91 mg of H2NC6H4OH
C4H11NO3.HCl White, crystals or crystalline powder
p-Aminophenol hydrochloride See 4-aminophenol hy-
4-(Aminomethyl)benzoic acid C8H9NO2 A white pow-
drochloride.
der.
Purity—Dissolve 10 mg of 4-(aminomethyl)benzoic acid in 4-Aminophenol hydrochloride HOC6H4NH2.HCl
100 mL of water, and use this as the sample solution. Pipet 1 White to pale colored crystals. Freely soluble in water and in
mL of the sample solution, add water to make exactly 20 ethanol (95). Melting point: about 3069C (with decomposi-
mL, and use this solution as the standard solution. Perform tion).
the test with exactly 20 mL each of the sample solution and Content: not less than 99.0z. Assay—Weigh accurately
standard solution as directed under Liquid Chromatography about 0.17 g of 4-aminophenol hydrochloride, dissolve in 50
<2.01> according to the operating conditions as directed in mL of acetic acid for nonaqueous titration and 5 mL of mer-
the Purity (5) under Tranexamic Acid, and determine each cury (II) acetate TS for nonaqueous titration, and titrate
peak area by the automatic integration method: each area of <2.50> with 0.1 mol/L perchloric acid-1,4-dioxane VS (indi-
the peak other than 4-(aminomethyl)benzoic acid obtained cator: 1 mL of p-naphtholbenzeine TS). Perform a blank de-
from the sample solution is not larger than the peak area of termination, and make any necessary correction.
4-(aminomethyl)benzoic acid obtained from the standard so-
Each mL of 0.1 mol/L perchloric acid-1,4-dioxane VS
lution.
＝ 14.56 mg of C6H8NOCl
1-Amino-2-methylnaphthalene C11H11N Pale yellow to
Storage—Preserve in tight, light-resistant containers.
pale brown, masses or liquid.
Aminopropylsilanized silica gel for pretreatment Pre-
2-Aminomethylpiperidine C6H14N A colorless or light
pared for pretreatment.
yellowish, clear liquid, having an amine like characteristic
odor. Aminopyrine C13H17N3O White to pale yellow, crystals
Identification—Determine the infrared absorption spec- or crystalline powder.
trum as directed in the liquid film method under Infrared Melting point <2.60>: 107 – 1099C
Spectrophotometry <2.25>: it exhibits absorption at the wave
6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate
numbers of about 3280 cm－1, 1600 cm－1, 1440 cm－1, 1120
C14H11N3O4 Prepared for biochemistry or amino acid
cm－1 and 840 cm－1.
analysis.
Purity Related substances—Perform the test with 0.8 mL
of 2-aminomethylpiperidine as directed under Gas Chroma- L-2-Aminosuberic acid C8H15NO4 White, crystals or
tography <2.02>. Determine each peak area by the automatic crystalline powder. Odorless.
integration method, and calculate these amounts by the area Optical rotation <2.49> [a]20
D : ＋19.1 – ＋20.19(after dry-
percentage method: the total amount of the peaks other than ing, 0.1 g, 5 mol/L hydrochloric acid TS, 100 mm).
2-aminomethylpiperidine is not more than 1.5z. Loss on drying <2.41>: not more than 0.3z (1 g, 1059C,
Operating conditions 2 hours).
Detector: A hydrogen flame-ionization detector. Assay—Weigh accurately about 0.3 g of L-2-aminosuberic
Column: A glass column 3 mm in inside diameter and 2 m acid, previously dried, add exactly 6 mL of formic acid to
in length, packed with siliceous earth for gas chromatogra- dissolve, then add exactly 50 mL of acetic acid (100), and
phy (150 – 180 mm) coated with 10z of polyethylene glycol titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
20M for gas chromatography and 2z of potassium hydrox- metric titration). Perform a blank determination in the same
ide. manner, and make any necessary correction.
Column temperature: 1009C at beginning, and raise to
Each mL of 0.1 mol/L perchloric acid VS
2009C at a rate 109 C per minute after injection.
＝ 18.92 mg of C8H15NO4
Carrier gas: Nitrogen.
Flow rate: Adjust so that the retention time of 2- Amiodarone hydrochloride for assay C25H29I2NO3.HCl
aminomethylpiperidine is about 5 minutes. [Same as the monograph Amiodarone Hydrochloride. When
Time span of measurement: About 2 times as long as the dried, it contains not less than 99.5z of amiodarone hydro-
retention time of 2-aminomethylpiperidine. chloride (C25H29I2NO3.HCl).]
1-Amino-2-naphthol-4-sulfonic acid C10H9NO4S Ammonia-ammonium acetate buffer solution (pH 8.0)

To ammonium acetate TS add ammonia TS dropwise to Ammonium amidosulfate TS Dissolve 1 g of ammonium

adjust the pH to 8.0. amidosulfate in water to make 40 mL.
Ammonia-ammonium acetate buffer solution (pH 8.5) Ammonium amminetrichloroplatinate for liquid chroma-
Dissolve 50 g of ammonium acetate in 800 mL of water and tography Cl3H7N2Pt To 20 g of cisplatin add 600 mL of 6
200 mL of ethanol (95), and add ammonia solution (28) to mol/L hydrochloric acid TS, and heat under a reflux con-
adjust the pH to 8.5. denser for 4 – 6 hours to boil while stirring. After cooling,
evaporate the solvent, and dry the orange residue at room
Ammonia-ammonium chloride buffer solution (pH 8.0)
temperature under reduced pressure. To the residue so ob-
Dissolve 1.07 g of ammonium chloride in water to make 100
tained add 300 mL of methanol, and heat at about 509 C to
mL, and adjust the pH to 8.0 by adding diluted ammonia TS
dissolve. Filter, separate insoluble yellow solids, and wash
(1 in 30).
the solids with 10 mL of methanol. Combine the filtrate and
Ammonia-ammonium chloride buffer solution (pH 10.0) the washing, heat at about 509C, and add slowly 100 mL of
Dissolve 70 g of ammonium chloride in water, add 100 mL ethyl acetate while stirring. Cool the mixture to room tem-
of ammonia solution (28), dilute with water to make 1000 perature avoiding exposure to light, and allow to stand at
mL, and add ammonia solution (28) dropwise to adjust the －109C for 1 hour. Filter the mixture to take off the formed
pH to 10.0. crystals, wash the crystals with 100 mL of acetone, combine
the washing to the filtrate, and evaporate to dryness to ob-
Ammonia-ammonium chloride buffer solution (pH 10.7)
tain orange crystals. If necessary, repeat the purification
Dissolve 67.5 g of ammonium chloride in water, add 570 mL
procedure described above to take off the insoluble crystals.
of ammonia solution (28), and dilute with water to make
To the orange crystals obtained add 300 to 500 mL of a mix-
1000 mL.
ture of acetone and methanol (5:1), and heat at about 509 C
Ammonia-ammonium chloride buffer solution (pH 11.0) while stirring to dissolve. Filter while hot to take off the in-
Dissolve 53.5 g of ammonium chloride in water, add 480 mL soluble crystals, wash the crystals with the mixture, and
of ammonia solution (28), and dilute with water to make combine the filtrate and washing. Repeat the procedure
1000 mL. several times, and evaporate to dryness. Suspense the crys-
tals so obtained in 50 mL of acetone, filter, wash the crystals
Ammonia copper TS To 0.5 g of cupric carbonate
with 20 mL of acetone, and dry the crystals at room temper-
monohydrate add 10 mL of water, triturate, and add 10 mL
ature under reduced pressure. It is a yellow-brown crystalline
of ammonia solution (28).
powder.
Ammonia-ethanol TS To 20 mL of ammonia solution Identification—Determine the infrared absorption spec-
(28) add 100 mL of ethanol (99.5). trum of the substance to be examined, previously dried at
809C for 3 hours, as directed in the potassium bromide disk
Ammonia gas NH3 Prepare by heating ammonia solu-
method under Infrared Spectrophotometry <2.25>: it exhibits
tion (28).
absorption at the wave numbers of about 3480 cm－1, 3220
Ammonia-saturated 1-butanol TS To 100 mL of 1- cm－1, 1622 cm－1, 1408 cm－1 and 1321 cm－1.
butanol add 60 mL of diluted ammonia solution (28) (1 in Purity Related substances—Cisplatin Conduct this
100), shake vigorously for 10 minutes, and allow to stand. procedure using light-resistant vessels. Dissolve 10 mg in
Use the upper layer. N, N-dimethylformamide to make exactly 10 mL, and use
this solution as the sample solution. Separately, dissolve 10
Ammonia solution (28) NH3 [K 8085, Ammonia
mg of Cisplatin in N, N-dimethylformamide to make exactly
Water, Special class, Density: about 0.90 g/mL, Content:
50 mL. Pipet 5 mL of this solution, add N, N-dimethylfor-
28 – 30z]
mamide to make exactly 20 mL, and use this solution as the
Ammonia TS To 400 mL of ammonia solution (28) add standard solution. Perform the test with exactly 40 mL each
water to make 1000 mL (10z). of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
1 mol/L Ammonia TS To 65 mL of ammonia solution
lowing conditions, and determine the peak area of cisplatin
(28) add water to make 1000 mL.
by the automatic integration method: the peak area from the
13.5 mol/L Ammonia TS To exactly 9 mL of water add sample solution is not larger than that from the standard so-
ammonia solution (28) to make exactly 50 mL. lution.
Operating conditions
Ammonia water See ammonia TS.
Proceed as directed in the operating conditions in the
1 mol/L Ammonia water See 1 mol/L ammonia TS. Assay under Cisplatin.
System suitability
13.5 mol/L Ammonia water See 13.5 mol/L ammonia
System performance: When the procedure is run with
TS.
40 mL of the standard solution under the above operating
Ammonia water, strong See ammonia solution (28). conditions, the number of theoretical plates and the symme-
try factor of the peak of cisplatin are not less than 2500 and
Ammonium acetate CH3COONH4 [K 8359, Special
not more than 2.0, respectively.
class]
System repeatability: When the test is repeated 6 times
Ammonium acetate TS Dissolve 10 g of ammonium ace- with 40 mL of the standard solution under the above operat-
tate in water to make 100 mL. ing conditions, the relative standard deviation of the peak
area of cisplatin is not more than 5.0z.
0.5 mol/L Ammonium acetate TS Dissolve 38.5 g of am-
monium acetate in water to make 1000 mL. Ammonium aurintricarboxylate See aluminon.
Ammonium amidosulfate NH4OSO2NH2 [K 8588, Ammonium carbonate [K 8613, Special class]
Special class]
Ammonium carbonate TS Dissolve 20 g of ammonium

Ammonium tartrate See L-ammonium tartrate. retention time of amygdalin.

System suitability
L-Ammonium tartrate C4H12N2O6 [K 8534, (＋) Am-
System performance and system repeatability: Proceed as
monium tartrate, Special class]
directed in the system suitability in the Assay (3) under
Ammonium thiocyanate NH4SCN [K 9000, Special Keishibukuryogan Extract.
class] Test for required detectability: Pipet 1 mL of the standard
solution, and add the mobile phase to make exactly 20 mL.
Ammonium thiocyanate-cobalt (II) nitrate TS Dissolve
Confirm that the peak area of amygdalin obtained with 10
17.4 g of ammonium thiocyanate and 2.8 g of cobalt (II) ni-
mL of this solution is equivalent to 3.5 to 6.5z of that ob-
trate hexahydrate in water to make 100 mL.
tained with 10 mL of the standard solution.
Ammonium thiocyanate TS Dissolve 8 g of ammonium
Amygdalin for component determination See amygdalin
thiocyanate in water to make 100 mL (1 mol/L).
for assay.
Ammonium vanadate See ammonium vanadate (V).
Amygdalin for thin-layer chromatography C20H27NO11
Ammonium vanadate (V) NH4VO3 [K 8747, Special A white, odorless powder. Soluble in water, sparingly solu-
class] ble in methanol, and practically insoluble in ethanol (99.5).
Identification—Determine the absorption spectrum of a
Amosulalol hydrochloride for assay C18H24N2O5S.HCl
solution of amygdalin for thin-layer chromatography in
[Same as the monograph Amosulalol Hydrochloride. It con-
methanol (1 in 1000) as directed under Ultraviolet-visible
tains not less than 99.0z of amosulalol hydrochloride
Spectrophotometry <2.24>: it exhibits maxima between 250
(C18H24N2O5S.HCl), calculated on the anhydrous basis.]
nm and 254 nm, between 255 nm and 259 nm, between 261
Amoxicillin See amoxicillin hydrate. nm and 265 nm, and between 267 nm and 271 nm.
Purity Related substances—Dissolve 5 mg of amygdalin
Amoxicillin hydrate C16H19N3O5S.3H2O [Same as the
for thin-layer chromatography in 2 mL of methanol, and use
namesake monograph]
this solution as the sample solution. Pipet 1 mL of the sam-
Amphoteric electrolyte solution for pH 3 to 10 Ex- ple solution, add methanol to make exactly 100 mL, and use
tremely pale yellow liquid. Mixture consisting of multiple this solution as the standard solution. Perform the test with
types of molecules, buffer capacity is 0.35 mmol/pH-mL. 10 mL each of the sample solution and standard solution as
Forms a pH gradient over a pH range of 3 to 10 when mixed directed in the Identification under Peach Kernel: any spot
with polyacrylamide gel and placed in an electric field. other than the principal spot at the R f value of about 0.3 ob-
tained from the sample solution is not more intense than the
Amphoteric electrolyte solution for pH 6 to 9 Forms a
spot obtained from the standard solution.
pH gradient over a pH range of 6 to 9 when mixed with poly-
acrylamide gel and placed in an electric field. Prepare by n-Amyl alcohol CH3(CH2)4OH Clear, colorless liquid,
diluting a 0.35 mmol/pH-mL buffer capacity solution about having a characteristic odor. Sparingly soluble in water, and
20-fold with water. Almost colorless. miscible with ethanol (95) and with diethyl ether.
Refractive index <2.45> n 20
D : 1.409 – 1.411
Amphoteric electrolyte solution for pH 8 to 10.5 Ex-
Specific gravity <2.56> d 20
4 : 0.810 – 0.820
tremely pale yellow liquid. Mixture consisting of multiple
Distilling range <2.57>: 135 – 1409C, not less than 95
types of molecules, buffer capacity is 0.35 mmol/pH-mL.
volz.
Forms a pH gradient over a pH range of 8 to 10.5 when mix-
ed with polyacrylamide gel and placed in an electric field. t-Amyl alcohol (CH3)2C(OH)CH2CH3 Clear, colorless
liquid, having a characteristic odor. Miscible with t-butyl
Ampiroxicam for assay C20H21N3O7S [Same as the
alcohol and with 2-butanone, and freely soluble in water.
monograph Ampiroxicam]
Specific gravity <2.56> d 20
20: 0.808 – 0.815
Amygdalin for assay C20H27NO11 Amygdalin for thin- Purity Acid and ester—To 20 mL of t-amyl alcohol add
layer chromatography. However, it meets the following re- 20 mL of ethanol (95) and 5.0 mL of 0.1 mol/L sodium hy-
quirements: droxide VS, and heat gently under a reflux condenser in a
Absorbance <2.24> E 11zcm (263 nm): 5.2 – 5.8 [20 mg, meth- water bath for 10 minutes. Cool, add 2 drops of phenol-
anol, 20 mL; separately determine the water <2.48> (5 mg, phthalein TS, and titrate <2.50> with 0.1 mol/L hydrochloric
coulometric titration) and calculate on the anhydrous basis]. acid VS. Perform a blank determination: not more than 1.25
Purity Related substances—Dissolve 5 mg of amygdalin mL of 0.1 mol/L sodium hydroxide VS is consumed.
for assay in 10 mL of the mobile phase, and use this solution Nonvolatile residue—Evaporate 50 mL of t-amyl alcohol,
as the sample solution. Pipet 1 mL of the sample solution, and dry at 1059C for 1 hour: the residue is not more than
add the mobile phase to make exactly 100 mL, and use this 1.6 mg.
solution as the standard solution. Perform the test with ex- Distilling range <2.57>: 100 – 1039C, not less than 95
actly 10 mL each of the sample solution and standard solu- volz.
tion as directed under Liquid Chromatography <2.01> ac-
tert-Amyl alcohol See t-amyl alcohol.
cording to the following conditions, and determine each
peak area by the automatic integration method: the total Amyl alcohol, iso See 3-methyl-1-butanol.
area of the peaks other than amygdalin from the sample so-
Anesthetic ether See ether, anesthetic.
lution is not larger than the peak area of amygdalin from the
standard solution. Anhydrous caffeine See caffeine, anhydrous.
Operating conditions
Anhydrous cupric sulfate See copper (II) sulfate.
Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in Anhydrous dibasic sodium phosphate See disodium
the Assay (3) under Keishibukuryogan Extract. hydrogen phosphate, anhydrous.
Time span of measurement: About 3 times as long as the

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
204 General Tests / 9.41 Reagents, Test Solutions JP XVII
Anhydrous dibasic sodium phosphate for pH determina- retention time of 14-anisoylaconine.
tion See disodium hydrogen phosphate for pH determina- System suitability
tion. Test for required detectability: Pipet 1 mL of the standard
solution, and add the mobile phase to make exactly 20 mL.
Anhydrous hydrazine for amino acid analysis Prepared
Confirm that the peak area of 14-anisoylaconine obtained
for amino acid analysis.
from 20 mL of this solution is equivalent to 3.5 to 6.5z of
Anhydrous lactose C12H22O11 [Same as the monograph that of 14-anisoylaconine obtained from 20 mL of the stand-
Anhydrous Lactose] ard solution.
System performance: When the procedure is run with 20
Anhydrous potassium carbonate See potassium carbon-
mL of aconitum monoester alkaloids standard TS for assay
ate.
under the above operating conditions, benzoylmesaconine,
Anhydrous sodium acetate See sodium acetate, anhy- benzoylhypaconine and 14-anisoylaconine are eluted in this
drous. order with the resolution between these peaks being not less
than 4, respectively.
Anhydrous sodium carbonate See sodium carbonate,
System repeatability: When the test is repeated 6 times
anhydrous.
with 20 mL of aconitum monoester alkaloids standard TS for
Anhydrous sodium sulfate See sodium sulfate, anhy- assay under the above operating conditions, the relative
drous. standard deviations of the peak areas of benzoylmesaconine,
benzoylhypaconine and 14-anisoylaconine are not more than
Anhydrous sodium sulfite See sodium sulfite, anhy-
1.5z, respectively.
drous.
14-Anisoylaconine hydrochloride for component determi-
Aniline C6H5NH2 [K 8042, Special class]
nation See 14-anisoylaconine hydrochloride for assay.
Animal tissue peptone See peptone, animal tissue.
Anode solution A for water determination Dissolve
p-Anisaldehyde See 4-methoxybenzaldehyde. 100 g of diethanolamine in 900 mL of a mixture of methanol
for water determination and chloroform for water determi-
p-Anisaldehyde-acetic acid TS See 4-methoxyhenzalde-
nation (1:1), pass dried sulfur dioxide gas through this solu-
hyde-acetic acid TS.
tion while cooling until the mass increase of the solution
p-Anisaldehyde-sulfuric acid TS See 4-methoxybenzalde- reaches 64 g. Then add 20 g of iodine, and add water until
hyde-sulfuric acid TS. the color of the solution changes from brown to yellow. To
600 mL of this solution add 400 mL of chloroform for water
Anisole C7H8O A colorless liquid. Boiling point: about
determination.
1559C.
Specific gravity <2.56> d 20
20: 0.995 – 1.001. Anthrone C14H10O Light yellow, crystals or crystalline
powder.
14-Anisoylaconine hydrochloride for assay
Melting point <2.60>: 154 – 1609C
C33H47NO11.HCl White, crystalline powder or powder.
Preserve in a light-resistant tight container.
Freely soluble in methanol, sparingly soluble in water and in
ethanol (99.5). Melting point: about 2109C (with decomposi- Anthrone TS Dissolve 35 mg of anthrone in 100 mL of
tion). sulfuric acid.
Absorbance <2.24> E 11zcm (258 nm): 276 – 294 (5 mg calcu-
Anti-A type antibody for blood typing Conforms to the
lated on the anhydrous basis, methanol, 200 mL).
requirements of antibody for blood typing.
Purity (1) Related substances—To 1.0 mg of 14-
anisoylaconine hydrochloride for assay add exactly 1 mL of Anti-B type antibody for blood typing Conforms to the
ethanol (99.5). Perform the test with 5 mL of this solution as requirements of antibody for blood typing.
directed in the Identification under Processed Aconite Root:
Antibody fragment (Fab?) Purify E. coli protein anti-
any spot other than the principle spot with an Rf value of
body by affinity chromatography using Staphylococcus
about 0.5 does not appear.
aureus protein A as a ligand, and fractionate IgG. Digest this
(2) Related substances—Dissolve 5.0 mg of 14-anisoy-
fraction using pepsin, remove the pepsin and Fc fragment by
laconine hydrochloride for assay in 5 mL of the mobile
gel filtration chromatography, and obtain F(ab?)2 fragment
phase, and use this solution as the sample solution. Pipet 1
after removing undigested IgG by affinity chromatography
mL of the sample solution, add the mobile phase to make ex-
with protein A as ligand. Reduce this with 2-mercaptoethyla-
actly 50 mL, and use this solution as the standard solution.
mine.
Perform the test with exactly 20 mL each of the sample solu-
tion and standard solution as directed under Liquid Chroma- Anti-bradykinin antibody A colorless to light brown,
tography <2.01> according to the following conditions. De- clear solution prepared by dissolving rabbit origin anti-
termine each peak area of both solutions by the automatic bradykinin antibody in 0.04 mol/L phosphate buffer solu-
integration method: the total area of the peaks other than tion (pH 7.0) containing 1 mg/mL of bovine serum albumin.
14-anisoylaconine obtained from the sample solution is not Performance test—To a suitable amount of anti-bradyki-
larger than the peak area of 14-anisoylaconine obtained nin antibody to be tested add 0.04 mol/L phosphate buffer
from the standard solution. solution (pH 7.0) containing 1 mg/mL bovine serum albu-
Operating conditions min to make a 1 volz solution. Perform the test with 0.1
Column, column temperature, mobile phase and flow mL of this solution as directed in the Purity (2) under Kal-
rate: Proceed as directed in the operating conditions in the lidinogenase, and determine the absorbances at 490 – 492
Assay (3) under Goshajinkigan Extract. nm, A1 and A2, of the standard solution (1) and the standard
Detector: An ultraviolet absorption photometer (wave- solution (7): the value, A2 – A1, is not less than 1.
length: 245 nm).
Anti-bradykinin antibody TS To 0.15 mL of anti-
Time span of measurement: About 4 times as long as the

bradykinin antibody, 15 mg of bovine serum albumin, 2.97 Freund's complete adjuvant. Inject intracutaneously three
mg of sodium dihydrogen phosphate dihydrate, 13.5 mg of 2-mL portions of the emulsion to a healthy rabbit weighed
disodium hydrogen phosphate dodecahydrate and 13.5 mg between 2.5 kg and 3.0 kg in a week interval. Collect the
of sodium chloride add water to make 15 mL, and lyophi- blood from the rabbit at 7 to 10 days after the last injection,
lize. Dissolve this in 15 mL of water. Prepare before use. and prepare the anti-serum.
Performance test—Dissolve 1.0 g of agar in 100 mL of
Anti-E. coli protein antibody stock solution Taking E.
boric acid-sodium hydroxide buffer solution (pH 8.4) by
coli protein stock solution as the immunogen, mix with
warming, and pour the solution into a Petri dish to make a
Freund's complete adjuvant, and immunize rabbits by sub-
depth of about 2 mm. After cooling, bore three of a pair-
cutaneous injection at 3 week intervals to obtain antiserum.
well 2.5 mm in diameter with a space of 6 mm each other. In
Treat the antiserum obtained by ammonium sulfate precipi-
one of the wells of each pair-well, place 10 mL of anti-
tation.
urokinase serum, and in each another well, place 10 mL of a
Protein concentration: Dilute anti-E. coli protein antibody
solution of Urokinase containing 30,000 Units per mL in iso-
stock solution with 0.05 mol/L tris hydrochloride buffer so-
tonic sodium chloride solution, 10 mL of human serum and
lution (pH 7.5), measure the absorbance at 280 nm using
10 mL of human urine, respectively, and allow to stand over-
0.05 mol/L tris hydrochloride buffer solution (pH 7.5) as a
night: a precipitin line appears between anti-urokinase serum
control as direct under Ultraviolet-visible Spectrophotome-
and urokinase, and not appears between anti-urokinase se-
try <2.24>, and determine the protein concentration (absor-
rum and human serum or human urine.
bance 1.0 ＝ 0.676 mg/mL).
a-Apooxytetracycline C22H22N2O8 Yellow-brown to
Anti-interferon alfa antiserum Antiserum prepared by
green powder.
immunizing rabbits with interferon alfa, which is capable of
Melting point <2.60>: 200 – 2059C
reacting specifically with interferon alfa to neutralize 10,000
Units or more of interferon alfa in 1 mL. b-Apooxytetracycline C22H22N2O8 Yellow-brown to
brown powder.
Antimony (III) chloride SbCl3 [K 8400, Special class]
Purity Related substances—Dissolve 8 mg of b-apoox-
Antimony (III) chloride TS Wash chloroform with an ytetracycline in 5 mL of 0.01 mol/L sodium hydroxide TS,
equal volume of water twice or three times, add freshly add 0.01 mol/L hydrochloric acid TS to make 100 mL, and
ignited and cooled potassium carbonate, and allow to stand use this solution as the sample solution. Proceed the test with
overnight in a well-closed container protected from light. 20 mL of the sample solution as directed in the Purity (2)
Separate the chloroform layer, and distil it, preferably with under Oxytetracycline Hydrochloride, determine each peak
protection from light. With this chloroform, wash the sur- area by the automatic integration method, and calculate the
face of antimony (III) chloride until the rinsing solution amounts of them by the area percentage method: the total
becomes clear, add the chloroform to this antimony (III) amount of the peaks other than b-apooxytetracycline is not
chloride to make a saturated solution, and place in light- more than 10z.
resistant, glass-stoppered bottles. Prepare before use.
Aprindine hydrochloride for assay C22H30N2.HCl
Antimony trichlorid See antimony (III) chloride. [Same as the monograph Aprindine Hydrochloride. When
dried, it contains not less than 99.5z of aprindine hydro-
Antimony trichlorid TS See antimony (III) chlorid TS.
chloride (C22H30N2.HCl).]
Antipyrine C11H12N2O [Same as the namesake mono-
Aprotinin A clear and colorless liquid containing aproti-
graph]
nin extracted from the lung or parotid gland of a healthy cat-
Anti-rabbit antibody-coated wells Wells of a polystyrene tle. The pH is between 5.0 and 7.0.
microplate coated with goat origin anti-rabbit IgG antibody. Content: not less than 15,000 KIE Units and not more
than 25,000 KIE Units of aprotinin per mL.
Anti-thrombin III A white powder.
Assay—
Water <2.48>: not more than 5z.
(i) Trypsin solution: Weigh an amount of crystalline trypsin
Content: not less than 80z and not more than 130z of
equivalent to about 250 FIP Units of trypsin according to the
the labeled amount.
labeled FIP Units, and dissolve in 0.001 mol/L hydrochloric
Anti-thrombin III TS Dissolve 10 unit of anti-thrombin acid TS to make exactly 10 mL. Prepare before use, and
III in 10 mL of water. preserve in ice.
(ii) Sample solution: Dilute a suitable quantity of aprotinin
Anti-ulinastatin rabbit serum To a suitable amount of
with sodium tetraborate-calcium chloride buffer solution
Ulinastatin having the specific activity of more than 3000
(pH 8.0) so that each mL of the solution contains 800 KIE
Units per mg protein add isotonic sodium chloride solution
Units of aprotinin, and use this solution as the sample solu-
so that each mL of the solution contains about 1 mg of pro-
tion.
tein. To 1 mL of this solution add 1 mL of Freund's com-
(iii) Apparatus: Use a glass bottle as a reaction reservoir, 20
plete adjuvant, and emulsify completely. Intracutaneously,
mm in inside diameter and 50 mm in height, equipped with a
inject the emulsion so obtained into a rabbit weighing about
rubber stopper for attachment to a glass/silver-silver chlo-
2 kg. Repeat the injection at least 4 times at one-week inter-
ride electrode, a nitrogen-induction tube and an exhaust
vals, and draw the blood of the animal from the carotid
port. Fix the reaction reservoir in a thermostat, and keep the
artery after the antibody titer reaches 16 times or more.
temperature of the bath at 25 ± 0.19C by means of a precise
Separate the serum after the blood has coagulated. Preserve
thermoregulator.
at below －209 C.
(iv) Procedure: To 5.0 mL of N-a-benzoyl-L-arginine ethyl
Anti-urokinase serum Take Urokinase containing not ester TS add 45.0 mL of sodium tetraborate-calcium chloride
less than 140,000 Unit per mg of protein, dissolve in isotonic buffer solution (pH 8.0), and use this solution as the sub-
sodium chloride solution to make a solution containing 1 mg strate solution. Pipet 1 mL of the trypsin solution, add so-
of protein per mL, and emulsify with an equal volume of dium tetraborate-calcium chloride buffer solution (pH 8.0)

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
206 General Tests / 9.41 Reagents, Test Solutions JP XVII
to make exactly 10 mL, and use this solution as the test solu- Optical rotation <2.49> [a]20
D : ＋103.0 – ＋105.59 Weigh
tion I. Transfer 10.0 mL of the substrate solution to the accurately about 5 g of L-arabinose, previously dried at
reaction reservoir, adjust the pH of the solution to 8.00 by 1059C for 2 hours, dissolve in 30 mL of water, add 0.4 mL
adding dropwise 0.1 mol/L sodium hydroxide VS while stir- of ammonia TS, and add water to make exactly 50 mL.
ring and passing a current of nitrogen, add exactly 1 mL of Allow to stand for 1 hour, and determine using a 100-mm
the test solution I previously allowed to stand at 25 ± 0.19C cell.
for 10 minutes, then immediately add dropwise 0.1 mol/L Melting point <2.60>: 155 – 1609C
sodium hydroxide VS by a 50-mL micropipet (minimum
Arbutin for assay C12H16O7 Use arbutin for thin-layer
graduation of 1 mL), while stirring, to keep the reaction solu-
chromatography meeting the following additional specifica-
tion at pH 8.00, and read the amount of 0.1 mol/L sodium
tions.
hydroxide VS consumed and the reaction time when the pH
Absorbance <2.24> E 11zcm (280 nm): 70 – 76 [4 mg, previ-
reached 8.00. Continue this procedure up to 6 minutes.
ously dried in a desiccator (in vaccum, silica gel) for 12
Separately, pipet 2 mL of the trypsin solution and 1 mL of
hours, water, 100 mL].
the sample solution, add sodium tetraborate-calcium chlo-
Purity Related substances—Dissolve 40 mg of arbutin
ride buffer solution (pH 8.0) to make exactly 10 mL, and use
for assay in 100 mL of water, and use this solution as the
this solution as the test solution II. Transfer 10.0 mL of the
sample solution. Pipet 1 mL of the sample solution, add
substrate solution to the reaction reservoir, adjust the pH of
water to make exactly 100 mL, and use this solution as the
the solution to 8.00, while stirring and passing a current of
standard solution (1). Perform the test with exactly 10 mL
nitrogen, add exactly 1 mL of the test solution II, previously
each of the sample solution and standard solution (1) as di-
allowed to stand at 25 ± 0.19C for 10 minutes, and proceed
rected under Liquid Chromatography <2.01> according to
in the same manner. Separately, transfer 10.0 mL of the sub-
the following conditions, and determine each peak area of
strate solution to the reaction reservoir, adjust the pH of the
the both solutions by the automatic integration method: the
solution to 8.00, while stirring and passing a current of nitro-
total area of the peaks other than arbutin from the sample
gen, add 1 mL of sodium tetraborate-calcium chloride buffer
solution is not larger than the peak area of arbutin from the
solution (pH 8.0), previously allowed to stand at 25 ± 0.19 C
standard solution (1).
for 10 minutes, and perform a blank determination in the
Operating conditions
same manner.
Proceed the operating conditions in the Assay under
(v) Calculation: Plot the amount of consumption (mL) of 0.1
Bearberry Leaf except detection sensitivity and time span of
mol/L sodium hydroxide VS against the reaction time
measurement.
(minutes), select linear reaction times, t1 and t2, designate the
Detection sensitivity: Pipet 1 mL of the standard solution
corresponding consumption amount of 0.1 mol/L sodium
(1), add water to make exactly 20 mL, and use this solution
hydroxide VS as v1 and v2, respectively, and designate mmol
as the standard solution (2). Adjust the detection sensitivity
of sodium hydroxide consumed per minute as D.
so that the peak area of arbutin obtained from 10 mL of the
v2 － v1 1 standard solution (2) can be measured by the automatic in-
D ( mmol NaOH/minute) ＝ × ×f
t2 － t1 10 tegration method and the peak height of arbutin obtained
from 10 mL of the standard solution (1) is about 20z of the
f: Factor of 0.1 mol/L sodium hydroxide VS
full scale.
KIE Units per mL of aprotinin to be tested Time span of measurement: About 3 times as long as the
retention time of arbutin, beginning after the solvent peak.
2(DA － D0) － (DB － D0)
＝ × n × 32.5
L Arbutin for component determination See arbutin for
assay.
L: Amount (mL) of the sample solution added to the test
solution II Arbutin for thin-layer chromatography C12H16O7
n: Dilution coefficient of aprotinin to be tested Colorless to white, crystals or crystalline powder, and odor-
DA: mmol of sodium hydroxide consumed in 1 minute less. Freely soluble in water, soluble in methanol, sparingly
when the test solution I is used soluble in ethanol (95), and practically insoluble in ethyl ace-
DB: mmol of sodium hydroxide consumed in 1 minute tate and in chloroform.
when the test solution II is used Melting point <2.60>: 199 – 2019C
D0: mmol of sodium hydroxide consumed in 1 minute Purity Related substances—Dissolve 1.0 mg of arbutin
when the solution for blank determination is used for thin-layer chromatography in exactly 1 mL of a mixture
32.5: Equivalent coefficient for calculation of KIE Units of ethanol (95) and water (7:3). Perform the test with 20 mL
from FIP Units of this solution as directed in the Identification (2) under
Bearberry Leaf: any spot other than the main spot with an
One KIE Unit means an amount of aprotinin making a
R f value of about 0.4 does not appear.
reduction of 50z off the potency of 2 Units of kalli-
dinogenase at pH 8.0 and room temperature for 2 hours. Arecoline hydrobromide for thin-layer chromatography
Storage—Preserve in a light-resistant, hermetic container C8H13NO2.HBr White crystals. Freely soluble in water,
and in a cold place. soluble in methanol, and practically insoluble in diethyl
ether.
Aprotinin TS Measure an appropriate amount of aproti-
Melting point <2.60>: 169 – 1719C
nin, and dissolve in 0.05 mol/L phosphate buffer solution
Purity Related substances—Dissolve 5 mg of arecoline
(pH 7.0) to prepare a solution containing 50 KIE Units per
hydrobromide for thin-layer chromatography in exactly 1
mL.
mL of methanol. Perform the test with 10 mL of this solution
Aqua regia Add 1 volume of nitric acid to 3 volumes of as directed in the Identification under Areca: any spot other
hydrochloric acid. Prepare before use. than the principal spot at the R f value of about 0.6 does not
appear.
L-Arabinose C5H10O5 A white crystalline powder.

L-Arginine C6H14N4O2 White, crystals or crystalline add 5 mL of a mixture of methanol and water (3:2), shake
powder. It has a characteristic odor. for 10 minutes, centrifuge, and use the supernatant liquid as
Optical rotation <2.49> [a]20 D : ＋26.9 – ＋27.99(After dry- the sample solution. Perform the test with the sample solu-
ing, 4 g, 6 mol/L hydrochloric acid TS, 50 mL, 200 mm). tion as directed under Thin-layer Chromatography <2.03>.
Loss on drying <2.41>: not more than 0.50z (1 g, 1059C, Spot 10 mL of the sample solution on a plate of octadecyl-
3 hours). silanized silica gel for thin-layer chromatography. Develop
Content: not less than 98.0z and not more than 102.0z. the plate with a mixture of methanol and water (4:1) to a dis-
Assay—Weigh accurately about 0.15 g of L-arginine, previ- tance of about 7 cm, and air-dry the plate. Spray evenly
ously dried, dissolve in 3 mL of formic acid, add 50 mL of dilute sulfuric acid on the plate, heat at 1059
C for 5 minutes,
acetic acid (100), and titrate <2.50> with 0.1 mol/L perchloric and examine under ultraviolet light (main wavelength: 365
acid VS until the color of the solution changes to green nm): two green fluorescent spots appear at R f values of
through yellow (indicator: 10 drops of p-naphtholbenzein about 0.3 and about 0.4 (eupatilin and jaceosidin).
TS). Perform a blank determination in the same manner and
Asarinin for thin-layer chromatography C20H18O6
make any necessary correction.
White, crystals or crystalline powder. Slightly soluble in
Each mL of 0.1 mol/L perchloric acid VS methanol and in ethanol (99.5), and practically insoluble in
＝ 8.710 mg of C6H14N4O2 water. Melting point: 118 – 1229C.
Identification—Determine the absorption spectrum of a
L-Arginine hydrochloride C6H14N4O2.HCl [Same as the
solution in methanol (3 in 200,000) as directed under Ultravi-
namesake monograph]
olet-visible Spectrophotometry <2.24>: it exhibits maxima be-
Argon Ar [K 1105, First class] tween 234 nm and 238 nm, and between 285 nm and 289 nm.
Purity Related substances—Dissolve 1 mg of asarinin for
Aristolochic acid I for crude drugs purity test
thin-layer chromatography in 1 mL of methanol, and per-
C17H11NO7 Yellow crystalline powder. Melting point:
form the test with 1 mL of this solution as directed in the
about 2809C (with decomposition).
Identification (7) under Shoseiryuto Extract: no spot other
Absorbance <2.24> E 11zcm (318 nm): 384 – 424 (1 mg,
than the principal spot (R f value is about 0.4) appears.
methanol, 100 mL).
Purity Related substances—Dissolve 1.0 mg of aris- (E )-Asarone C12H16O3 White powder. Freely soluble in
tolochic acid I for crude drugs purity test in 100 mL of methanol and in ethanol (99.5) and practically insoluble in
diluted methanol (3 in 4), and use this solution as the sample water. Melting point: about 609C.
solution. Pipet 1 mL of the sample solution, add diluted Identification—Determine the infrared absorption spec-
methanol (3 in 4) to make exactly 100 mL, and use this solu- trum of (E )-asarone as directed in the potassium bromide
tion as the standard solution. Perform the test with exactly disk method under Infrared Spectrophotometry <2.25>, it ex-
10 mL each of the sample solution and standard solution as hibits absorption at the wave numbers of about 2990 cm－1,
directed under Liquid Chromatography <2.01> according to 2940 cm－1, 2830 cm－1, 1609 cm－1, 1519 cm－1, 1469 cm－1,
the following conditions, and determine each peak area by 1203 cm－1, 1030 cm－1, 970 cm－1 and 860 cm－1.
the automatic integration method: the total area of the peaks Purity Related substances—Dissolve 2 mg of (E )-asa-
other than aristolochic acid I obtained from the sample solu- rone in 10 mL of methanol, and use this solution as the sam-
tion is not larger than the peak area of aristolochic acid I ple solution. Pipet 1 mL of the sample solution, add metha-
obtained from the standard solution. nol to make exactly 10 mL, and use this solution as the
Operating conditions standard solution. Perform the test with exactly 10 mL each
Detector, column, column temperature, mobile phase, and of the sample solution and standard solution as directed
flow rate: Proceed as directed in the operating conditions in under Liquid Chromatography <2.01> according to the fol-
the Purity (5) under Asiasarum Root. lowing conditions. Determine each peak area of both solu-
Time span of measurement: About 3 times as long as the tions by the automatic integration method: the total area of
retention time of aristolochic acid I, beginning after the sol- the peaks other than (E )-asarone obtained from the sample
vent peak. solution is not larger than the peak area of (E )-asarone ob-
System suitability tained from the standard solution.
Proceed as directed in the system suitability in the Purity Operating conditions
(5) under Asiasarum Root. Detector, column, column temperature, mobile phase and
flow rate: Proceed as directed in the operating conditions in
Arsenazo III C22H18As2N4O14S2 [K 9524, Special class]
the Assay under Perilla Herb.
Arsenazo III TS Dissolve 0.1 g of arsenazo III in water Time span of measurement: About 3 times as long as the
to make 50 mL. retention time of (E )-asarone, beginning after the solvent
peak.
Arsenic-free zinc See zinc for arsenic analysis.
System suitability
Arsenic trioxide As2O3 [K 8044, Diarsenic trioxide, System performance: Proceed as directed in the system
Special class] suitability in the Assay under Perilla Herb.
Arsenic trioxide TS Add 1 g of arsenic trioxide to 30 mL Ascorbic acid See L-ascorbic acid.
of a solution of sodium hydroxide (1 in 40), dissolve by heat-
L-Ascorbic acid C6H8O6 [K 9502, L(＋)-Ascorbic
ing, cool, and add gently acetic acid (100) to make 100 mL.
Acid, Special class]
Arsenic (III) trioxide See arsenic trioxide.
Ascorbic acid for iron limit test See L-ascorbic acid.
Arsenic (III) trioxide TS See arsenic trioxide TS.
0.012 g/dL L-Ascorbic acid-hydrochloric aicd TS Dis-
Artemisia･argyi for purity test Powder of the leaf and solve 15 mg of L-ascorbic acid in 25 mL of methanol, add
twig of Artemisia argyi H. L áeveill áe et Vaniot. carefully 100 mL of hydrochloric acid, and mix. Prepare be-
Identification—To 0.5 g of artemisia･argyi for purity test fore use.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
208 General Tests / 9.41 Reagents, Test Solutions JP XVII
0.02 g/dL L-Ascorbic acid-hydrochloric acid TS Dissolve ditions, the number of theoretical plates and the symmetry
25 mg of L-ascorbic acid in 25 mL of methanol, add care- factor of the peak of atractylenolide III are not less than
fully 100 mL of hydrochloric acid, and mix. Prepare before 5000 and not more than 1.5, respectively.
use. System repeatability: When the test is repeated 6 times
with 10 mL of the standard solution under the above operat-
0.05 g/dL L-Ascorbic acid-hydrochloric acid TS Dissolve
ing conditions, the relative standard deviation of the peak
50 mg of L-ascorbic acid in 30 mL of methanol, add care-
area of atractylenolide III is not more than 1.5z.
fully hydrochloric acid to make 100 mL. Prepare before use.
Atractylenolide III for thin-layer chromatography
L-Asparagine monohydrate C4H8N2O3.H2O [K8021,
C15H20O3 White, crystals or crystalline powder. Freely
Special class]
soluble in methanol, soluble in ethanol (99.5), and practi-
DL-Aspartic acid C4H7NO4 A white crystalline powder cally insoluble in water. Melting point: 193 – 1969 C.
that is sparingly soluble in water. Melting point: 270 to Identification—(1) Determine the absorption spectrum of
2719C. a solution of atractylenolide III for thin-layer chromatogra-
phy in methanol (1 in 100,000) as directed under Ultraviolet-
L-Aspartic acid C4H7NO4 [K 9045, Special class]
visible Spectrophotometry <2.24>: it exhibits a maximum be-
Aspartic acid See L-aspartic acid. tween 217 nm and 221 nm.
(2) Determine the infrared absorption spectrum of atrac-
Aspirin C9H8O4 [Same as the namesake monograph]
tylenolide III for thin-layer chromatography as directed in
Astragaloside IV for thin-layer chromatography the potassium bromide disk method under Infrared Spectro-
C41H68O14 A white powder. Sparingly soluble in methanol, photometry <2.25>: it exhibits absorption at the wave num-
very slightly soluble in ethanol (99.5), and practically insolu- bers of about 3350 cm－1, 1742 cm－1, 1641 cm－1 and 1384
ble in water. cm－1.
Optical rotation <2.49> [a]20 D : ＋19 – ＋269(10 mg dried Purity Related substances—Dissolve 2 mg of atrac-
with silica gel for 24 hours, methanol, 2 mL, 50 mm). tylenolide III for thin-layer chromatography in 2 mL of
Purity Related substances—Dissolve 1 mg of astragalo- methanol, and use this solution as the sample solution. Pipet
side IV for thin-layer chromatography in 1 mL of methanol. 1 mL of the sample solution, add methanol to make exactly
Proceed the test with 5 mL of this solution as directed in the 50 mL, and use this solution as the standard solution. Per-
Identification (4) under Hochuekkito Extract: no spot ap- form the test with these solutions as directed under Thin-lay-
pears other than the principal spot of around R f value of er Chromatography <2.03>. Proceed the test with 5 mL each
0.4. of the sample solution and standard solution as directed in
the Identification (3) under Tokishakuyakusan Extract: the
Atractylenolide III for assay C15H20O3 Use atrac-
spot other than the principal spot with an Rf value of about
tylenolide III for thin-layer chromatography. It meets the
0.5 obtained from the sample solution is not more intense
following additional specifications.
than the spot obtained from the standard solution.
Absorbance <2.24> E 11zcm (219 nm): 446 – 481 (5 mg, meth-
anol, 500 mL). Atractylodin for assay C13H10O White to pale yellow-
Purity Related substances—Dissolve 5 mg of atrac- ish crystals. Freely soluble in methanol and in ethanol (99.5),
tylenolide III for assay in 50 mL of methanol, and use this and practically insoluble in water. Melting point: about
solution as the sample solution. To exactly 1 mL of the sam- 549C.
ple solution add methanol to make exactly 100 mL, and use Identification—Conduct this procedure without exposure
this solution as the standard solution. Perform the test with to light, using light-resistant vessels. Determine the absorp-
exactly 10 mL each of the sample solution and standard solution spectrum of a solution of atractylodin for assay in meth-
tion as directed under Liquid Chromatography <2.01> ac- anol (1 in 250,000) as directed under Ultraviolet-visible Spec-
cording to the following conditions, and determine each trophotometry <2.24>: it exhibits maxima between 256 nm
peak area by the automatic integration method: the total and 260 nm, between 270 nm and 274 nm, between 332 nm
area of the peaks other than atractylenolide III obtained and 336 nm and between 352 nm and 356 nm.
from the sample solution is not larger than the peak area of Absorbance <2.24> E 11zcm (272 nm): 763 – 819 (2 mg, meth-
atractylenolide III obtained from the standard solution. anol, 250 mL). Conduct this procedure without exposure to
Operating conditions light, using light-resistant vessels.
Column, column temperature and mobile phase: Proceed Purity Related substances—
as directed in the operating conditions in the Assay (3) under (i) Conduct this procedure without exposure to light,
Tokishakuyakusan Extract. using light-resistant vessels. Dissolve 2 mg of atractylodin
Detector: An ultraviolet absorption photometer (wave- for assay in 2 mL of methanol, and use this solution as the
length: 220 nm). sample solution. To exactly 1 mL of the sample solution add
Flow rate: Adjust so that the retention time of atrac- methanol to make exactly 100 mL, and use this solution as
tylenolide III is about 11 minutes. the standard solution. Perform the test with these solutions
Time span of measurement: About 5 times as long as the as directed under Thin-layer Chromatography <2.03>. Spot
retention time of atractylenolide III, beginning after the sol- 10 mL each of the sample solution and standard solution on a
vent peak. plate of silica gel for thin-layer chromatography and imme-
System suitability diately develop the plate with a mixture of hexane and ace-
Test for required detectability: To exactly 1 mL of the tone (7:1) to a distance of about 10 cm, and air-dry the plate.
standard solution add methanol to make exactly 20 mL. Spray evenly vanillin-sulfuric acid-ethanol TS for splaying
Confirm that the peak area of atractylenolide III obtained on the plate, and heat at 1059C for 5 minutes: the spot other
with 10 mL of this solution is equivalent to 3.5 to 6.5z of than the principle spot which appears at an Rf value of
that obtained with 10 mL of the standard solution. about 0.4 obtained from the sample solution is not more in-
System performance: When the procedure is run with 10 tense than the spot obtained from the standard solution.
mL of the standard solution under the above operating con- (ii) Conduct this procedure without exposure to light,

using light-resistant vessels. Dissolve 5 mg of atractylodin chloroform and diethylamine (9:1) to a distance of about 10
for assay in 250 mL of methanol, and use this solution as the cm, air-dry the plate, and spray evenly hydrogen hexachloro-
sample solution. To exactly 1 mL of the sample solution add platinate (IV)-potassium iodide TS on the plate: any spot
methanol to make exactly 100 mL, and use this solution as other than the principle spot at the R f value of about 0.4
the standard solution. Perform the test with exactly 20 mL does not appear.
each of the sample solution and standard solution as directed
A-type erythrocyte suspension Prepare a suspension con-
under Liquid Chromatography <2.01> according to the fol-
taining 1 volz of erythrocyte separated from human A-type
lowing conditions, and determine each peak area by the au-
blood in isotonic sodium chloride solution.
tomatic integration method: the total area of the peaks other
than atractylodin obtained from the sample solution is not Avidin-biotin TS To 15 mL of phosphate-buffered so-
larger than the peak area of atractylodin obtained from the dium chloride TS add 2 drops each of avidin TS and
standard solution. biotinylated peroxidase TS, and mix.
Operating conditions
Azelastine hydrochloride for assay C22H24ClN3O.HCl
Detector, column, column temperature and mobile phase:
[Same as the monograph Azelastine Hydrochloride]
Proceed as directed in the operating conditions in the Assay
(4) under Tokishakuyakusan Extract. Azelnidipine for assay C33H34N4O6 [Same as the mono-
Flow rate: Adjust so that the retention time of atrac- graph Azelnidipine. When dried, it contains not less than
tylodin is about 13 minutes. 99.5z of azelnidipine (C33H34N4O6).]
Time span of measurement: About 5 times as long as the
2,2?-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) di-
retention time of atractylodin, beginning after the solvent
ammonium salt C18H16N4O6S4.(NH4)2 A bluish green
peak.
crystalline powder.
System suitability
Melting point <2.60>: about 3309C (with decomposition).
Test for required detectability: To exactly 1 mL of the
standard solution add methanol to make exactly 20 mL. 2,2?-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) di-
Confirm that the peak area of atractylodin obtained with 20 ammonium salt TS Dissolve 5.3 g of citric acid monohy-
mL of this solution is equivalent to 3.5 to 6.5z of that ob- drate in water to make 500 mL. To this solution add a solu-
tained with 20 mL of the standard solution. tion prepared by dissolving 7.1 g of anhydrous disodium
System performance: Put a suitable amount of the stand- hydrogen phosphate in water to make 500 mL to adjust to
ard solution in a colorless vessel, and expose to ultraviolet pH 4.3. To 20 mL of this solution add 15 mg of 2,2?-
light (main wavelength: 365 nm) for about 1 minute. When azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammoni-
the procedure is run with 20 mL of this solution under the um salt. To this solution add 14 mL of hydrogen peroxide TS
above operating conditions, a peak of an isomer is found in before use.
addition to the peak of atractylodin, and the isomer and
Baicalin for thin-layer chromatography C21H18O11
atractylodin are eluted in this order with the resolution be-
Light yellow, crystals or powder. Slightly soluble in metha-
tween these peaks being not less than 1.5.
nol and in ethanol (99.5), and practically insoluble in water.
System repeatability: When the test is repeated 6 times
Identification Determine the infrared absorption spec-
with 20 mL of the standard solution under the above operat-
trum of baicalin to be examined as directed in the potassium
ing conditions, the relative standard deviation of the peak
bromide disk method under Infrared Spectrophotometry
area of atractylodin is not more than 1.5z.
<2.25>: it exhibits absorption at the wave numbers of about
Atractylodin TS for assay Conduct this procedure with- 3390 cm－1, 1662 cm－1, 1492 cm－1, 1068 cm－1 and 685 cm－1.
out exposure to light, using light-resistant vessels. Weigh ac- Purity Related substance—Dissolve 1 mg of baicalin to
curately about 5 mg of atractylodin for assay, and dissolve in be examined in exactly 1 mL of methanol. Perform the test
methanol to make exactly 1000 mL. with 10 mL of this solution as directed in the Identification
(2) under Scutellaria Root: any spot other than the principal
Atropine sulfate See atropine sulfate hydrate.
spot with an R f value of about 0.4 does not appear.
Atropine sulfate for assay See atropine sulfate hydrate
Baicalin hydrate for thin-layer chromatography See bai-
for assay.
calin for thin-layer chromatography‚
Atropine sulfate for thin-layer chromatography See
Balsam Canada balsam for microscopy. Before use,
atropine sulfate hydrate for thin-layer chromatography.
dilute to a suitable concentration with xylene.
Atropine sulfate hydrate (C17H23NO3)2.H2SO4.H2O
Bamethan sulfate (C12H19NO2)2.H2SO4 [Same as the
[Same as the namesake monograph]
namesake monograph]
Atropine sulfate hydrate for assay (C17H23NO3)2.H2SO4.
Barbaloin for assay C21H22O9 Use barbaloin for thin-
H2O [Same as the monograph Atropine Sulfate Hydrate.
layer chromatography meeting the following additional spe-
When dried, it contains not less than 99.0z of atropine
cifications.
sulfate [(C17H23NO3)2.H2SO4].]
Absorbance <2.24> E 11zcm (360 nm): 260 – 290 [10 mg
Atropine sulfate hydrate for thin-layer chromatography dried in a desiccator (in vacuum, phosphorus (V) oxide) for
(C17H23NO3)2.H2SO4.H2O Use atropine sulfate hydrate for not less than 24 hours, methanol, 500 mL].
assay meeting the following additional specification. To Purity Related substances—Dissolve 10 mg of the sub-
about 50 mg of the substance to be examined, dissolve in stance to be tested in 10 mL of methanol, and use this solu-
ethanol (95) to make 10 mL, and use this solution as the tion as the sample solution. Pipet 1 mL of the sample solu-
sample solution. Perform the test with the sample solution as tion, add methanol to make exactly 100 mL, and use this
directed under Thin-layer Chromatography <2.03>. Spot 50 solution as the standard solution (1). Perform the test with
mL of the sample solution on a plate of silica gel for thin- exactly 20 mL each of the sample solution and standard solu-
layer chromatography, develop the plate with a mixture of tion (1) as directed under Liquid Chromatography <2.01> ac-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
210 General Tests / 9.41 Reagents, Test Solutions JP XVII
cording to the following conditions, and measure each peak Barium chloride See barium chloride dihydrate.
area of the both solutions by the automatic integration
Barium chloride dihydrate BaCl2.2H2O [K 8155, Spe-
method: the total area of the peaks other than barbaloin
cial class]
from the sample solution is not larger than the peak area of
barbaloin from the standard solution (1). Barium chloride TS Dissolve 12 g of barium chloride di-
Operating conditions hydrate in water to make 100 mL (0.5 mol/L).
Proceed the operating conditions in the Assay under Aloe
Barium hydroxide See barium hydroxide octahydrate.
except detector, detection sensitivity and time span of meas-
urement. Barium hydroxide octahydrate Ba(OH)2.8H2O
Detector: An ultraviolet absorption photometer (wave- [K 8577, Special class] Store in tightly stoppered containers.
length: 300 nm).
Barium hydroxide TS Saturate barium hydroxide octa-
Detection sensitivity: Pipet 1 mL of the standard solution
hydrate in freshly boiled and cooled water (0.25 mol/L).
(1), add methanol to make exactly 20 mL, and use this solu-
Prepare before use.
tion as the standard solution (2). Adjust the detection sensi-
tivity so that the peak area of barbaloin obtained from 20 mL Barium nitrate Ba(NO3)2 [K 8565, Special class]
of the standard solution (2) can be measured by the auto-
Barium nitrate TS Dissolve 6.5 g of barium nitrate in
matic integration method and the peak height of barbaloin
water to make 100 mL (0.25 mol/L).
obtained from 20 mL of the standard solution (1) shows
about 20z of the full scale. Barium oxide BaO A white to yellowish or grayish white
Time span of measurement: About 3 times as long as the powder.
retention time of barbaloin, beginning after the solvent Identification (1) Dissolve 0.5 g of barium oxide in 15
peak. mL of water and 5 mL of hydrochloric acid, and add 10 mL
of dilute sulfuric acid: white precipitates appear.
Barbaloin for component determination See barbaloin
(2) Perform the test with barium oxide as directed under
for assay.
Flame Coloration Test <1.04> (1): a green color appears.
Barbaloin for thin-layer chromatography C21H22O9
Barium perchlorate Ba(ClO4)2 [K 9551, Special class]
Light yellow crystalline powder. Freely soluble in methanol,
practically insoluble in water. Becanamycin sulfate C18H37N5O10.xH2SO4 [Same as
Melting point <2.60>: 1489C the namesake monograph]
Purity Related substances—Dissolve 1.0 mg of barbaloin
Beclometasone dipropionate C28H37ClO7 [Same as the
for thin-layer chromatography in exactly 1 mL of methanol.
namesake monograph]
Perform the test with 20 mL of this solution as directed in the
Identification (2) under Aloe: any spot other than the princi- Benidipine hydrochloride C28H31N3O6.HCl [Same as
pal spot at the R f value of about 0.3 does not appear. the namesake monograph]
Barbital C8H12N2O3 [Same as the namesake mono- Benidipine hydrochloride for assay C28H31N3O6.HCl
graph] [Same as the monograph Benidipine Hydrochloride. When
dried, it contains not less than 99.5z of benidipine hydro-
Barbital buffer solution Dissolve 15 g of barbital sodium
chloride (C28H31N3O6.HCl)]
in 700 mL of water, adjust the pH to 7.6 with dilute hydro-
chloric acid, and filter. Benzaldehyde C6H5CHO [K 8857, First class]
Barbital sodium C8H11N2NaO3 White, odorless, crys- Benzalkonium chloride [Same as the namesake mono-
tals or crystalline powder, having a bitter taste. Freely solu- graph]
ble in water, slightly soluble in ethanol (95), and practically
Benzalphthalide C15H10O2 Yellow crystalline powder.
insoluble in diethyl ether.
Melting point: 99 – 1029C.
pH <2.54>—The pH of a solution of 1.0 g of barbital so-
dium in 200 mL of water is between 9.9 and 10.3. Benz[a]anthracene C18H12 White to yellow, crystalline
Loss on drying <2.41>: not more than 1.0z (1 g, 1059C, powder or powder. Practically insoluble in water, in metha-
4 hours). nol and in ethanol (99.5). Melting point: 158 – 1639 C.
Content: not less than 98.5z. Assay—Weigh accurately Identification Perform the test with benz[a]anthracene
about 0.5 g of barbital sodium, previously dried, transfer to as directed in the Purity: the mass spectrum of the main peak
a separator, dissolve in 20 mL of water, add 5 mL of ethanol shows a molecular ion peak (m/z 228) and a fragment ion
(95) and 10 mL of dilute hydrochloric acid, and extract with peak (m/z 114).
50 mL of chloroform. Then extract with three 25-mL por- Purity Related substances—Dissolve 3.0 mg of
tions of chloroform, combine the total extract, wash with benz[a]anthracene in methanol to make 100 mL, and use this
two 5-mL portions of water, and extract the washings with solution as the sample solution. Perform the test with 1 mL
two 10-mL portions of chloroform. Combine the chlo- of this solution as directed under Gas Chromatography
roform extracts, and filter into a conical flask. Wash the <2.02> according to the following conditions, and determine
filter paper with three 5-mL portions of chloroform, com- each peak area by the automatic integration method. Calcu-
bine the filtrate and the washings, add 10 mL of ethanol late the amounts of these peaks by the area percentage
(95), and titrate <2.50> with 0.1 mol/L potassium hydroxide- method: the total amount of the peaks other than
ethanol VS until the color of the solution changes from benz[a]anthracene is not more than 2.0z.
yellow to purple through light purple (indicator: 2 mL of Operating conditions
alizarin yellow GG-thymolphthalein TS). Perform a blank Detector: A mass spectrophotometer (EI).
determination in the same manner. Mass scan range: 15.00 – 300.00.
Time of measurement: 12 – 30 minutes.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS
Column: A fused silica column 0.25 mm in inside diameter
＝ 20.62 mg of C8H11N2NaO3

and 30 m in length, coated inside with 5z diphenyl-95z per minute, and maintain at 3209C for 3 minutes.
dimethylpolysiloxane for gas chromatography in thickness Injection port temperature: A constant temperature of
of 0.25 – 0.5 mm. about 2509C.
Column temperature: Inject at a constant temperature of Interface temperature: A constant temperature of about
about 459C, raise the temperature to 2409C at a rate of 409C 3009C.
per minute, maintain at 2409C for 5 minutes, raise to 3009C Carrier gas: Helium.
at a rate of 49 C per minute, raise to 3209C at a rate of 109C Flow rate: Adjust so that the retention time of benzo[a]py-
per minute, and maintain at 3209C for 3 minutes. rene is about 22 minutes.
Injection port temperature: At a constant temperature of Split ratio: Splitless.
about 2509C. System suitability
Interface temperature: At a constant temperature of Test for required detectability: Pipet 1 mL of the sample
3009C. solution, add methanol to make exactly 10 mL. Confirm
Carrier gas: Helium. that the peak area of benzo[a]pyrene obtained from 1 mL of
Flow rate: Adjust so that the retention time of this solution is equivalent to 5 to 15z of that of benzo[a]py-
benz[a]anthracene is about 15 minutes. rene obtained from 1 mL of the sample solution.
Split ratio: Splitless.
p-Benzoquinone C6H4O2 Yellow to yellow-brown,
System suitability
crystals or crystalline powder, having a pungent odor. Solu-
Test for required detectability: Pipet 1 mL of the sample
ble in ethanol (95) and in diethyl ether, slightly soluble in
solution, and add methanol to make exactly 10 mL. Confirm
water. It is gradually changed to a blackish brown color by
that the peak area of benz[a]anthracene obtained from 1 mL
light.
of this solution is equivalent to 5 to 15z of that of
Melting point <2.60>: 111 – 1169C
benz[a]anthracene obtained from 1 mL of the standard solu-
Content: not less than 98.0z. Assay—Weigh accurately
tion.
about 0.1 g of p-benzoquinone, place in an iodine bottle,
Benzene C6H6 [K 8858, Special class] add exactly 25 mL of water and 25 mL of diluted sulfuric
acid (1 in 15), dissolve 3 g of potassium iodide by shaking,
Benzethonium chloride for assay C27H42ClNO2 [Same
and titrate <2.50> with 0.1 mol/L sodium thiosulfate VS (in-
as the monograph Benzethonium Chloride. When dried, it
dicator: 3 mL of starch TS). Perform a blank determination.
contains not less than 99.0z of benzethonium chloride
(C27H42ClNO2).] Each mL of 0.1 mol/L sodium thiosulfate VS
＝ 5.405 mg of C6H4O2
Benzoic acid C6H5COOH [K 8073, Special class]
p-Benzoquinone TS Dissolve 1 g of p-benzoquinone in 5
Benzoin C6H5CH(OH)COC6H5 White to pale yellow,
mL of acetic acid (100), and add ethanol (95) to make 100
crystals or powder.
mL.
Melting point <2.60>: 132 – 1379
C
N-a -Benzoyl-L-arginine ethyl ester hydrochloride
Benzophenone C6H5COC6H5 Colorless crystals, having
C15H22N4O3.HCl White, crystals or crystalline powder.
a characteristic odor.
Freely soluble in water and in ethanol (95), and slightly solu-
Melting point <2.60>: 48 – 509C
ble in diethyl ether.
Benzo[a]pyrene C20H12 Light yellow to green-yellow, Optical rotation <2.49> [a]20 D : －15.5 – －17.09 (2.5 g,
crystalline powder or powder. Practically insoluble in water, water, 50 mL, 100 mm).
in methanol and in ethanol (99.5). Melting point: 176 – Melting point <2.60>: 129 – 1339C
1819C. Purity (1) Clarity and color of solution—Dissolve 0.1 g
Identification—Perform the test with benzo[a]pyrene as of N-a-benzoyl-L-arginine ethyl ester hydrochloride in 20 mL
directed in the Purity: the mass spectrum of the main peak of water: the solution is clear and colorless.
shows a molecular ion peak (m/z 252) and a fragment ion (2) Related substances—Weigh 0.10 g of N-a-benzoyl-L-
peak (m/z 125). arginine ethyl ester hydrochloride, dissolve in 6 mL of water,
Purity Related substances—Dissolve 3.0 mg of ben- add 4 mL of hydrochloric acid, heat in a boiling water bath
zo[a]pyrene in methanol to make 100 mL, and use this solu- for 5 minutes to decompose, and use this solution as the
tion as the sample solution. Perform the test with 1 mL of sample solution. Perform the test with the sample solution as
this solution as directed under Gas Chromatography <2.02> directed under Paper Chromatography. Spot 5 mL of the
under the following conditions, and determine each peak sample solution on a chromatographic filter paper. Develop
area by the automatic integration method. Calculate the with a mixture of water, acetic acid (100) and 1-butanol
amounts of these peaks by the area percentage method: the (5:4:1) to a distance of about 30 cm, and air-dry the paper.
total amount of the peaks other than benzo[a]pyrene is not Spray evenly a solution of ninhydrin in acetone (1 in 50)
more than 3.0z. upon the paper, and heat at 909C for 10 minutes: only one
Operating conditions purple spot appears.
Detector: A mass spectrophotometer (EI). Content: not less than 99.0z. Assay—Weigh accurately
Mass scan range: 15.00 – 300.00. about 0.6 g of N-a-benzoyl-L-arginine ethyl ester hydrochlo-
Time of measurement: 12 – 30 minutes. ride, dissolve in 50 mL of water, neutralize with 0.1 mol/L
Column: A fused silica column 0.25 mm in inside diameter sodium hydroxide VS, if necessary, and titrate <2.50> with
and 30 m in length, coated inside with 5z diphenyl-95z 0.1 mol/L silver nitrate VS (indicator: 4 drops of
dimethylpolysiloxane for gas chromatography in thickness dichlorofluorescein TS).
of 0.25–0.5 mm.
Each mL of 0.1 mol/L silver nitrate VS
Column temperature: Inject at a constant temperature of
＝ 34.28 mg of C15H22N4O3.HCl
about 459C, raise the temperature to 2409C at a rate of 409C
per minute, maintain at 2409C for 5 minutes, raise to 3009C N-a-Benzoyl-L-arginine ethyl ester TS Dissolve 70 mg of
at a rate of 49 C per minute, raise to 3209C at a rate of 109C N-a-benzoyl-L-arginine ethyl ester hydrochloride in freshly

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
212 General Tests / 9.41 Reagents, Test Solutions JP XVII
boiled and cooled water to make exactly 10 mL. that of benzoylhypaconine obtained from 20 mL of the
standard solution.
N-a-Benzoyl-L-arginine-4-nitroanilide hydrochloride
System performance: When the procedure is run with 20
C19H22N6O4.HCl Light yellow crystalline powder.
mL of aconitum monoester alkaloids standard TS for assay
Optical rotation <2.49> [a]20
D : ＋45.5 – ＋48.09(after dry-
under the above operating conditions, benzoylmesaconine,
ing, 0.5 g, N, N-dimethylformamide, 25 mL, 100 mm).
benzoylhypaconine and 14-anisoylaconine are eluted in this
Purity Related substances—Dissolve 0.20 g of N-a-ben-
order with the resolution between these peaks being not less
zoyl-L-arginine-4-nitroanilide hydrochloride in 10 mL of
than 4, respectively.
N, N-dimethylformamide, and use this solution as the sample
System repeatability: When the test is repeated 6 times
solution. Perform the test with this solution as directed
with 20 mL of aconitum monoester alkaloids standard TS for
under Thin-layer Chromatography <2.03>. Spot 10 mL of the
assay under the above operating conditions, the relative
sample solution on a plate of silica gel for thin-layer chroma-
standard deviations of the peak areas of benzoylmesaconine,
tography, develop the plate with a mixture of 1-butanol,
benzoylhypaconine and 14-anisoylaconine are not more than
water and acetic acid (100) (4:1:1) to a distance of about 10
1.5z, respectively.
cm, and air-dry the plate. Exposure the plate to a vapor of
iodine: only one spot appears. Benzoylhypaconine hydrochloride for component determi-
nation See benzoylhypaconine hydrochloride for assay.
N-a-Benzoyl-L-arginine-4-nitroanilide TS Dissolve 0.1 g
of N-a-benzoyl-L-arginine-4-nitroanilide hydrochloride in N-Benzoyl-L -isoleucyl-L -glutamyl(g -OR)-glycyl- L -arginyl-
water to make 100 mL. p-nitroanilide hydrochloride An equal amount mixture of
two components, R ＝ H and R ＝ CH3. A white powder.
Benzoyl chloride C6H5COCl A clear and colorless fum-
Slightly soluble in water.
ing liquid. Specific gravity: about 1.2 g/mL.
Absorbance <2.24> E 11zcm (316 nm): 166 – 184 (10 mg,
Identification—Determine the infrared absorption spec-
water, 300 mL).
trum as directed in the liquid film method as directed under
Infrared Spectrophotometry <2.25>: it exhibits absorption at Benzoylmesaconine hydrochloride for assay Benzoyl-
the wave numbers of about 1775 cm－1, 1596 cm－1, 1450 mesaconine hydrochloride for thin-layer chromatography
cm－1, 1307 cm－1, 1206 cm－1, 873 cm－1, 776 cm－1 and 671 meeting the following additional specifications.
cm－1. Purity Related substances—Dissolve 5.0 mg of benzoyl-
mesaconine hydrochloride for assay in 5 mL of the mobile
Benzoylhypaconine hydrochloride for assay
phase, and use this solution as the sample solution. Pipet 1
C31H43NO9.HCl White, crystals or crystalline powder.
mL of the sample solution, add the mobile phase to make ex-
Freely soluble in methanol, soluble in water, and sparingly
actly 50 mL, and use this solution as the standard solution.
soluble in ethanol (99.5). Melting point: about 2309C (with
Perform the test with exactly 20 mL each of the sample solu-
decomposition).
tion and standard solution as directed under Liquid Chroma-
Absorbance <2.24> E 11zcm (230 nm): 225 – 240 (5 mg calcu-
tography <2.01> according to the following conditions. De-
lated on the anhydrous basis, methanol, 200 mL).
termine each peak area of both solutions by the automatic
Purity (1) Related substances—To 1.0 mg of benzoyl-
integration method: the total area of the peaks other than
hypaconine hydrochloride for assay add exactly 1 mL of
benzoylmesaconine obtained from the sample solution is not
ethanol (99.5). Perform the test with 5 mL of this solution as
larger than the peak area of benzoylmesaconine obtained
directed in the Identification under Processed Aconite Root:
from the standard solution.
no spot other than the principal spot with an Rf value of
Operating conditions
about 0.5 appears.
Column, column temperature, mobile phase and flow
(2) Related substance—Dissolve 5.0 mg of benzoyl-
rate: Proceed as directed in the operating conditions in the
hypaconine hydrochloride for assay in 5 mL of the mobile
Assay (3) under Goshajinkigan Extract.
phase, and use this solution as the sample solution. Pipet 1
Detector: An ultraviolet absorption photometer (wave-
mL of the sample solution, add the mobile phase to make ex-
length: 245 nm).
actly 50 mL, and use this solution as the standard solution.
Time span of measurement: About 6 times as long as the
Perform the test with exactly 20 mL each of the sample solu-
retention time of benzoylmesaconine.
tion and standard solution as directed under Liquid Chroma-
System suitability
tography <2.01> according to the following conditions. De-
Test for required detectability: Pipet 1 mL of the standard
termine each peak area of both solutions by the automatic
solution, and add the mobile phase to make exactly 20 mL.
integration method: the total area of the peaks other than
Confirm that the peak area of benzoylmesaconine obtained
benzoylhypaconine obtained from the sample solution is not
from 20 mL of this solution is equivalent to 3.5 to 6.5z of
larger than the peak area of benzoylhypaconine obtained
that of benzoylmesaconine obtained from 20 mL of the
from the standard solution.
standard solution.
Operating conditions
System performance: When the procedure is run with 20
Column, column temperature, mobile phase and flow
mL of aconitum monoester alkaloids standard TS for assay
rate: Proceed as directed in the operating conditions in the
under the above operating conditions, benzoylmesaconine,
Assay (3) under Goshajinkigan Extract.
benzoylhypaconine and 14-anisoylaconine are eluted in this
Detector: An ultraviolet absorption photometer (wave-
order with the resolution between these peaks being not less
length: 245 nm).
than 4, respectively.
Time span of measurement: About 5 times as long as the
System repeatability: When the test is repeated 6 times
retention time of benzoylhypaconine.
with 20 mL of aconitum monoester alkaloids standard TS for
System suitability
assay under the above operating conditions, the relative
Test for required detectability: Pipet 1 mL of the standard
standard deviations of the peak areas of benzoylmesaconine,
solution, and add the mobile phase to make exactly 20 mL.
benzoylhypaconine and 14-anisoylaconine are not more than
Confirm that the peak area of benzoylhypaconine obtained
1.5z, respectively.
from 20 mL of this solution is equivalent to 3.5 to 6.5z of

Benzoylmesaconine hydrochloride for component deter- residual solvent-free basis.]

mination See benzoylmesaconine hydrochloride for assay.
Beraprost sodium C24H29NaO5 [Same as the namesake
Benzoylmesaconine hydrochloride for thin-layer chroma- monograph]
tography C31H43NO10.HCl White, crystals or crystalline
Beraprost sodium for assay C24H29NaO5 [Same as the
powder. Soluble in water and in ethanol (99.5) and sparingly
monograph Beraprost Sodium. When dried it contains not
soluble in methanol. Melting point: about 2509 C (with de-
less than 99.0z of beraprost sodium (C24H29NaO5).]
composition).
Absorbance <2.24> E 11zcm (230 nm): 217 – 231 (5 mg calcu- Berberine chloride See berberin chloride hydrate.
lated on the anhydrous basis, methanol, 200 mL).
Berberine chloride hydrate C20H18ClNO4.xH2O [Same
Purity Related substances—Dissolve 1.0 mg of benzoy-
as the namesake monograph]
lmesaconine hydrochloride for thin-layer chromatography in
exactly 1 mL of ethanol (99.5). Perform the test with 5 mL of Berberine chloride for thin-layer chromatography See
this solution as directed in the Identification under Processed barberine chloride hydrate for thin-layer chromatography.
Aconite Root: no spot other than the principal spot with an
Berberine chloride hydrate for thin-layer chromatography
R f value of about 0.4 appears.
C20H18ClNO4.xH2O [Same as the monograph Berberine
Benzoyl peroxide, 25z water containing (C6H5CO)2O2 Chloride Hydrate] or berberine chloride hydrate meeting the
White, moist, crystals or powder. Soluble in diethyl ether following requirements. Yellow, crystals or crystalline pow-
and in chloroform, and very slightly soluble in water and in der. Sparingly soluble in methanol, slightly soluble in
ethanol (95). Melting point: 103 – 1069C (dried substance) ethanol (99.5), and very slightly soluble in water.
(with decomposition). Identification Determine the absorption spectrum of a
Loss on drying <2.41>: not more than 30z (0.1 g, in vacu- solution of the substance to be examined (1 in 100,000) as di-
um, silica gel, constant mass). rected under Ultraviolet-visible Spectrophotometry <2.24>: it
exhibits maxima between 226 nm and 230 nm, between 261
Benzyl alcohol C6H5CH2OH Clear and colorless liq-
nm and 265 nm, and between 342 nm and 346 nm.
uid, having a characteristic odor.
Purity Related substances—Dissolve 10 mg of the sub-
Specific gravity <2.56> d 20
20: 1.045 – 1.050.
stance to be examined in 10 mL of methanol, and use this
Preserve in a light-resistant tight container.
solution as the sample solution. Pipet 1 mL of the sample
Benzyl benzoate C6H5COOCH2C6H5 A colorless oily solution, add methanol to make exactly 100 mL, and use this
liquid. Congealing point: about 189 C. Boiling point: about solution as the standard solution. Perform the test with 10
3239C. mL each of the sample solution and standard solution as di-
Specific gravity <2.56> d 20
20: 1.118 – 1.123. rected in the Identification (2) under Phellodendron Bark:
Preserve in a light-resistant tight container. any spot other than the principal spot with an R f value of
about 0.3 obtained from the sample solution is not more in-
Benzyl parahydroxybenzoate C14H12O3 White, fine cry-
tense than the spot obtained from the standard solution.
stals or crystalline powder. Freely soluble in ethanol (95),
and very slightly soluble in water. Bergenin for thin-layer chromatography C14H16O9
Melting point <2.60>: 109 – 1129 C White, crystals or crystalline powder. Freely soluble in
Residue on ignition <2.44>: not more than 0.1z. methanol, slightly soluble in ethanol (99.5), very slightly
Content: not less than 99.0z. Assay—Weigh accurately soluble in water, and practically insoluble in diethyl ether.
about 1 g of benzyl parahydroxybenzoate, add exactly 20 Identification—Determine the absorption spectrum of a
mL of 1 mol/L sodium hydroxide VS, heat at about 709 C solution of bergenin for thin-layer chromatography in meth-
for 1 hour, and immediately cool in ice. Titrate <2.50> the anol (1 in 50,000) as directed under Ultraviolet-visible Spec-
excess sodium hydroxide with 0.5 mol/L sulfuric acid VS up trophotometry <2.24>: it exhibits maxima between 217 nm
to the second equivalent point (potentiometric titration). and 221 nm, and between 273 nm and 277 nm, and a mini-
Perform a blank determination. mum between 241 nm and 245 nm.
Purity Related substances—Dissolve 1.0 mg of bergenin
Each mL of 1 mol/L sodium hydroxide VS
for thin-layer chromatography in 1 mL of methanol. Per-
＝ 228.2 mg of C14H12O3
form the test with 20 mL of this solution as directed in the
Benzylpenicillin benzathin See benzylpenicillin benza- Identification under Mallotus Bark: no spot other than the
thine hydrate. principal spot at the R f value of about 0.5 appears.
Benzylpenicillin benzathine hydrate Betahistine mesilate C8H12N2.2CH4O3S [Same as the
(C16H18N2O4S)2.C16H20N2.4H2O [Same as the namesake namesake monograph]
monograph]
Betahistine mesilate for assay C8H12N2.2CH4O3S
Benzylpenicillin potassium C16H17KN2O4S [Same as the [Same as the monograph Betahistine Mesilate. When dried,
namesake monograph] it contains not less than 99.0z of betahistine mesilate
(C8H12N2.2CH4O3S).]
Benzyl p-hydroxybenzoate See benzyl parahydroxyben-
zoate. Betamipron C10H11NO3 [Same as the namesake mono-
graph]
p-Benzylphenol C6H5CH2C6H4OH White to pale yel-
lowish white, crystals or crystalline powder. Betamipron for assay C10H11NO3 [Same as the mono-
Melting point <2.60>: 80 – 859C graph Betamipron. It contains not less than 99.5z of be-
tamipron (C10H11NO3), calculated on the anhydrous basis.]
Bepotastine besilate for assay C21H25ClN2O3.C6H6O3S
[Same as the monograph Bepotastine Besilate. However, it Bezafibrate for assay C19H20ClNO4 [Same as the
contains not less than 99.5z of bepotastine besilate monograph Bezafibrate. When dried it contains not less than
(C21H25ClN2O3.C6H6O3S), calculated on the anhydrous and 99.0z of bezafibrate (C19H20ClNO4).]

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
214 General Tests / 9.41 Reagents, Test Solutions JP XVII
BGLB Dissolve 10 g of peptone and 10 g of lactose graph]
monohydrate in 500 mL of water, add 200 mL of fresh ox
Bile salts See Microbial Limit Test for Crude Drugs
bile or a solution prepared by dissolving 20 g of dried ox bile
<5.02>.
powder in 200 mL of water and adjusted the pH to between
7.0 and 7.5, then add water to make 975 mL, and again Biotin-labeled elderberry lectin A solution of elderberry
adjust to pH 7.4. Then add 13.3 mL of a solution of brilliant lectin labeled with biotin, dissolved in appropriate buffer so-
green (1 in 1000) and water to make 1000 mL in total lution.
volume, and filter through absorbent cotton. Dispense 10
2-(4-Biphenylyl)propionic acid C15H14O2 Light yellow-
mL portions of the filtrate into tubes for fermentation, and
ish white powder.
sterilize by autoclaving at 1219C for not more than 20
Melting point <2.60>: 145 – 1489C
minutes, then cool quickly, or sterilize fractionally on each
Purity—Dissolve 1 mg of 2-(4-biphenylyl) propionic acid
of three successive days for 30 minutes at 1009 C.
in a mixture of water and acetonitrile (11:9) to make 50 mL.
a-BHC (a-Hexachlorocyclohexane) C6H6Cl6 Perform the test with 20 mL of this solution as directed under
Melting point <2.60>: 157 – 1599 C Liquid Chromatography <2.01> according to the operating
Purity Related substances—Dissolve 10 mg of a-BHC in conditions of the Related substances in the Purity (3) under
5 mL of acetone for purity of crude drug, and add hexane Flurbiprofen. Determine each peak area of the solution in
for purity of crude drug to make exactly 100 mL. Pipet 1 mL about twice as long as the retention time of the main peak by
of this solution, add hexane for purity of crude drug to make the automatic integration method, and calculate the amount
exactly 100 mL, and use this solution as the sample solution. of 2-(4-biphenylyl)propionic acid by the area percentage
Pipet 1 mL of the sample solution, add hexane for purity of method: it is not less than 98.0z.
crude drug to make exactly 100 mL, and use this solution as Content: not less than 98.0z. Assay—Weigh accurately
the standard solution (1). Perform the test with exactly 1 mL about 0.5 g of 2-(4-biphenilyl)propionic acid, previously
each of the sample solution and standard solution (1) as di- dried in vacuum over silica gel for 4 hours, dissolve in 50 mL
rected under Gas Chromatography <2.02> according to the of ethanol (95), and titrate <2.50> with 0.1 mol/L sodium
following conditions, and measure each peak area from hydroxide VS (indicator: 3 drops of phenolphthalein TS).
these solutions by the automatic integration method: the Perform a blank determination, and make any necessary
total peak area other than a-BHC from the sample solution correction.
is not larger than the peak area of a-BHC from the standard
Each mL of 0.1 mol/L sodium hydroxide VS
solution (1).
＝ 22.63 mg of C15H14O2
Operating conditions
Proceed the operating conditions in 4. Purity 4.3. under 2,2?-Bipyridyl C10H8N2 [K 8486, Special class]
Crude Drugs Test <5.01> except detection sensitivity and time
Bis(cis-3,3,5-trimethylcyclohexyl) phthalate
span of measurement.
C6H4[COOC6H8(CH3)3]2 White crystalline powder.
Detection sensitivity: Pipet 1 mL of the standard solution
Melting point <2.60>: 91 – 949C
(1), add hexane for purity of crude drug to make exactly 20
mL, and use this solution as standard solution (2). Adjust Bisdemethoxycurcumin C19H16O4 Yellow to orange
the detection sensitivity so that the peak area of a-BHC ob- crystalline powder. Sparingly soluble in methanol, slightly
tained from 1 mL of the standard solution (2) can be meas- soluble in ethanol (99.5), and practically insoluble in water.
ured by the automatic integration method, and the peak Melting point: 213 – 2179C.
height of a-BHC obtained from 1 mL of the standard solu- Identification—Determine the absorption spectrum of a
tion (1) is about 20z of the full scale. solution of bisdemethoxycurcumin in methanol (1 in
Time span of measurement: About twice as long as the 400,000) as directed under Ultraviolet-visible Spectropho-
retention time of a-BHC, beginning after the solvent peak. tometry <2.24>: it exhibits a maximum between 413 nm and
417 nm.
b-BHC ( b-Hexachlorocyclohexane) C6H6Cl6
Purity Related substances—(1) Dissolve 4 mg of bis-
Melting point <2.60>: 308 – 3109C
demethoxycurcumin in 2 mL of methanol, and use this
Purity Related substances—Proceed as directed in the
solution as the sample solution. Pipet 1 mL of the sample
Purity under a-BHC using the following standard solution
solution, add methanol to make exactly 20 mL, and use this
(1).
solution as the standard solution. Perform the test with these
Standard solution (1): Pipet 2 mL of the sample solution,
solutions as directed under Thin-layer Chromatography
and add hexane for purity of crude drug to make exactly 100
<2.03>. Spot 5 mL each of the sample solution and standard
mL.
solution on a plate of silica gel for thin-layer chromatogra-
g-BHC (g-Hexachlorocyclohexane) C6H6Cl6 phy. Develop the plate with a mixture of dichloromethane
Melting point <2.60>: 112 – 1149
C and methanol (19:1) to a distance of about 10 cm, and air-
Purity Related substances—Proceed as directed in the dry the plate. Examine under ultraviolet light (main wave-
Purity under a-BHC. length: 365 nm): the spots other than the principal spot at R f
value of about 0.3 obtained from the sample solution are not
d-BHC (d-Hexachlorocyclohexane) C6H6Cl6
more intense than the spot obtained from the standard solu-
Melting point <2.60>: 137 – 1409C
tion.
Purity Related substances—Proceed as directed in the
(2) Dissolve 1.0 mg of bisdemethoxycurcumin in 5 mL of
Purity under a-BHC using the following standard solution
methanol, and use this solution as the sample solution. Pipet
(1).
1 mL of this solution, add methanol to make exactly 20 mL,
Standard solution (1): Pipet 5 mL of the sample solution,
and use this solution as the standard solution. Perform the
and add hexane for purity of crude drug to make exactly 100
test with exactly 10 mL each of the sample solution and
mL.
standard solution as directed under Liquid Chromatography
Bifonazole C22H18N2 [Same as the namesake mono- <2.01> under the following conditions. Determine each peak

area from both solutions by the automatic integration Iopamidol.

method: the total area of the peaks other than bisdemethox- System suitability
ycurcumin obtained from the sample solution is not larger Proceed the system suitability in the Purity (6) under
than the peak area of bisdemethoxycurcumin obtained from Iopamidol.
the standard solution.
Bismuth nitrate See bismuth nitrate pentahydrate.
Operating conditions
Column, column temperature, mobile phase and flow Bismuth nitrate pentahydrate Bi(NO3)3.5H2O [K 8566,
rate: Proceed as directed in the operating conditions in the Special class]
Assay under Turmeric.
Bismuth nitrate-potassium iodide TS Dissolve 0.35 g of
Detector: A visible absorption photometer (wavelength:
bismuth nitrate pentahydrate in 4 mL of acetic acid (100)
422 nm).
and 16 mL of water (solution A). Dissolve 8 g of potassium
Time span of measurement: About 4 times as long as the
iodide in 20 mL of water (solution B). To 20 mL of a mix-
retention time of bisdemethoxycurcumin, beginning after the
ture of solution A and solution B (1:1) add 80 mL of dilute
solvent peak.
sulfuric acid and 0.2 mL of hydrogen peroxide (30). Prepare
System suitability
before use.
System performance and system repeatability: Proceed as
directed in the operating conditions in the Assay under Bismuth nitrate TS Dissolve 5.0 g of bismuth nitrate
Turmeric. pentahydrate in acetic acid (100) to make 100 mL.
Test for required detectability: Pipet 1 mL of the standard
Bismuth potassium iodide TS Dissolve 10 g of L-tartaric
solution, and add methanol to make exactly 20 mL. Confirm
acid in 40 mL of water, add 0.85 g of bismuth subnitrate,
that the peak area of bisdemethoxycurcumin obtained from
shake for 1 hour, add 20 mL of a solution of potassium
10 mL of this solution is equivalent to 3.5 to 6.5z of that of
iodide (2 in 5), shake thoroughly, allow to stand for 24
bisdemethoxycurcumin from 10 mL of the standard solution.
hours, and filter (solution A). Separately, dissolve 10 g of
4,4?-Bis(diethylamino)benzophenone [(C2H5)2NC6H4]2CO L-tartaric acid in 50 mL of water, add 5 mL of solution A,
Light yellow crystals. and preserve in a light-resistant, glass-stoppered bottle.
Content: not less than 98z. Assay—Weigh accurately
Bismuth sodium trioxide NaBiO3 A yellow-brown
0.25 g of 4,4?-bis(diethylamino)benzophenone, dissolve in 50
powder.
mL of acetic acid (100), and titrate <2.50> with 0.1 mol/L
Identification—(1) To 10 mg of bismuth sodium trioxide
perchloric acid VS (potentiometric titration). Perform a
add 5 mL of a solution of manganese (II) nitrate hexahy-
blank titration in the same manner, and make any necessary
drate (4 in 125) and 1 mL of diluted nitric acid (1 in 3), and
correction.
shake vigorously for 10 seconds: a red-purple color is deve-
Each mL of 0.1 mol/L perchloric acid VS loped.
＝ 16.22 mg of C21H28N2O (2) Dissolve 10 mg of bismuth sodium trioxide in 2 mL
of diluted hydrochloric acid (1 in 2): this solution responds
N, N?-Bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-hydroxya-
to the Qualitative Tests <1.09> (1) for sodium salt.
cetylamino-2,4,6-triiodoisophthalamide C16H20I3N3O8
White crystalline powder. Bismuth subnitrate [Same as the namesake monograph]
Identification (1) Heat 0.1 g of N, N?-bis[2-hydroxy-1-
Bismuth subnitrate TS Dissolve 10 g of L-tartaric acid in
(hydroxymethyl)ethyl]-5-hydroxyacetylamino-2,4,6-triio-
40 mL of water, add 0.85 g of bismuth subnitrate, stir for 1
doisophthalamide over free flame: a purple colored gas
hour, then add 20 mL of a solution of potassium iodide (2 in
evolves.
5), and shake well. After standing for 24 hours, filter, and
(2) Determine the infrared absorption spectrum of
preserve the filtrate in a light-resistant bottle.
N, N?- bis[2 - hydroxy - 1 - (hydroxymethyl)ethyl] - 5 - hydroxya-
cetylamino-2,4,6-triiodoisophthalamide according to the Bismuth subnitrate-potassium iodide TS for spraying,
potassium bromide disk method under Infrared Spectropho- dilute Dissolve 10 g of L-tartaric acid in 50 mL of water,
tometry <2.25>: it exhibits absorption at the wave numbers of and add 5 mL of bismuth subnitrate TS.
about 3390 cm－1, 3230 cm－1, 2882 cm－1, 1637 cm－1, 1540
Bismuth sulfite indicator Prepared for microbial test.
cm－1, 1356 cm－1 and 1053 cm－1.
Purity—Dissolve 0.10 g of N, N?-bis[2-hydroxy-1-(hydrox- Bisoprolol fumarate for assay (C18H31NO4)2.C4H4O4
ymethyl)ethyl]-5-hydroxyacetylamino-2,4,6-triiodoisophtha- [Same as the monograph Bisoprolol Fumarate. However,
lamide in 10 mL of water, and use this solution as the sample when dried, it contains not less than 99.0z of bisoprolol
solution. Pipet 1 mL of the sample solution, add water to fumarate [(C18H31NO4)2.C4H4O4]. Also, when performing
make exactly 100 mL, and use this solution as the standard the Purity (2) under Bisoprolol Fumarate, the total area of
solution. Perform the test with exactly 20 mL each of the the peaks other than bisoprolol from the sample solution is
sample solution and standard solution as directed under not larger than 1/5 times the peak area of bisoprolol from
Liquid Chromatography <2.01> according to the following the standard solution].
conditions. Determine each peak area of both solutions by Purify as follows if needed.
automatic integration method: the total area of the peaks Purification method—Dissolve, with heating, 2 g of Bi-
other than N, N?-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5- soprolol Fumarate in 200 mL of ethyl acetate, add 0.5 g of
hydroxyacetylamino-2,4,6-triiodoisophthalamide obtained activated carbon, shake well, and filter using a glass filter
from the sample solution is not larger than 3 times of the (G4). Place the filtrate in ice water for 2 hours while occa-
peak area of N, N?-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5- sional shaking. Collect the crystals that precipitate out using
hydroxyacetylamino-2,4,6-triiodoisophthalamide obtained a glass filter (G3). Dry the crystals obtained in vacuum at
from the standard solution. 809C for 5 hours using phosphorus (V) oxide as a dessicant.
Operating conditions
Bis-(1-phenyl-3-methyl-5-pyrazolone) C20H18B4O2
Proceed the operating conditions in the Purity (6) under
White to pale yellow, crystals or crystalline powder. It dis-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
216 General Tests / 9.41 Reagents, Test Solutions JP XVII
solves in mineral acids and in alkali hydroxides, and it does solve 19.0 g of sodium tetraborate decahydrate in 900 mL of
not dissolve in water, in ammonia TS, or in organic solvents. water, adjust the pH to exactly 9.0 with 1 mol/L hydrochlo-
Melting point: not below 3009 C. ric acid TS, and add water to make 1000 mL.
Residue on ignition <2.44>: not more than 0.1z.
Borax See sodium tetraborate decahydrate.
Nitrogen content <1.08>: 15.5 – 16.5z
Boric acid H3BO3 [K 8863, Special class]
Bis(1,1-trifluoroacetoxy)iodobenzene C10H5F6IO4
Prepared for amino acid analysis or biochemistry. Boric acid-magnesium chloride buffer solution (pH 9.0)
Dissolve 3.1 g of boric acid in 210 mL of dilute sodium hy-
Bis-trimethyl silyl acetamide CH3CON[Si(CH3)3]2
droxide TS, and add 10 mL of a solution of magnesium
Colorless liquid.
chloride hexahydrate (1 in 50) and water to make 1000 mL.
Refractive index <2.45> n 20
D : 1.414 – 1.418
Adjust the pH to 9.0, if necessary.
Specific gravity <2.56> d 20
20: 0.825 – 0.835
Boiling point <2.57>: 71 – 739C Boric acid-methanol buffer solution Weigh exactly 2.1 g
of boric acid, dissolve in 28 mL of sodium hydroxide TS,
1,4-Bis(trimethylsilyl)benzene-d4, for nuclear magnetic
and dilute with water to exactly 100 mL. Mix equal volumes
resonance spectroscopy See 1,4-BTMSB-d4 for nuclear
of this solution and methanol, and shake.
magnetic resonance spectroscopy.
Boric acid-potassium chloride-sodium hydroxide buffer
Bitter orange peel [Same as the namesake monograph]
solution (pH 9.0) To 50 mL of 0.2 mol/L boric acid-0.2
Block buffer solution Dissolve 4 g of blocking agent in mol/L potassium chloride TS for buffer solution add 21.30
100 mL of water, and add 100 mL of 0.01 mol/L phosphate mL of 0.2 mol/L sodium hydroxide VS and water to make
buffer-sodium chloride TS (pH 7.4). 200 mL.
Blocking agent Powder whose main ingredient is bovine- Boric acid-potassium chloride-sodium hydroxide buffer
derived lactoprotein. For immunological research purposes. solution (pH 9.2) To 50 mL of 0.2 mol/L boric acid-0.2
mol/L potassium chloride TS for buffer solution add 26.70
Blocking TS for epoetin alfa Used for Western blotting.
mL of 0.2 mol/L sodium hydroxide VS and water to make
Blocking TS for nartograstim test Dissolve 1.0 g of bo- 200 mL.
vine serum albumin in phosphate-buffered sodium chloride
Boric acid-potassium chloride-sodium hydroxide buffer
TS to make 100 mL.
solution (pH 9.6) To 50 mL of 0.2 mol/L boric acid-0.2
Blood agar medium Sterilize 950 mL of heart infusion mol/L potassium chloride TS for buffer solution add 36.85
agar medium under increased pressure. Allow the media to mL of 0.2 mol/L sodium hydroxide VS and water to make
cool to about 509C, add 50 mL of horse or sheep defibrinat- 200 mL.
ed blood, dispense in sterilized Petri dishes, and make them
Boric acid-potassium chloride-sodium hydroxide buffer
as plate media.
solution (pH 10.0) To 50 mL of 0.2 mol/L boric acid-0.2
1z blood suspension Wash a defibrinated animal blood mol/L potassium chloride TS for buffer solution add 43.90
in isotonic solution, and make it into suspension to contain 1 mL of 0.2 mol/L sodium hydroxide VS and water to make
volz. Prepare before use. 200 mL.
Blotting TS Dissolve 5.81 g of 2-amino-2-hydrox- 0.2 mol/L Boric acid-0.2 mol/L potassium chloride TS for
ymethyl-1,3-propanediol, 2.93 of glycine and 0.38 g of so- buffer solution Dissolve 12.376 g of bodic acid and
dium lauryl sulfate in a suitable amount of water, add 200 14.911 g of potassium chloride in water to make 1000 mL.
mL of methanol, and add water to make 1000 mL.
Boric acid-sodium hydroxide buffer solution (pH 8.4)
Blue tetrazolium C40H32Cl2N8O2 3,3?-Dianisole-bis- Dissolve 24.736 g of boric acid in 0.1 mol/L sodium hydrox-
[4,4?-(3,5-diphenyl) tetrazolium chloride] Light yellow ide VS to make exactly 1000 mL.
crystals. Freely soluble in methanol, in ethanol (95) and in
Borneol acetate C12H20O2 A white to pale brown solid,
chloroform, slightly soluble in water, and practically insolu-
or colorless to pale brown, clear liquid. Very soluble in
ble in acetone and in ether. Melting point: about 2459C (with
methanol and in ethanol (99.5), and practically insoluble in
decomposition).
water.
Absorbance <2.24> E 11zcm (252 nm): not less than 826
Identification Determine the infrared absorption spec-
(methanol).
trum of borneol acetate as directed in the liquid film method
Blue tetrazolium TS, alkaline To 1 volume of a solution under Infrared Spectrophotometry <2.25>: it exhibits absorp-
of blue tetrazolium in methanol (1 in 200) add 3 volumes of a tion at the wave numbers of about 2950 cm－1, 1736 cm－1,
solution of sodium hydroxide in methanol (3 in 25). Prepare 1454 cm－1 and 1248 cm－1.
before use. Purity Related substances—Dissolve 50 mg of borneol
acetate in 5 mL of methanol, and use this solution as the
Borane-pyridine complex C5H8BN
sample solution. Pipet 1 mL of the sample solution, add
Content: not less than 80z. Assay—Accurately weigh
methanol to make exactly 20 mL, and use this solution as the
about 30 mg of borane-pyridine complex, dissolve in 40 mL
standard solution. Perform the test with these solutions as
of 0.05 mol/L iodide solution, add 10 mL of diluted sulfuric
directed under Thin-layer Chromatography <2.03>. Spot 5
acid (1 in 6), and titrate <2.50> with 0.1 mol/L sodium thio-
mL each of the sample solution and standard solution on a
sulfate VS (indicator: starch TS). Perform a blank determi-
plate of silica gel for thin-layer chromatography. Develop
nation, and make any necessary correction.
the plate with a mixture of hexane, diethyl ether and metha-
Each mL of 0.1 mol/L sodium thiosulfate VS nol (15:5:1) to a distance of about 7 cm, and air-dry the
＝ 1.549 mg of C5H8BN plate. Spray evenly 4-methoxybenzaldehyde-sulfuric acid TS
on the plate, and heat at 1059C for 10 minutes: the spot at
Borate-hydrochloric acid buffer solution (pH 9.0) Dis-

an R f value of about 0.7 obtained from the sample solution dium hydrogen phosphate and 0.2 g of potassium dihydro-
is not more intense than the spot obtained from the standard gen phosphate in water to make 1000 mL. To this solution
solution. add a solution of bovine serum albumin dissolved 1.0 g in 10
mL of water.
Boron trifluoride BF3 Colorless gas, having an irritat-
ing odor. Bovine serum albumin-sodium chloride-phosphate buffer
Boiling point <2.57>: －100.39C solution (pH 7.2) Dissolve 10.75 g of disodium hydrogen
Melting point <2.60>: －127.19C phosphate dodecahydrate, 7.6 g of sodium chloride and
1.0 g of bovine serum albumin in water to make 1000 mL.
Boron trifluoride-methanol TS A solution containing
Adjust to pH 7.2 with dilute sodium hydroxide TS or diluted
14 g/dL of boron trifluoride (BF3: 67.81) in methanol.
phosphoric acid (1 in 10) before use.
Bovine activated blood coagulation factor X A protein
Bovine serum albumin TS for nartograstim test Dissolve
obtained from bovine plasma. It has an activity to decom-
0.5 g of bovine serum albumin and 0.5 mL of polysorbate 20
pose prothrombin specifically and limitedly and produce
in phosphate-buffered sodium chloride TS to make 500 mL.
thrombin. It does not contain thrombin and plasmin. It con-
tains not less than 500 Units per mg protein. One unit indi- Bovine serum albumin TS for secretin Dissolve 0.1 g of
cates an amount of the factor X which hydrolyzes 1 mmol of bovine serum albumin, 0.1 g of L-cysteine hydrochloride
N-benzoyl-L-isoleucyl-L-glutamyl(g-OR)-glycyl-L-arginyl-p- monohydrate, 0.8 g of L-alanine, 0.01 g of citric acid mono-
nitroanilide in 1 minute at 259C. hydrate, 0.14 g of disodium hydrogen phosphate dodecahy-
drate and 0.45 g of sodium chloride in 100 mL of water for
Bovine serum Serum obtained from blood of bovine. In-
injection.
terleukin-2 dependent cell growth suppression substance is
removed by heat at 569 C for 30 minutes before use Bovine serum albumin TS for Secretin RS Dissolve 0.1 g
of bovine serum albumin, 0.8 g of L-alanine, 0.01 g of citric
Bovine serum albumin Obtained from cattle serum as
acid monohydrate, 0.14 g of disodium hydrogen phosphate
Cohn's fifth fraction. Contains not less than 95z of albu-
dodecahydrate and 0.45 g of sodium chloride in 100 mL of
min.
water for injection.
Bovine serum albumin for assay White or yellowish,
Bradykinin C50H73N15O11 A white powder. Freely solu-
crystals or crystalline powder.
ble in water and in acetic acid (31), and practically insoluble
Take 50 mg of bovine serum albumin containing 99z or
in diethyl ether.
more albumin in glass ampoules and put them in the desicca-
Optical rotation <2.49> [a]20
D : －80 – －909(15 mg, water, 5
tor, whose humidity is adjusted to 31zRH at 259 C with cal-
mL, 100 mm).
cium chloride-saturated solution, for 2 weeks, and then take
Purity Related substances—Dissolve 2.0 mg of bradyki-
out and seal them immediately.
nin in 0.2 mL of water, and use this solution as the sample
Protein content: not less than 88z. Assay—Weigh accu-
solution. Perform the test with the sample solution as di-
rately about 0.1 g of bovine serum albumin for assay, dis-
rected under Thin-layer Chromatography <2.03>. Spot 5 mL
solve in water, and add water to make exactly 20 mL. Put
of the sample solution on a plate of cellulose for thin-layer
exactly 3 mL of the solution in the Kjeldahl frask, and deter-
chromatography. Develop the plate with a mixture of 1-
mine protein content following Nitrogen Determination
butanol, water, pyridine and acetic acid (31) (15:12:10:3) to
<1.08>.
a distance of about 10 cm, and dry the plate at 609 C. Spray
Each mL of 0.005 mol/L sulfuric acid VS evenly a solution of ninhydrin in 1-butanol (1 in 1000) on the
＝ 0.8754 mg protein plate, and heat at 609C for 30 to 60 minutes: any spot other
than the principal spot arisen from bradykinin does not ap-
Storage—Store at 49C or lower.
pear.
Bovine serum albumin for gel filtration molecular mass
Brilliant green C27H34N2O4S Fine, glistening, yellow
marker Albumin obtained from bovine serum. For gel
crystals. It dissolves in water and in ethanol (95). The wave-
filtration chromatography.
length of absorption maximum: 623 nm.
Bovine serum albumin for test of ulinastatin White crys-
Bromine Br [K 8529, Special class]
talline powder obtained from bovine serum by a purification
method which does not denature albumin and other serum Bromine-acetic acid TS Dissolve 10 g of sodium acetate
proteins. It contains not less than 99z of albumin. trihydrate in acetic acid (100) to make 100 mL, add 5 mL of
bromine, and shake. Preserve in light-resistant containers,
0.1z Bovine serum albumin-acetate buffer solution Dis-
preferably in a cold place.
solve 0.1 g of bovine serum albumin in a solution of sodium
acetate trihydrate (1 in 100) to make exactly 100 mL, and Bromine-carbon tetrachloride TS To 0.1 g of bromine
adjust to pH 4.0 with 1 mol/L hydrochloric acid TS. add carbon tetrachloride to make 100 mL, and dilute a 2 mL
portion of this solution with carbon tetrachloride to make
Bovine serum albumin-isotonic sodium chloride solution
100 mL. Prepare before use.
Dissolve 0.1 g of bovine serum albumin in 100 mL of iso-
tonic sodium chloride solution. Prepare before use. Bromine-cyclohexane TS Dissolve 0.1 g of bromine in
cyclohexane to make 100 mL. To 2 mL of this solution add
1 w/vz Bovine serum albumin-phosphate buffer-sodium
cyclohexane to make 10 mL. Prepare before use.
chloride TS Dissolve 1 g of bovine serum albumin in 100
mL of 0.01 mol/L phosphate buffer-sodium chloride TS (pH Bromine-sodium hydroxide TS To 100 mL of a solution
7.4). of sodium hydroxide (3 in 100) add 0.2 mL of bromine. Pre-
pare before use.
0.1 w/vz Bovine serum albumin-sodium chloride-
phosphate buffer solution Dissolve 8.0 g of sodium chlo- Bromine TS Prepare by saturating water with bromine
ride, 0.2 g of potassium chloride, 1.15 g of anhydrous diso- as follows: Transfer 2 to 3 mL of bromine to a glass-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
218 General Tests / 9.41 Reagents, Test Solutions JP XVII
stoppered bottle, the stopper of which should be lubricated Bromophenol blue TS, dilute Dissolve 50 mg of bromo-
with petrolatum, add 100 mL of cold water, insert the stop- phenol blue in 100 mL of ethanol (99.5). Prepare before use.
per, and shake.
Bromophenol blue TS (pH 7.0) Mix 10 mL of bromo-
Storage—Preserve in light-resistant containers, preferably
phenol blue TS and 10 mL of ethanol (95), and adjust the
in a cold place.
pH to 7.0 with diluted dilute sodium hydroxide TS (1 in 10).
Bromocresol green C21H14Br4O5S [K 8840, Special
N-Bromosuccinimide C4H4BrNO2 [K 9553, Special
class]
class]
Bromocresol green-crystal violet TS Dissolve 0.3 g of
N-Bromosuccinimide TS Dissolve 1 g of N-bromosuc-
bromocresol green and 75 mg of crystal violet in 2 mL of
cinimide in 1000 mL of water.
ethanol (95), and dilute with acetone to make 100 mL.
Bromothymol blue C27H28Br2O5S [K 8842, Special
Bromocresol green-methyl red TS Dissolve 0.15 g of
class]
bromocresol green and 0.1 g of methyl red in 180 mL of
ethanol (99.5), and add water to make 200 mL. Bromothymol blue-sodium hydroxide TS To 0.2 g of
powdered bromothymol blue add 5 mL of dilute sodium hy-
Bromocresol green-sodium hydroxide-acetic acid-sodium
droxide TS and a small quantity of water, dissolve by shak-
acetate TS To 0.25 g of bromocresol green add 15 mL of
ing in a water bath at 509
C, then add water to make 100 mL.
water and 5 mL of dilute sodium hydroxide TS, then add a
small quantity of acetic acid-sodium acetate buffer solution Bromothymol blue-sodium hydroxide-ethanol TS Dis-
(pH 4.5), dissolve while shaking, and add acetic acid-sodium solve 50 mg of bromothymol blue in 4 mL of diluted 0.2
acetate buffer solution (pH 4.5) to make 500 mL. Wash 250 mol/L sodium hydroxide TS (1 in 10) and 20 mL of ethanol
mL of the solution with two 100 mL portions of dichloro- (95), and add water to make 100 mL.
methane. Filter if necessary.
Bromothymol blue TS Dissolve 0.1 g of bromothymol
Bromocresol green-sodium hydroxide-ethanol TS Dis- blue in 100 mL of dilute ethanol, and filter if necessary.
solve 50 mg of bromocresol green in 0.72 mL of 0.1 mol/L
Bromovalerylurea C6H11BrN2O2 [Same as the name-
sodium hydroxide VS and 20 mL of ethanol (95), and add
sake monograph]
water to make 100 mL.
Test for sensitivity—To 0.2 mL of the bromocresol green- Brotizolam for assay C15H10BrClN4S [Same as the
sodium hydroxide-ethanol TS add 100 mL of freshly boiled monograph Brotizolam. When dried, it contains not less
and cool water: the solution is blue, and not more than 0.2 than 99.0z of brotizolam (C15H10BrClN4S).]
mL of 0.02 mol/L hydrochloric acid VS is required to
Brucine See brucine n-hydrate.
change the color of this solution to yellow.
Color change: pH 3.6 (yellow) to pH 5.2 (blue). Brucine dihydrate See brucine n-hydrate.
Bromocresol green-sodium hydroxide TS Triturate 0.2 g Brucine n-hydrate C23H26N2O4.nH2O [K 8832, Special
of bromocresol green with 2.8 mL of 0.1 mol/L sodium hy- class]
droxide VS in a mortar, add water to make 200 mL, and
1,4-BTMSB-d4 for nuclear magnetic resonance spectros-
filter if necessary.
copy C12H18D4Si2 1,4-Bis(trimethylsilyl)benzene-d4 that the
Bromocresol green TS Dissolve 50 mg of bromocresol traceability to the international unit system was secured.
green in 100 mL of ethanol (95), and filter if necessary.
B-type erythrocyte suspension Prepare a suspension con-
Bromocresol purple C21H16Br2O5S [K 8841, Special taining 1 volz of erythrocyte separated from human B-type
class] blood in isotonic sodium chloride solution.
Bromocresol purple-dipotassium hydrogenphosphate- Bucillamine C7H13NO3S2 [Same as the namesake
citric acid TS Mix 30 mL of bromocresol purple-sodium monograph]
hydroxide TS and 30 mL of dibasic potassium phosphate-
Bucillamine for assay C7H13NO3S2 [Same as the mono-
citric acid buffer solution (pH 5.3), and wash with three
graph Bucillamine. However, when dried, it contains not less
60-mL portions of chloroform.
than 99.0z of bucillamine (C7H13NO3S2). Furthermore, it
Bromocresol purple-sodium hydroxide TS Triturate conforms to the following test.]
0.4 g of bromocresol purple with 6.3 mL of dilute sodium Purity Related substances—Dissolve 60 mg of bucilla-
hydroxide TS in a mortar, add water to make 250 mL, and mine for assay in 20 mL of a mixture of water and methanol
filter if necessary. (1:1) and use this solution as the sample solution. Pipet 1 mL
of the sample solution, add the mixture of water and metha-
Bromocresol purple TS Dissolve 50 mg of bromocresol
nol (1:1) to make exactly 100 mL, and use this solution as the
purple in 100 mL of ethanol (95), and filter if necessary.
standard solution. When the test is performed according to
Bromophenol blue C19H10Br4O5S [K 8844, Special the Purity (3) under Bucillamine, the total area of the peaks
class] other than bucillamine from the sample solution is not larger
than the peak area of bucillamine from the standard solu-
Bromophenol blue-potassium biphthalate TS Dissolve
tion.
0.1 g of bromophenol blue in potassium biphthalate buffer
solution (pH 4.6) to make 100 mL. Bufalin for assay C24H34O4.xH2O White, odorless crys-
talline powder.
Bromophenol blue TS Dissolve 0.1 g of bromophenol
Absorbance <2.24> E 11zcm (300 nm): 143 – 153 (10 mg,
blue in 100 mL of dilute ethanol, and filter if necessary.
methanol, 250 mL). Use the sample dried in a desiccator
0.05z Bromophenol blue TS Dissolve 10 mg of bromo- (silica gel) for 24 hours for the test.
phenol blue in water to make 20 mL. Purity Related substances—Dissolve 40 mg of bufalin

for assay in 5 mL of chloroform and use this solution as the 2.70 g in 100 mL of water, 50 mL of dithiothreitol in solution
sample solution. Pipet 1 mL of the sample solution, add containing 30.9 mg in 1 mL of water and 420 mL of water.
chloroform to make exactly 100 mL, and use this solution as
Buffer solution for epoetin alfa sample Dissolve 1.2 g of
the standard solution. Perform the test with these solutions
2-amino-2-hydroxymethyl-1,3-propanediol and 3.2 g of so-
as directed under Thin-layer Chromatography <2.03>. Spot 5
dium lauryl sulfate in a suitable amount of water, adjust to
mL each of the sample solution and standard solution on a
pH 6.8 with 6 mol/L hydrochloric acid TS, 1 mol/L hydro-
plate of silica gel for thin-layer chromatography. Develop
chloric acid TS or 0.1 mol/L hydrochloric acid TS, add
the plate with a mixture of cyclohexane, acetone and chlo-
32 mg of bromophenol blue and 16 mL of glycerin, and add
roform (4:3:3) to a distance of about 14 cm, and air-dry.
water to make 40 mL. Before use, dissolve 50 mg of
Spray evenly sulfuric acid, and heat at 1009 C for 2 to 3
dithiothreitol in 10 mL of this solution.
minutes: any spot other than the principal spot obtained
from the sample solution is not larger and not more intense Buffer solution for filgrastim sample Dissolve 1.2 g of 2-
than the spot obtained from the standard solution. amino-2-hydroxymethyl-1,3-propanediol and 3.2 g of so-
Content: not less than 99.0z. Assay—Weigh accurately dium lauryl sulfate in a suitable amount of water, adjust to
about 10 mg of bufalin for assay, previously dried in a desic- pH 6.8 with 6 mol/L hydrochloric acid TS, 1 mol/L hydro-
cator (silica gel) for 24 hours, dissolve in methanol to make chloric acid TS or 0.1 mol/L hydrochloric acid TS, add
exactly 10 mL, and use this solution as the sample solution. 32 mg of bromophenol blue and 16 mL of glycerin, and add
Perform the test with 20 mL of the sample solution as di- water to make 40 mL.
rected under Liquid Chromatography <2.01> according to
Buffer solution for nartograstim sample Mix 0.8 mL of
the following conditions. Determine the peak area by the au-
sodium lauryl sulfate solution (1 in 10), 0.5 mL of 0.5 mol/L
tomatic integration method, and calculate the amount of
tris buffer solution (pH 6.8), 0.4 mL of glycerin and 0.1 mL
bufalin by the area percentage method.
of bromophenol blue solution (1 in 200). Prepare before use.
Operating conditions
Detector: An ultraviolet absorption photometer (wave- Buffer solution for SDS polyacrylamide gel electrophore-
length: 300 nm). sis Dissolve 3.0 g of 2-amino-2-hydroxymethyl-1,3-
Column: A stainless steel column 4 to 6 mm in inside di- propanediol, 14.4 g of glycine and 1.0 g of sodium lauryl sul-
ameter and 15 to 30 cm in length, packed with octadecyl- fate in water to make 1000 mL.
silanized silica gel for liquid chromatography (5 to 10 mm in
Buformin hydrochloride for assay C6H15N5.HCl [Same
particle diameter).
as the monograph Buformin Hydrochloride. When dried, it
Column temperature: A constant temperature of about
contains not less than 99.5z of buformin hydrochloride
409 C.
(C6H15N5.HCl).]
Mobile phase: A mixture of water and acetonitrile (1:1).
Flow rate: Adjust so that the retention time of bufalin is n-Butanol See 1-butanol.
about 6 minutes.
sec-Butanol See 2-butanol.
Selection of column: Dissolve 10 mg each of bufalin for
assay, cinobufagin for assay and resibufogenin for assay in 1-Butanol CH3(CH2)2CH2OH [K 8810, Special class]
methanol to make 200 mL. Proceed with 20 mL of this solu-
2-Butanol CH3CH2CH(OH)CH3 [K 8812, Special
tion according to the above conditions. Use a column giving
class]
elution of bufalin, cinobufagin and resibufogenin in this
order and completely resolving these peaks. 2-Butanone CH3COC2H5 [K 8900, Special class]
Detection sensitivity: Pipet 1 mL of the sample solution,
Butenafine hydrochloride for assay C23H27N.HCl
add methanol to make exactly 100 mL, and use this solution
[Same as the monograph Butenafine Hydrochloride]
as the standard solution (1). Pipet 1 mL of this solution, add
methanol to make exactly 20 mL, and use this solution as the N-t-Butoxycarbonyl-L-glutamic acid-a-phenyl ester
standard solution (2). Adjust the detection sensitivity so that C16H21NO6 White powder.
the peak area of bufalin obtained from 20 mL of the stand- Melting point <2.60>: 95–1049C
ard solution (2) can be measured by the automatic integra- Purity Related substances—Dissolve 10 mg of N-t-
tion method, and the peak height of bufalin from 20 mL of butoxycarbonyl-L-glutamic acid-a-phenyl ester in 5 mL of
the standard solution (1) is about 20z of the full scale. dilute ethanol, and use this solution as the sample solution.
Time span of measutrement: About twice as long as the Pipet 1 mL of the sample solution, add dilute ethanol to
retention time of bufalin, beginning after the solvent peak. make exactly 50 mL, and use this solution as the standard
solution. Perform the test with these solutions as directed
Bufalin for component determination See bufalin for
under Thin-layer Chromatography <2.03>. Spot 10 mL each
assay.
of the sample solution and standard solution on three plates
Buffer solution for celmoleukin Combine 12.5 mL of 0.5 of silica gel with fluorescent indicator for thin-layer chroma-
mol/L tris buffer solution (pH 6.8), 10 mL of sodium lauryl tography. Develop the first plate with a mixture of chlo-
sulfate solution (1→10), 10 mL of glycerin, and 17.5 mL of roform, ethyl acetate and acetic acid (100) (25:25:1), the
water, shake, and then add and dissolve 5 mg of bromophe- second plate with a mixture of benzene, 1,4-dioxane and
nol blue. acetic acid (100) (95:25:4), and the third plate with a mixture
Storage—Store in a cool place, shielded from light. of chloroform, methanol and acetic acid (100) (45:4:1) to a
distance of about 12 cm, and air-dry these plates. Examine
Buffer solution for enzyme digestion Dissolve 0.30 g of
under ultraviolet light (main wavelength: 254 nm): the spots
urea in a mixture of 100 mL of 2-amino-2-hydroxymethyl-
other than the principal spot obtained from the sample solu-
1,3-propanediol solution containing 6.06 g in 100 mL of
tion are not more intense than the spot from the standard so-
water, 100 mL of 2-amino-2-hydroxymethyl-1,3-propanediol
lution in all plates.
hydrochloride solution containing 7.88 g in 100 mL of water,
100 mL of methylamine hydrochloride solution containing Butyl acetate CH3COOCH2CH2CH2CH3 [K 8377,

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
220 General Tests / 9.41 Reagents, Test Solutions JP XVII
Special class] butyl parahydroxybenzoate obtained from the standard solu-
tion.
n-Butyl acetate See butyl acetate
Operating conditions
t-Butyl alcohol (CH3)3COH A crystalline solid, having Detector, column, column temperature, mobile phase, and
a characteristic odor. A colorless liquid at above an ordinary flow rate: Proceed as directed in the operating conditions in
temperature. Specific gravity d 20
20: about 0.78; Boiling point: the Assay under Butyl Parahydroxybenzoate.
about 839 C; Melting point: about 259 C. Time span of measurement: About 1.5 times as long as the
Identification—Determine the infrared absorption spec- retention time of butyl parahydroxybenzoate.
trum as directed in the liquid film method as directed under System suitability
Infrared Spectrophotometry <2.25>: it exhibits absorption at Test for required detectability: Pipet 1 mL of the standard
the wave numbers of about 3370 cm－1, 2970 cm－1, 1471 solution, and add methanol to make exactly 20 mL. Confirm
cm－1, 1202 cm－1, 1022 cm－1, 913 cm－1 and 749 cm－1. that the peak area of butyl parahydroxybenzoate obtained
with 10 mL of this solution is equivalent to 3.5 to 6.5z of
n-Butylamine CH3CH2CH2CH2NH2 A colorless liquid,
that obtained with 10 mL of the standard solution.
having an amine-like, characteristic odor. Miscible with
System performance: When the procedure is run with 10
water, with ethanol (95) and with diethyl ether. The solution
mL of the standard solution under the above operating con-
in water shows alkalinity and rapidly absorbs carbon dioxide
ditions, the number of theoretical plates and the symmetry
from the air.
factor of the peak of butyl parahydroxybenzoate are not less
Specific gravity <2.56> d 20
20: 0.740 – 0.747
than 2500 and not more than 2.0, respectively.
Distilling range <2.57>: 76.5 – 799 C, not less than 96
System repeatability: When the test is repeated 6 times
volz.
with 10 mL of the standard solution under the above operat-
Butyl benzoate C6H5COOCH2CH2CH2CH3 A clear ing conditions, the relative standard deviation of the peak
and colorless liquid. area of butyl parahydroxybenzoate is not more than 5.0z.
Refractive index <2.45> n 20
D : 1.495 – 1.500
Butyrolactone C4H6O2 Clear, colorless to practically
Specific gravity <2.56> d 20
20: 1.006 – 1.013
colorless liquid.
n-Butylboronic acid C4H11BO2 White flakes. Specific gravity <2.56> d 25
4 : 1.128 – 1.135
Melting point <2.60>: 90 – 929C Boiling point <2.57>: 198 – 2089 C
n-Butyl chloride See 1-chlorobutane. Cadmium acetate See cadmium acetate dihydrate.
n-Butyl formate HCOO(CH2)3CH3 Clear and colorless Cadmium acetate dihydrate Cd(CH3COO)2.2H2O
liquid, having a characteristic odor. White, crystals or crystalline powder.
Specific gravity <2.56> d 20
20: 0.884 – 0.904 Identification—(1) Dissolve 0.2 g of cadmium acetate di-
hydrate in 20 mL of water, and use this solution as the sam-
tert-Butyl methyl ether (CH3)3COCH3 Clear colorless
ple solution. To 10 mL of the sample solution add 2 mL of
liquid, having a specific odor.
iron (III) chloride TS: a red-brown color is produced.
Refractive index <2.45> n 20
D : 1.3689
(2) To 10 mL of the sample solution obtained in (1) add
Specific gravity <2.56> d 20
4 : 0.7404
1 mL of sodium sulfide TS: a yellow precipitate is produced.
Butyl parahydroxybenzoate
Cadmium ground metal Cd [H 2113, First class]
HOC6H4COOCH2CH2CH2CH3 [Same as the namesake
monograph] Cadmium-ninhydrin TS Dissolve 50 mg of cadmium ace-
tate dihydrate in 5 mL of water and 1 mL of acetic acid
Butyl parahydroxybenzoate for resolution check
(100), add 2-butanone to make 50 mL, and dissolve 0.1 g of
C11H14O3 Colorless crystals or a white crystalline powder.
ninhydrin in this solution. Prepare before use.
Very soluble in methanol, freely soluble in ethanol (95) and
in acetone, and practically insoluble in water. Melting point: Cadralazine for assay C12H21N5O3 [Same as the mono-
68 – 719 C. graph Cadralazine. When dried, it contains not less than
Identification—Determine the infrared absorption spec- 99.0z of cadralazine (C12H21N5O3).]
trum of butyl parahydroxybenzoate for resolution check as
Caffeine See caffeine hydrate.
directed in the potassium bromide disk method under In-
frared Spectrophotometry <2.25>, and compare the spectrum Caffeine hydrate C8H10N4O2.H2O [Same as the name-
with the Reference Spectrum of Butyl Parahydroxybenzoate sake monograph]
or the spectrum of Butyl Parahydroxybenzoate RS: both
Caffeine, anhydrous C8H10N4O2 [Same as the mono-
spectra exhibit similar intensities of absorption at the same
graph Anhydrous Caffeine]
wave numbers.
Purity Related substances—Dissolve 50 mg of butyl Calcium acetate monohydrate (CH3COO)2Ca.H2O
parahydroxybenzoate for resolution check in 2.5 mL of [K 8364, Special class]
methanol, and add the mobile phase to make 50 mL. To 10
Calcium carbonate CaCO3 [K 8617, Special class]
mL of this solution add the mobile phase to make 100 mL,
and use this solution as the sample solution. Pipet 1 mL of Calcium carbonate for assay CaCO3 [Same as the
the sample solution, add the mobile phase to make exactly 10 monograph Precipitated Calcium Carbonate. When dried, it
mL, and use this solution as the standard solution. Perform contains not less than 99.0z of calcium carbonate
the test with exactly 10 mL each of the sample solution and (CaCO3).]
standard solution as directed under Liquid Chromatography
Calcium chloride See calcium chloride dihydrate.
<2.01> according to the following conditions. Determine each
peak area by the automatic integration method: total area of Calcium chloride dihydrate CaCl2.2H2O [K 8122, Spe-
the peaks other than butyl parahydroxybenzoate obtained cial class]
from the sample solution is not larger than the peak area of

Calcium chloride dihydrate for assay See calcium chlo- Candesartan cilexetil C33H34N6O6 [Same as the name-
ride hydrate for assay. sake monograph]
Calcium chloride for drying CaCl2 [K 8124, For dry- Candesartan cilexetil for assay C33H34N6O6 [Same as
ing] the monograph Candesartan Cilexetil. It contains not less
than 99.5z of candesartan cilexetil (C33H34N6O6), calculated
Calcium chloride hydrate for assay CaCl2.2H2O [Same
on the anhydrous basis, and when performed the test as di-
as the monograph, Calcium Chloride Hydrate. It contains
rected in the Purity (2) under Candesartan Cilexetil, the total
not less than 99.0z of calcium chloride hydrate (CaCl2.
area of the peaks other than candesartan cilexetil obtained
2H2O).]
from the sample solution is not larger than 1/2 times the
Calcium chloride for water determination CaCl2 peak area of candesartan cilexetil obtained from the stand-
[K 8125, For water determination] ard solution.]
Calcium chloride TS Dissolve 7.5 g of calcium chloride Caprylic acid CH3(CH2)6COOH A clear and colorless,
dihydrate in water to make 100 mL (0.5 mol/L). oily liquid, having a slight unpleasant odor. Freely soluble in
ethanol (95) and in chloroform, and very slightly soluble in
Calcium gluconate for thin-layer chromatography See
water.
calcium gluconate hydrate for thin-layer chromatography.
Refractive index <2.45> n 20
D : 1.426 – 1.430
Calcium gluconate hydrate for thin-layer chromatography Specific gravity <2.56> d 20
4 : 0.908 – 0.912
[Same as the monograph Calcium Gluconate Hydrate. When Distilling range <2.57>: 238 – 2429C, not less than 95
the test is performed as directed in the Identification (1) volz.
under Calcium Gluconate Hydrate, any spot other than the
(E )-Capsaicin for assay C18H27NO3 Use (E )-capsaicin
principal spot at the R f value of about 0.4 does not appear.]
for thin-layer chromatography meeting the following addi-
Calcium hydroxide Ca(OH)2 [K 8575, Special class] tional specifications.
Absorbance <2.24> E 11zcm (281 nm): 97 – 105 (10 mg, meth-
Calcium hydroxide for pH determination Calcium hy-
anol, 200 mL). Use the sample dried in a desiccator (in vacu-
droxide prepared for pH determination.
um, phosphorus (V) oxide, 409 C) for 5 hours for the test.
Calcium hydroxide pH standard solution See pH Deter- Purity Related substances—Dissolve 10 mg of (E )-cap-
mination <2.54>. saicin for assay in 50 mL of methanol, and use this solution
as the sample solution. Pipet 1 mL of the sample solution,
Calcium hydroxide TS To 3 g of calcium hydroxide add
add methanol to make exactly 100 mL, and use this solution
1000 mL of cold distilled water, and occasionally shake the
as the standard solution. Perform the test with exactly 20 mL
mixture vigorously for 1 hour. Allow to stand, and use the
each of the sample solution and standard solution as directed
supernatant liquid (0.04 mol/L).
under Liquid Chromatography <2.01> according to the fol-
Calcium nitrate See calcium nitrate tetrahydrate. lowing conditions, and determine each peak area from these
solutions by the automatic integration method: the total area
Calcium nitrate tetrahydrate Ca(NO3)2.4H2O
of the peaks other than capsaicin from the sample solution is
[K 8549, Special class]
not larger than the peak area of capsaicin from the standard
Calcium oxide CaO [K 8410, Special class] solution.
Operating conditions
Calcium pantothenate C18H32CaN2O10 [Same as the
Detector, column, column temperature, mobile phase, and
namesake monograph]
flow rate: Proceed the operating conditions in the Assay
Calcium paraaminosalicylate hydrate for assay under Capsicum.
C7H5CaNO3.3 1/2 H2O [Same as the monograph Calcium Time span of measurement: About 3 times as long as the
Paraaminosalicylate Hydrate. It contains not less than retention time of capsaicin, beginning after the solvent peak.
99.0z of calcium paraaminosalicylate (C7H5CaNO3), calcu- System suitability
lated on the anhydrous basis.] System performance, and system repeatability: Proceed
the system suitability in the Assay under Capsicum.
Camphor C10H16O [Same as the monograph d-Cam-
Test for required detectability: Pipet 1 mL of the standard
phor or dl-Camphor]
solution, and add methanol to make exactly 20 mL. Confirm
d-Camphorsulfonic acid C10H16O4S White, crystals or that the peak area of capsaicin obtained from 20 mL of this
crystalline powder, having a characteristic odor. Very solu- solution is equivalent to 3.5 to 6.5z of that of capsaicin ob-
ble in water, and soluble in chloroform. tained from 20 mL of the standard solution.
Purity Clarity and color of solution—Dissolve 1.0 g of
(E )-Capsaicin for component determination See (E )-cap-
d-camphorsulfonic acid in 10 mL of water: the solution is
saicin for assay.
clear and colorless or pale yellow.
Loss on drying <2.41>: not more than 2.0z (1 g, 1059C, Capsaicin for thin-layer chromatography See (E )-capsai-
5 hours). cin for thin-layer chromatography.
Content: not less than 99.0z, calculated on the dried
(E )-Capsaicin for thin-layer chromatography C18H27NO3
basis. Assay—Weigh accurately about 4 g of d-camphor-
White crystals, having a strong irritative odor. Very soluble
sulfonic acid, dissolve in 50 mL of water, and titrate <2.50>
in methanol, freely soluble in ethanol (95) and in diethyl
with 1 mol/L sodium hydroxide VS (indicator: 3 drops of
ether, and practically insoluble in water.
methyl red TS). Perform a blank determination in the same
Melting point <2.60>: 65 – 709C
manner.
Purity Related substances—Dissolve 20 mg of (E )-cap-
Each mL of 1 mol/L sodium hydroxide VS saicin for thin-layer chromatography in 2 mL of methanol,
＝ 232.3 mg of C10H16O4S and use this solution as the sample solution. Pipet 1 mL of
the sample solution, add methanol to make exactly 100 mL,

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
222 General Tests / 9.41 Reagents, Test Solutions JP XVII
and use this solution as the standard solution. Perform the graph Carvedilol]
test with 10 mL each of the sample solution and standard so-
Casein, milk A white to light yellow, powder or grain.
lution as directed in the Identification under Capsicum: any
Identification—Determine the infrared absorption spec-
spot other than the principal spot (R f value is about 0.5)
trum as directed in the potassium bromide disk method as di-
from the sample solution is not more intense than the spot
rected under Infrared Spectrophotometry <2.25>: it exhibits
from the standard solution.
absorption at the wave numbers of about 1650 cm－1, 1540
Carbazochrome C10H12N4O3 Yellow-red to red, crys- cm－1 and 1250 cm－1.
tals or crystalline powder. Melting point: about 2229C (with
Casein (milk origin) See casein, milk.
decomposition).
Content: not less than 98.0z. Assay—Dissolve about Casein peptone See peptone, casein.
0.2 g of carbazochrome, weighed accurately, in 20 mL of
Castor oil [Same as the namesake monograph]
acetic acid (100) by heating, add 80 mL of acetic anhydride,
cool, and titrate <2.50> with 0.1 mol/L perchloric acid VS Catechol C6H4(OH)2 White crystals.
(potentiometric titration). Perform a blank determination, Melting point <2.60>: 104 – 1079C
and make any necessary correction. Preserve in a light-resistant tight container.
Each mL of 0.1 mol/L perchloric acid VS Cefadroxil C16H17N3O5S [Same as the namesake mono-
＝ 23.62 mg of C10H12N4O3 graph]
Carbazochrome sodium sulfonate for component determi- Cefatrizine propylene glycolate C18H18N6O5S2.C3H8O2
nation See carbazochrome sodium sulfonate trihydrate. [Same as the namesake monograph]
Carbazochrome sodium sulfonate trihydrate Cefcapene pivoxil hydrochloide hydrate
C10H11N4NaO5S.3H2O [Same as the monograph Car- C23H29N5O8S2.HCl.H2O [Same as the namesake mono-
bazochrome Sodium Sulfonate Hydrate. It contains graph]
not less than 99.0z of carbazochrome sodium sulfonate
Cefdinir lactam ring-cleavage lactones C14H15N5O6S2 A
(C10H11N4NaO5S), calculated on the anhydrous basis, and
mixture of 4 diastereoisomers. A white to yellow powder.
meets the following additional requirement.]
Identification—Determine the infrared absorption spec-
Water <2.48>: 14.0 – 15.0z.
trum of cefdinir lactam ring-cleavage lactones as directed in
Carbazole C12H9N White to nearly white foliaceous or the paste method under Infrared Spectrophotometry <2.25>:
plate-like crystals or crystalline powder. Freely soluble in it exhibits absorption at the wave numbers of about 1743
pyridine and in acetone, slightly soluble in ethanol (99.5), cm－1, 1330 cm－1, 1163 cm－1 and 1047 cm－1.
and practically insoluble in water. It readily sublimes when Content: not less than 90z. Assay—Dissolve about 5 mg
heated. of cefdinir lactam ring-cleavage lactones in 5 mL of 0.1
Melting point <2.60>: 243 – 2459 C mol/L phosphate buffer solution (pH 7.0), and use this solu-
Purity Clarity and color of solution—To 0.5 g of carba- tion as the sample solution. Perform the test with 5 mL of the
zole add 20 mL of ethanol (99.5), and dissolve by warming: sample solution as directed in the operating conditions of the
the solution is clear. Purity (2) under Cefdinir, and calculate the areas of each
Residue on ignition: Not more than 0.1z (1 g). peak by the automatic integration method. Determine the
percent of the total peak area of 4 cefdinir lactam ring-
Carbazole TS Dissolve 0.125 g of carbazole in ethanol
cleavage lactones to the total area of all peaks.
(99.5) to make 100 mL.
Cell suspension solution for teceleukin Centrifuge for 5
L-Carbocisteine for assay C5H9NO4S [Same as the
minutes at 1000 rpm culture medium of NK-7 cells that have
monograph L-Carbocisteine. When dried, it contains not less
been cultured statically for 2 to 4 hours. Remove the super-
than 99.0z of L-carbocisteine (C5H9NO4S).]
natant by aspiration, and add potency measuring medium
0.1 mol/L Carbonate buffer solution (pH 9.6) Dissolve for teceleukin to a cell concentration of 2 to 4 × 105 cells/
3.18 g of anhydrous sodium carbonate and 5.88 g of sodium mL.
hydrogen carbonate in water to make 1000 mL.
Celmoleukin for liquid chromatography
Carbon dioxide CO2 [Same as the namesake mono- C693H1118N178O203S7 [Same as the monograph Celmoleukin
graph] (Genetical Recombination). However, contains 0.5 to 1.5 mg
of protein per mL, polymers amount for 0.5z or less, and
Carbon disulfide CS2 [K 8732, Special class]
conforms to the following test].
Preserve in tightly stoppered containers in a dark, cold
Identification (1) When the amino acid sequence is in-
place, remote from fire.
vestigated using the Edman technique and liquid chromatog-
Carbonic anhydrase White powder. Derived from bo- raphy, the amino acids are detected in the following se-
vine RBC. Molecular weight about 29,000. quence: alanine, proline, threonine, serine, serine, serine,
threonine, lysine, lysine, threonine, glutamine, leucine,
Carbon monoxide CO A toxic, colorless gas. Prepare
glutamine, leucine, and glutamic acid. Also, based on the
by passing the gas generated by reacting formic acid with sul-
results of the protein content determination test, place an
furic acid through a layer of sodium hydroxide TS. Carbon
amount of celmoleukin equivalent to about 0.3 mg in a hy-
monoxide from a metal cylinder may be used.
drolysis tube, evaporate to dryness under vacuum, and then
Carbon tetrachloride CCl4 [K 8459, Special class] add 100 mL of hydrazine anhydride for amino acid sequence
analysis. Reduce the internal pressure of the hydrolysis tube
Carboplatin C6H12N2O4Pt [Same as the namesake
by heating for 6 hours at about 1009C. After evaporating to
monograph]
dryness under vacuum, add 250 mL of water to dissolve the
Carvedilol for assay C24H26N2O4 [Same as the mono- residue. To this add 200 mL of benzaldehyde, shake occa-

sionally, leave for one hour, centrifuge, and remove the Cerium (IV) diammonium nitrate TS Dissolve 6.25 g of
aqueous layer. Add 250 mL of water to the benzaldehyde cerium (IV) diammonium nitrate in 160 mL of diluted dilute
layer, shake, centrifuge, combine the aqueous layers, and nitric acid (9 in 50). Use within 3 days.
evaporate to dryness under vacuum. Threonine is detected
Cerium (IV) sulfate tetrahydrate Ce(SO4)2・4H2O
when amino acid analysis is conducted using the postcolumn
[K 8976, Special class]
technique with ninhydrin on a solution of the residue dis-
solved by adding 100 mL of 0.02 mol/L hydrochloric acid Cerium (IV) tetraammonium sulfate dihydrate
TS. Ce(SO4)2.2(NH4)2SO4.2H2O [K 8977, Tetraammonium
(2) Add 1 mL of protein digestive enzyme solution to 1 cerium (IV) sulfate dihydrate, Special class]
mL of celmoleukin for liquid chromatography, shake, and
Cerium (IV) tetraammonium sulfate-phosphoric acid TS
leave for 18 to 24 hours at 379C. Pipet 1 mL of this solution
Dissolve 0.1 g of cerium (IV) tetraammonium sulfate dihy-
and add 25 mL of trifluoroacetic acid (1 in 10). To another 1
drate in diluted phosphoric acid (4 in 5) to make 100 mL.
mL, add 10 mL of 2-mercaptoethanol, leave for 30 minutes
at 379C, and then add 25 mL of trifluoroacetic acid (1 in 10). Cerium (IV) tetraammonium sulfate TS Dissolve 6.8 g of
Perform Liquid Chromatography <2.01> on these two solu- cerium (IV) tetraammonium sulfate dihydrate in diluted sul-
tions separately under the conditions outlined in Celmoleu- furic acid (3 in 100) to make 100 mL.
kin (Genetical Recombination), Identification (4). Repeated-
Cerous nitrate See cerium (III) nitrate hexahydrate.
ly pipet the celmoleukin derived peak fraction that elutes and
when the test is performed according to Celmoleukin (Genet- Cerous nitrate TS See cerium (III) nitrate TS.
ical Recombination), Identification (2), except for the lysines
Cesium chloride CsCl White, crystals or crystalline
in positions 9 and 49 from the amino terminal amino acid, a
powder. Very soluble in water, and freely soluble in ethanol
peptide estimated from the complete primary structure is de-
(99.5).
tected.
Loss on drying <2.41>: Not more than 1.0z (1 g, 1109C,
Cephaeline hydrobromate C28H38N2O4.2HBr A white 2 hours).
or light-yellow crystalline powder. Content: not less than 99.0z. Assay—Weigh accurately
Purity—Dissolve 10 mg of cephaeline hydrobromate in 10 about 0.5 g, previously dried, and dissolve in water to make
mL of the mobile phase, and use this solution as the sample exactly 200 mL. Pipet 20 mL of this solution, add 30 mL of
solution. Pipet 1 mL of the sample solution, add the mobile water, and titrate <2.50> with 0.1 mol/L silver nitrate VS
phase to make exactly 10 mL, and use this solution as the (indicator: fluorescein sodium TS).
standard solution. Perform the test with exactly 10 mL each
Each mL of 0.1 mol/L silver nitrate VS
of the sample solution and standard solution as directed in
＝ 16.84 mg of CsCl
the Assay under Ipecac: when measure the peak areas for 2
times the retention time of emetine, the total area of the Cesium chloride TS To 25.34 g of cesium chloride add
peaks other than cephaeline obtained from the sample solu- water to make 1000 mL.
tion is not larger than the peak area of cephaeline obtained
Cetanol [Same as the namesake monograph]
from the standard solution.
Cetirizine hydrochloride for assay C21H25ClN2O3.2HCl
Ceric ammonium sulfate See cerium (IV) tetraammoni-
[Same as the monograph Cetirizine Hydrochloride. When
um sulfate dihydrate.
dried, it contains not less than 99.5z of cetirizine hydro-
Ceric ammonium sulfate-phosphoric acid TS See cerium chloride (C21H25ClN2O3.2HCl).]
(IV) tetraammonium sulfate-phosphoric acid TS.
Cetrimide C17H38BrN White to pale yellowish white
Ceric ammonium sulfate TS See cerium (IV) tetraammo- powder, having a faint, characteristic odor.
nium sulfate TS. Purity Clarity of solution—Dissolve 1.0 g of cetrimide in
5 mL of water: the solution is clear.
Cerium (III) nitrate hexahydrate Ce(NO3)3.6H2O A
Content: not less than 96.0z. Assay—Weigh accurately
colorless or light yellow crystalline powder. It dissolves in
about 2 g of cetrimide, previously dried, and dissolve in
water.
water to make exactly 100 mL. Pipet 25 mL of this solution
Purity (1) Chloride <1.03>: not more than 0.036z.
into a separator, add 25 mL of chloroform, 10 mL of 0.1
(2) Sulfate <1.14>: not more than 0.120z.
mol/L sodium hydroxide VS and 10 mL of a freshly pre-
Content: not less than 98.0z. Assay—To about 1.5 g of
pared solution of potassium iodide (1 in 20), shake well,
cerium (III) nitrate hexahydrate, accurately weighed, add 5
allow to stand, and remove the chloroform layer. Wash the
mL of sulfuric acid, and heat it until white fumes are evolved
solution with three 10-mL portions of chloroform, take the
vigorously. After cooling, add 200 mL of water, 0.5 mL of
water layer, and add 40 mL of hydrochloric acid. After cool-
0.1 mol/L silver nitrate VS, dissolve 5 g of ammonium
ing, titrate with 0.05 mol/L potassium iodide VS until the
peroxodisulfate, dissolve, and boil it for 15 minutes. After
deep brown color of the solution almost disappears, add 2
cooling, add 2 drops of 1,10-phenanthroline TS, and titrate
mL of chloroform, and titrate <2.50> again until the red-pur-
<2.50> with 0.1 mol/L ammonium iron (II) sulfate VS until
ple color of the chloroform layer disappears. The end point
the pale blue color of the solution changes to red.
is reached when the red-purple color of the chloroform layer
Each mL of 0.1 mol/L ammonium iron (II) sulfate VS no more reappears within 5 minutes after the chloroform
＝ 43.42 mg of Ce(NO3)3.6H2O layer is decolorized. Perform a blank determination with 20
mL of water, 10 mL of a solution of potassium iodide (1 in
Cerium (III) nitrate TS Dissolve 0.44 g of cerium (III)
20) and 40 mL of hydrochloric acid.
nitrate hexahydrate in water to make 1000 mL.
Each mL of 0.05 mol/L potassium iodate VS
Cerium (IV) diammonium nitrate Ce(NH4)2(NO3)6
＝ 33.64 mg of C17H38BrN
[K 8556, Diammonium cerium (IV) nitrate, Special class]
Cetylpyridinium chloride monohydrate C21H38ClN.H2O

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
224 General Tests / 9.41 Reagents, Test Solutions JP XVII
White, powder or crystals. Odorless or having slightly a C (with decomposi-
diethyl ether. Melting point: about 2159
characteristic odor. tion).
Melting point <2.60>: 80 – 849C Purity Related substances—Dissolve 2 mg of chikuset-
Water <2.48>: 4.5 – 5.5z susaponin IV for thin-layer chromatography in 1 mL of
Residue on ignition <2.44>: Not more than 0.2z (1 g). methanol, and perform the test with 5 mL of this solution as
Content: 99.0 – 102.0z, calculated on the anhydrous directed in the Identification under Panax Japonicus Rhi-
basis. Assay—Weigh accurately about 0.2 g of cetylpyridini- zome: any spot other than the principal spot with an R f
um chloride monohydrate, dissolve in 75 mL of water, add value of about 0.4 does not appear.
10 mL of chloroform, 0.4 mL of bromophenol blue solution
Chloral hydrate CCl3CH(OH)2 [Same as the namesake
(1 in 2000) and 5 mL of freshly prepared sodium hydrogen
monograph]
carbonate solution (21 in 5000), and titrate <2.50> with 0.02
mol/L sodium tetraphenylboron VS until the blue color in Chloral hydrate TS Dissolve 5 g of chloral hydrate in 3
the chloroform layer disappears by vigorous shaking after mL of water.
adding 1 drop of the titrant.
Chloramine See sodium toluensulfonchloramide trihy-
Each mL of 0.02 mol/L sodium tetraphenylboron VS drate.
＝ 6.800 mg of C21H38ClN
Chloramine TS See sodium toluensulfonchloramide TS.
Chenodeoxycholic acid for thin-layer chromatography
Chloramphenicol C11H12Cl2N2O5 [Same as the mono-
C24H40O4 White, crystals or crystalline powder. Very solu-
graph Chloramphenicol]
ble in methanol and in acetic acid (100), freely soluble in
ethanol (95), soluble in acetone, sparingly soluble in ethyl Chlorauric acid See hydrogen tetrachloroaurate (III)
acetate, slightly soluble in chloroform, and practically in- tetrahydrate.
soluble in water. Melting point: about 1199C (recrystallize
Chlorauric acid TS See hydrogen tetrachloroaurate (III)
from ethyl acetate).
tetrahydrate TS.
Purity Related substances—Dissolve 25 mg of
chenodeoxycholic acid for thin-layer chromatography in a Chlordiazepoxide C16H14ClN3O [Same as the namesake
mixture of chloroform and ethanol (95) (9:1) to make exactly monograph]
250 mL and use this solution as the sample solution. Per-
Chlordiazepoxide for assay C16H14ClN3O [Same as the
form the test with this solution as directed under the Thin-
monograph Chlordiazepoxide. When dried, it contains not
layer Chromatography <2.03>. Spot 10 mL of the sample so-
less than 99.0z of C16H14ClN3O.]
lution on a plate of silica gel for thin-layer chromatography.
Develop the plate with a mixture of chloroform, acetone and Chlorhexidine hydrochloride C22H30Cl2N10.2HCl
acetic acid (100) (7:2:1) to a distance of about 10 cm, and [Same as the namesake monograph]
air-dry the plate. Dry the plate at 1209 C for 30 minutes,
Chlorinated lime [Same as the namesake monograph]
spray evenly a solution of phosphomolybdic acid n-hydrate
in ethanol (95) (1 in 5) immediately, and heat at 1209 C for 2 Chlorinated lime TS Triturate 1 g of chlorinated lime
or 3 minutes: any spot other than the principal spot at the R f with 9 mL of water, and filter. Prepare before use.
value of about 0.4 does not appear.
Chlorine Cl2 A yellow-green gas, having a suffocating
Content: not less than 98.0z. Assay—Weigh accurately
odor. It is heavier than air, and dissolves in water. Prepare
about 0.5 g of chenodeoxycholic acid for thin-layer chroma-
from chlorinated lime with hydrochloric acid. Chlorine from
tography, previously dried under reduced pressure (phospho-
a metal cylinder may be used.
rus (V) oxide) at 809C for 4 hours, and dissolve in 40 mL of
neutralized ethanol and 20 mL of water. Add 2 drops of phe- Chlorine TS Use a saturated solution of chlorine in
nolphthalein TS, and titrate <2.50> with 0.1 mol/L sodium water. Preserve this solution in fully filled, light-resistant,
hydroxide VS. Near the end point add 100 mL of freshly glass-stoppered bottles, preferably in a cold place.
boiled and cooled water, and titrate again.
Chloroacetic acid C2H3ClO2 [K 8899, Special class]
Each mL of 0.1 mol/L sodium hydroxide VS
p-Chloroaniline See 4-chloroaniline
＝ 39.26 mg of C24H40O4
4-Chloroaniline H2NC6H4Cl White, crystals or crystal-
0.5 volz Chicken erythrocyte suspension Centrifuge the
line powder. Freely soluble in ethanol (95) and in acetone,
blood taken from healthy chicken, discard the supernatant
and soluble in hot water.
liquid. To the residue add 0.01 mol/L phosphate buffer so-
Melting point <2.60>: 70 – 729C
lution to make 45 mL, suspend the cells, and centrifuge. Dis-
Residue on ignition <2.44>: not more than 0.1z (1 g).
card the supernatant liquid, and repeat the same procedure 3
times more. Suspend 5 mL of the middle layer of the residue 4-Chlorobenzenediazonium TS Dissolve 0.5 g of 4-chlo-
so obtained in 40 mL of 0.01 mol/L phosphate buffer solu- roaniline in 1.5 mL of hydrochloric acid, and add water to
tion, and centrifuge. Discard the supernatant liquid, suspend make 100 mL. To 10 mL of this solution add 10 mL of so-
3 mL of the middle layer of the residue in 10 mL of 0.01 dium nitrite TS and 5 mL of acetone. Prepare before use.
mol/L phosphate buffer solution, and centrifuge. Discard
p-Chlorobenzene sulfonamide See 4-chlorobenzene sul-
the supernatant liquid, and suspend 2 mL of the middle layer
fonamide.
of the residue in 18 mL of 0.01 mol/L phosphate buffer so-
lution. To 10 mL of this solution add 190 mL of 0.01 mol/L 4-Chlorobenzene sulfonamide ClC6H4SO2NH2 White
phosphate buffer solution, and stir to suspend. to pale yellow, odorless, crystalline powder. Dissolves in ace-
tone.
Chikusetsusaponin IV for thin-layer chromatography
Purity Related substances—Dissolve 0.60 g of 4-chlo-
C47H74O18 White crystalline powder. Freely soluble in
robenzene sulfonamide in acetone to make exactly 300 mL,
methanol and in ethanol (95), and practically insoluble in
and perform the test with 5 mL of this solution as directed in

the Purity (5) under Chlorpropamide: any spot other than C16H18O9 A white powder. Freely soluble in methanol and
the principal spot at the R f value of about 0.5 does not ap- in ethanol (99.5), and sparingly soluble in water. Melting
pear. point: about 2059C (with decomposition).
Purity Related substances—Dissolve 1.0 mg of (E )-
p-Chlorobenzoic acid See 4-chlorobenzoic acid.
chlorogenic acid for thin-layer chromatography in 2 mL of
4-Chlorobenzoic acid ClC6H4COOH White, crystals or methanol, and use this solution as the sample solution. Per-
powder. Sparingly soluble in ethanol (95), slightly soluble in form the test with the sample solution as directed under
chloroform, and practically insoluble in water. Thin-layer Chromatography <2.03>. Spot 10 mL of the sam-
Melting point <2.60>: 238 – 2429C ple solution on a plate of silica gel for thin-layer chromatog-
Content: not less than 99.0z. Assay—Weigh accurately raphy, develop the plate with a mixture of ethyl acetate,
about 0.3 g of 4-chlorobenzoic acid, dissolve in 30 mL of water and formic acid (6:1:1) to a distance of about 10 cm,
neutralized ethanol, and titrate <2.50> with 0.1 mol/L so- and air-dry the plate. Examine under ultraviolet light (main
dium hydroxide VS (indicator: 2 drops of phenolphthalein wavelength: 365 nm): no spot other than the principal spot
TS). with an R f value of about 0.5 appears.
Each mL of 0.1 mol/L sodium hydroxide VS p-Chlorophenol See 4-Chlorophenol.
＝ 15.66 mg of C7H5ClO2
4-Chlorophenol ClC6H4OH Colorless or pale red, crys-
1-Chlorobutane CH3(CH2)3Cl Clear and colorless liq- tals or crystalline mass, having a characteristic odor. Very
uid, miscible with ethanol (95) and with diethyl ether, practi- soluble in ethanol (95), in chloroform, in diethyl ether and in
cally insoluble in water. glycerin, and sparingly soluble in water. Melting point:
Refractive index <2.45> n 20
D : 1.401 – 1.045 about 439C.
Specific gravity <2.56> d 20
20: 0.884 – 0.890 Content: not less than 99.0z. Assay—Weigh accurately
Boiling point <2.57>: about 789C about 0.2 g of 4-chlorophenol, and dissolve in water to make
100 mL. Measure exactly 25 mL of this solution into an
Chlorobutanol C4H7Cl3O [Same as the namesake
iodine flask, add exactly 20 mL of 0.05 mol/L bromine VS
monograph]
and then 5 mL of hydrochloric acid, stopper immediately,
1-Chloro-2,4-dinitrobenzene C6H3(NO2)2Cl Light yel- shake occasionally for 30 minutes, and allow to stand for 15
low, crystals or crystalline powder. minutes. Add 5 mL of a solution of potassium iodide (1 in
Melting point <2.60>: 50 – 549C 5), stopper immediately, shake well, and titrate <2.50> with
Preserve in a light-resistant tight container. 0.1 mol/L sodium thiosulfate VS (indicator: 1 mL of starch
TS). Perform a blank determination.
3?-Chloro-3?-deoxythymidine for liquid chromatography
C10H13N2O4Cl Occurs as a white powder. Each mL of 0.05 mol/L bromine VS
Purity—Dissolve 10 mg of 3?-chloro-3?-deoxythymidine ＝ 3.214 mg of C6H5ClO
for liquid chromatography in the mobile phase to make 100
Preserve in tight, light-resistant containers.
mL. Perform the test with 10 mL of this solution as directed
in the Purity (3) under Zidovudine: a peak is not observed at (2-Chlorophenyl)-diphenylmethanol for thin-layer chro-
the retention time for zidovudine. matography C19H15ClO To 5 g of clotrimazole add 300
mL of 0.2 mol/L hydrochloric acid TS, boil for 30 minutes,
(2-Chloroethyl) diethylamine hydrochloride
cool, and extract with 100 mL of diethyl ether. Wash the
C6H14ClN.HCl White powder.
diethyl ether extract with two 10 mL portions of 0.2 mol/L
Content: not less than 95.0z. Assay—Weigh accurately
hydrochloric acid TS, then with two 10-mL portions of
about 0.2 g of (2-chloroethyl)diethylamine hydrochloride,
water. Shake the diethyl ether extract with 5 g of anhydrous
previously dried at 459C for 3 hours under reduced pressure,
sodium sulfate, and filter. Evaporate the diethyl ether of the
and dissolve in 15 mL of acetic acid (100). To this solution
filtrate, dissolve the residue in 200 mL of methanol by warm-
add 10 mL of a mixture of acetic acid (100) and mercury (II)
ing, and filter. Warm the filtrate, and add gradually 100 mL
acetate TS for nonaqueous titration (5:3), and titrate <2.50>
of water by stirring. Cool in an ice bath, filter the separated
with 0.1 mol/L perchloric acid VS (potentiometric titration).
crystals, and dry in a desiccator (phosphorus (V) oxide) for
Perform a blank determination in the same manner, and
24 hours. A white crystalline powder. Very soluble in
make any necessary correction.
dichloromethane, freely soluble in diethyl ether, soluble in
Each mL of 0.1 mol/L perchloric acid VS methanol, and practically insoluble in water.
＝ 17.21 mg of C6H14ClN.HCl Melting point <2.60>: 92 – 959C
Purity Related substances—Dissolve 10 mg of (2-chlo-
Chloroform CHCl3 [K 8322, Special class]
rophenyl)-diphenylmethanol for thin-layer chromatography
Chloroform, ethanol-free Mix 20 mL of chloroform in dichloromethane to make exactly 20 mL, and perform the
with 20 mL of water, gently shake for 3 minutes, separate test with 10 mL of this solution as directed in the Purity (7)
the chloroform layer, wash the layer again with two 20-mL under Clotrimazole: any spot other than the principal spot
portions of water, and filter it through dry filter paper. To does not appear.
the filtrate add 5 g of anhydrous sodium sulfate, shake for 5
Chloroplatinic acid See hydrogen hexachloroplatinate
minutes, allow the mixture to stand for 2 hours, and filter
(IV) hexahydrate.
through dry filter paper. Prepare before use.
Chloroplatinic acid-potassium iodide TS See hydrogen
Chloroform for water determination See Water Determi-
hexaclhoroplatinate (IV)-potassium iodide TS.
nation <2.48>.
Chloroplatinic acid TS See hydrogen hexachloro-
Chlorogenic acid for thin-layer chromatography See (E )-
platinate (IV) TS.
chlorogenic acid for thin-layer chromatography.
3-Chloro-1,2-propanediol C3H7ClO2 A clear and color-
(E )-Chlorogenic acid for thin-layer chromatography

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
226 General Tests / 9.41 Reagents, Test Solutions JP XVII
less viscous liquid. for 30 minutes, spray evenly a solution of phosphomolybdic
Purity Dissolve 0.20 g of 3-chloro-1,2-propanediol in acid n-hydrate in ethanol (95) (1 in 5) immediately, and heat
100 mL of diethyl ether, and use this solution as the sample at 1209C for 2 or 3 minutes: any spot other than the princi-
solution. Pipet 1 mL of the sample solution, add diethyl pal spot, having R f value of about 0.1, does not appear.
ether to make exactly 100 mL, and use this solution as the Content: not less than 98.0z. Assay—Weigh accurately
standard solution. Perform the test with exactly 5 mL each of about 0.5 g of cholic acid for thin-layer chromatography,
the sample solution and standard solution as directed under previously dried at 809C for 4 hours (in vacuum, phos-
Gas Chromatography <2.02> according to the following con- phorous (V) oxide), dissolve in 40 mL of neutralized ethanol
ditions, and determine each peak area by the automatic in- and 20 mL of water, add 2 drops of phenolphthalein TS, and
tegration method: the total area of the peaks other than 3- titrate with 0.1 mol/L sodium hydroxide VS until immedi-
chloro-1,2-propanediol obtained from the sample solution is ately before the end-point has been reached. Then add 100
not larger than 2 times the peak area obtained from the mL of freshly boiled and cooled water, and continue the
standard solution. titration <2.50>. Perform a blank determination in the same
Operating conditions manner, and make any necessary correction.
Proceed as directed in the operating conditions in the
Each mL of 0.1 mol/L sodium hydroxide VS
Purity (6) under Iohexol except the time span of meas-
＝ 40.86 mg of C24H40O5
urement.
Time span of measurement: About 5 times as long as the Choline chloride [(CH3)3NCH2CH2OH]Cl White crys-
retention time of 3-chloro-1,2-propanediol, beginning after talline powder.
the solvent peak. Melting point <2.60>: 303 – 3059C (with decomposition).
System suitability Water <2.48>: less than 0.1z.
System performance and system repeatability: Proceed as
Chromic acid-sulfuric acid TS Saturate chromium (VI)
directed in the system suitability in the Purity (6) under
oxide in sulfuric acid.
Iohexol.
Test for required detectability: To exactly 5 mL of the Chromium trioxide See chromium (VI) oxide.
standard solution add diethyl ether to make exactly 20 mL.
Chromium trioxide TS See chromium (VI) oxide TS.
Confirm that the peak area of 3-chloro-1,2-propanediol ob-
tained with 5 mL of this solution is equivalent to 20 to 30z Chromium (VI) oxide CrO3 A dark red-purple thin
of that obtained with 5 mL of the standard solution. needle-shaped or inner prism-like crystals, or light masses.
Identification—To 5 mL of a solution (1 in 50) add 0.2
Chlorotrimethylsilane (CH3)3SiCl A colorless or practi-
mL of lead (II) acetate TS: yellow precipitates appearwhich
cally colorless liquid, having a pungent odor. Evolves fumes
does not dissolve on the addition of acetic acid.
in a damp atmosphere. Very soluble in diethyl ether, and
reactable with water or with ethanol. Boiling point: about Chromium (VI) oxide TS Dissolve 3 g of chromium (VI)
589 C. oxide in water to make 100 mL.
Chlorphenesin carbamate for assay C10H12ClNO4 Chromogenic synthetic substrate Equal amount mixture
[Same as the monograph Chlorphenesin Carbamate. When of N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginyl-p-
dried, it contains not less than 99.0z of chlorphenesin car- nitroanilid hydrochloride and N-benzoyl-L-isoleucyl-g-
bamate (C10H12ClNO4).] metoxy glutamyl-glycyl-L-arginyl-p-nitroanilid hydrochlo-
ride. White to pale yellow, masses or powder. It is slightly
Chlorpheniramine maleate C16H19ClN2.C4H4O4 [Same
soluble in water.
as the namesake monograph]
Identification—Perform the test with the solution of chro-
Chlorpromazine hydrochloride for assay mogenic synthetic substrate (1 in 30,000) as directed under
C17H19ClN2S.HCl [Same as the monograph Chlorproma- Untraviolet-visible Spectrophotometry <2.24>: the absorp-
zine Hydrochloride] tion maximum at about 316 nm is observed.
Purity Free 4-nitroaniline: not more than 0.5z.
Chlorpropamide for assay C10H13ClN2O3S [Same as
Loss on drying <2.41>: not more than 5z (0.2 g, reduced
the monograph Chlorpropamide. When dried, it contains
pressure (0.3 kPa), calcium chloride, 30 to 409C, 18 hours).
not less than 99.0z of chlorpropamide (C10H13ClN2O3S).]
Content: not less than 95z and not more than 105z of
Cholesterol C27H45OH [Same as the namesake mono- the labeled amount.
graph]
Chromophore TS for teceleukin Mix 0.1 mL of diluted
Cholesterol benzoate C34H50O2 White crystalline pow- hydrogen peroxide (30) (1 in 20) with 10 mL of 0.2 mol/L
der. Melting point: 145 – 1529
C. citric acid buffer (pH 3.8) containing 0.2 mmol/L 3,3?,5,5?-
tetramethylbenzidine dihydrochloride dehydrate, and use
Cholic acid for thin-layer chromatography C24H40O5
immediately.
White, crystals or crystalline powder. Soluble in acetic acid
(100), sparingly soluble in acetone and in ethanol (95), and Chromotropic acid See disodium chromotropate dihy-
very slightly soluble in water. Melting point: about 1989C. drate.
Purity Related substances—Dissolve 25 mg of cholic
Chromotropic acid TS Dissolve 50 mg of disodium chro-
acid for thin-layer chromatography in acetone to make ex-
motropate dihydrate in the solution prepared by cautiously
actly 250 mL and use this solution as the sample solution.
adding 68 mL of sulfuric acid to 30 mL of water, cooling,
Perform the test with this solution as directed under the
then adding water to make 100 mL. Preserve in light-
Thin-layer Chromatography <2.03>. Spot 10 mL of the sam-
resistant containers.
ple solution on a plate of silica gel for thin-layer chro-
matography. Develop the plate with a mixture of chlo- Chromotropic acid TS, concentrated Suspend 0.5 g of
roform, acetone and acetic acid (100) (7:2:1) to a distance of disodium chromotropate dihydrate in 50 mL of sulfuric acid,
about 10 cm, and air-dry the plate. Dry the plate at 1209C centrifuge, and use the supernatant liquid. Prepare before

use.
Time after injection Mobile phase A Mobile phase B
Chymotrypsinogen for gel filtration molecular mass mar- of sample (min) (volz) (volz)
ker A chymotrypsinogen obtained from bovine spleen. For
gel filtration chromatography. 0 – 30 15 → 100 85 → 0
30 – 40 100 100
Cibenzoline succinate for assay C18H18N2.C4H6O4
[Same as the monograph Cibenzoline Succinate. When
dried, it contains not less than 99.0z of cibenzoline suc- Flow rate: 2.0 mL per minute.
cinate (C18H18N2.C4H6O4) and meets the following require- Time span of measurement: 40 minutes.
ment.] System suitability
Purity Related substances—Dissolve 0.10 g of cibenzo- Test for required detectability: To exactly 1 mL of the
line succinate for assay in 2 mL of methanol, and use this so- standard solution add water to make exactly 30 mL. Con-
lution as the sample solution. Pipet 1 mL of the sample solu- firm that the peak area of cilastatin obtained with 20 mL of
tion, and add methanol to make exactly 100 mL. To exactly this solution is equivalent to 2.3 to 4.5z of that obtained
1 mL of this solution add methanol to make exactly 10 mL, with 20 mL of the standard solution.
and use this solution as the standard solution. Perform the System performance: When the procedure is run with 20
test with these solutions as directed under Thin-layer Chro- mL of the standard solution under the above operating con-
matography <2.03>. Spot 10 mL each of the sample solution ditions: the retention time of cilastatin is about 20 minutes,
and standard solution on a plate of silica gel with fluorescent and the number of theoretical plates and the symmetry fac-
indicator for thin-layer chromatography. Develop the plate tor of the peak of cilastatin are not less than 10,000 and not
with a mixture of ethyl acetate, methanol and ammonia solu- more than 2.5, respectively.
tion (28) (20:3:2) to a distance of about 10 cm, air-dry the System repeatability: When the test is repeated 3 times
plate, and dry at 809C for 30 minutes. After cooling, exa- with 20 mL of the standard solution under the above operat-
mine under ultraviolet light (main wavelength: 254 nm): the ing conditions, the relative standard deviation of the peak
spot other than the principal spot obtained with the sample area of cilastatin is not more than 3.0z.
solution is not more intense than the spot obtained with the Residual solvent—Weigh accurately about 1 g of cilastatin
standard solution. On standing the plate for 30 minutes in ammonium for assay, dissolve in water to make exactly 100
the tank saturated with iodine vapor, the spot other than the mL, and use this solution as the sample solution. Separately,
principal spot with the sample solution is not more intense weigh accurately about 0.10 g of ethanol (99.5), add water to
than the spot with the standard solution. make exactly 100 mL, and use this solution as the standard
solution. Perform the test with exactly 1 mL each of the sam-
Cilastatin ammonium for assay C16H29N3O5S: 375.48 ple solution and standard solution as directed under Gas
A white crystalline powder. Chromatography <2.02> according to the following condi-
Purity Related substances—Dissolve 40 mg of the sub- tions. Determine the peak areas, AT and AS, of ethanol by
stance to be examined in 25 mL of water, and use this the automatic integration method, and calculate the amount
solution as the sample solution. Pipet 3 mL of the sample of ethanol (C2H5OH): not more than 0.5z.
solution, add water to make exactly 100 mL, and use this
solution as the standard solution. Perform the test with Amount (z) of ethanol (C2H5OH)
exactly 20 mL each of the sample solution and standard ＝ MS/MT × AT/AS × 100
solution as directed under Liquid Chromatography <2.01> MS: Amount (mg) of ethanol (99.5) taken
according to the following conditions, and determine the MT: Amount (mg) of cilastatine ammonium for assay
each peak area by the automatic integration method. Sepa- taken
rately, perform the test with 20 mL of water in the same
manner to correct any variance of the peak area caused the Operating conditions
variation of the baseline: the total area of the peaks other Detector: A hydrogen flame-ionization detector.
than cilastatin from the sample solution is not larger than Column: A fused silica column 0.5 mm in inside diameter
1/6 times the peak area of cilastatin from the standard solu- and 30 m in length, coated the inside with 5z diphenyl-95z
tion. dimethylpolysiloxane for gas chromatography in thickness
Operating conditions of 5 mm.
Detector: An ultraviolet absorption photometer (wave- Column temperature: Inject the sample at a constant tem-
length: 210 nm). perature of about 509C, keep on for 150 seconds, then raise
Column: A stainless steel column 4.6 mm in inside diame- to 709 C at the rate of 89C per minute, and keep this for 30
ter and 25 cm in length, packed with octadecylsilanized silica seconds.
gel for liquid chromatography (5 mm in particle diameter). Carrier gas: Helium.
Column temperature: A constant temperature of about Flow rate: Adjust so that the retention time of ethanol is
509 C. about 1 minute.
Mobile phase A: A mixture of diluted phosphoric acid (1 Split ratio: 5:1.
in 1000) and acetonitrile (7:3). System suitability
Mobile phase B: Diluted phosphoric acid (1 in 1000). Test for required detectability: To exactly 1 mL of the
Flowing of mobile phase: Control the gradient by mixing standard solution add water to make exactly 10 mL, and
the mobile phases A and B as directed in the following table. designate this the solution for system suitability test. To ex-
actly 1 mL of the solution for system suitability test add
water to make exactly 10 mL. Confirm that the peak area of
ethanol obtained with 1 mL of this solution is equivalent to 7
to 13z of that obtained with 1 mL of the solution for system
suitability test.
System performance: When the procedure is run with 1 mL

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
228 General Tests / 9.41 Reagents, Test Solutions JP XVII
of the standard solution under the above operating condi- Refractive index <2.45> n 20
D : 1.457 – 1.459
tions, the number of theoretical plates and the symmetry fac- Specific gravity <2.56> d 20
20: 0.920 – 0.930
tor of the peak of ethanol are not less than 1500 and not Purity (1) Related substances (i)—Dissolve 0.20 g of
more than 3.0, respectively. cineol for assay in 10 mL of hexane and use this solution as
System repeatability: When determine the peak area of the sample solution. Perform the test with the sample solu-
ethanol by repeating 6 times with 1 mL of the standard solution as directed under Thin-layer Chromatography <2.03>.
tion under the above operating conditions, the relative stand- Spot 5 mL of the sample solution on a plate of silica gel for
ard deviation of the peak area is not more than 2.0z. thin-layer chromatography, develop the plate with a mixture
Water <2.48>: not more than 0.5z (0.5 g, volumetric titra- of hexane and ethyl acetate (9:1) to a distance of about 10
tion, direct titration). cm, and air-dry. Spray evenly 4-methoxybenzaldehyde-
Residue on ignition <2.44>: not more than 0.5z (1 g). sulfuric acid TS, and heat at 1059C for 5 minutes: any spot
Content: not less than 99.0z of cilastatin ammonium other than the principal spot does not appear.
(C16H29N3O5S), calculated on the anhydrous and ethanol- (2) Related substances (ii)—Dissolve 0.10 g of cineol for
free basis. Assay—Weigh accurately about 0.5 g of cilastatin assay in 25 mL of hexane and use this solution as the sample
ammonium for assay, dissolve in 30 mL of methanol, and solution. Perform the test with 2 mL of the sample solution
add 5 mL of water. Adjust to pH 3.0 with 0.1 mol/L hydro- as directed under Gas Chromatography <2.02> according to
chloric acid TS, and titrate <2.50> with 0.1 mol/L sodium the following conditions. Measure each peak area by the
hydroxide VS from the first equivalence point to the second automatic integration method and calculate the amount of
equivalence point (potentiometric titration). cineol by the area percentage method: it is not less than
99.0z.
Each mL of 0.1 mol/L sodium hydroxide VS
Operating conditions
＝ 37.55 mg of C16H29N3O5S
Proceed the operating conditions in the Assay under Eu-
Cilazapril See cilazapril hydrate. calyptus Oil except detection sensitivity and time span of
measurement.
Cilazapril for assay See cilazapril hydrate for assay.
Detection sensitivity: Measure 1 mL of the sample solution
Cilazapril hydrate C22H31N3O5.H2O [Same as the and add hexane to make 100 mL. Adjust the detection sensi-
namesake monograph] tivity so that the peak height of cineol obtained from 2 mL of
this solution is 40 to 60z of the full scale.
Cilazapril hydrate for assay C22H31N3O5.H2O [Same as
Time span of measurement: About 3 times as long as the
the monograph Cilazapril Hydrate. It contains not less than
retention time of cineol, beginning after the solvent peak.
99.0z of cilazapril (C22H31N3O5), calculated on the anhy-
drous basis.] Cinnamaldehyde for thin-layer chromatography See (E )-
cinnamaldehyde for thin-layer chromatography.
Cinchonidine C19H22N2O White, crystals or crystalline
powder. Soluble in methanol, in ethanol (95) and in chlo- (E )-Cinnamaldehyde for thin-layer chromatography
roform, sparingly soluble in diethyl ether, and practically in- C9H8O A colorless or light yellow liquid, having a charac-
soluble in water. A solution of cinchonidine in ethanol (95) teristic aromatic odor. Very soluble in methanol and in
(1 in 100) is levorotatory. Melting point: about 2079C. ethanol (99.5), and practically insoluble in water.
Content: not less than 98.0z. Assay—Weigh accurately Absorbance <2.24> E 11zcm (285 nm): 1679 – 1943 (5 mg,
about 0.3 g of cinchonidine, dissolve in 20 mL of acetic acid methanol, 2000 mL).
(100), add 80 mL of acetic anhydride, and titrate <2.50> with Purity Related substances—Dissolve 10 mg of (E )-cin-
0.1 mol/L perchloric acid VS (indicator: 3 drops of crystal namaldehyde for thin-layer chromatography in 2 mL of
violet). Perform a blank determination in the same manner. methanol. Perform the test with 1 mL of this solution as
directed in the Identification (3) under Kakkonto Extract: no
Each mL of 0.1 mol/L perchloric acid VS
spot other than the principal spot (R f value is about 0.4)
＝ 14.72 mg of C19H22N2O
appears.
Cinchonine C19H22N2O White, crystals or powder.
Cinnamic acid C9H8O2 White crystalline powder, hav-
Identification—Dissolve 1 g in 20 mL of diluted hydro-
ing a characteristic odor.
chloric acid (1 in 4), and add 2 mL of potassium hexacyano-
Melting point <2.60>: 132 – 1359C
ferrate (II) TS: yellow precipitates appear, which are dis-
solved by heating, and crystals are formed after allowing to (E )-Cinnamic acid for assay C9H8O2 (E )-Cinnamic
cool. acid for thin-layer chromatography. It meets the require-
Purity Cinchonidine and quinine—To 1 g of cinchonine ment of the following (E )-Cinnamic acid for assay 1 or (E )-
add 30 mL of water, add diluted hydrochloric acid (2 in 3) Cinnamic acid for assay 2 (Purity value by quantitative
dropwise until the substance to be tested dissolves, and neu- NMR). The former is used after drying in a desiccator (silica
tralize with ammonia TS. To this solution add 10 mL of a gel) for 24 hours, and the latter is used with correction for its
solution of sodium tartrate dihydrate (1 in 2), boil, and allow amount based on the result obtained in the Assay.
to stand for 1 hour: no precipitates appear. 1) (E )-Cinnamic acid for assay 1
Content: not less than 98.0z. Assay—Weigh accurately Purity Related substances—Conduct this procedure
about 0.3 g of cinchonine, dissolve in 50 mL of acetic acid without exposure to light, using light-resistant vessels. Dis-
(100), and titrate <2.50> with 0.1 mol/L perchloric acid VS solve 10 mg of (E )-Cinnamic acid for assay 1 in 50 mL of the
(potentiometric titration). Perform a blank determination in mobile phase, and use this solution as the sample solution.
the same manner, and make any necessary correction. Pipet 1 mL of the sample solution, add the mobile phase to
make exactly 100 mL, and use this solution as the standard
Each mL of 0.1 mol/L perchloric acid VS
solution. Perform the test with exactly 10 mL each of the
＝ 14.72 mg of C19H22N2O
sample solution and standard solution as directed under
Cineol for assay C10H18O Clear and colorless liquid, Liquid Chromatography <2.01> according to the following
having a characteristic aroma. conditions, and determine each peak area by the automatic

integration method: the total area of the peaks other than Operating conditions
(E )-cinnamic acid obtained with the sample solution is not Apparatus: A nuclear magnetic resonance spectrometer
larger than the peak area of (E )-cinnamic acid obtained with having 1H resonance frequency of not less than 400 MHz.
the standard solution. Target nucleus: 1H.
Operating conditions Digital resolution: 0.25 or lower.
Detector, column, column temperature, mobile phase, and Measuring spectrum range: 20 ppm or upper, including
flow rate: Proceed as directed in the operating conditions in between －5 ppm and 15 ppm.
the Assay (1) under Ryokeijutsukanto Extract. Spinning: off.
Time span of measurement: About 6 times as long as the Pulse angle: 909 .
retention time of (E )-cinnamic acid. 13C decoupling: on.

System suitability Delay time: Repeating pulse waiting time not less than 60
System performance, and system repeatability: Proceed as seconds.
directed in the system suitability in the Assay (1) under Integrating times: 8 or more times.
Ryokeijutsukanto Extract. Dummy scanning: 2 or more times.
Test for required detectability: To exactly measured 1 mL Measuring temperature: A constant temperature between
of the standard solution add the mobile phase to make ex- 209C and 309 C.
actly 20 mL. Confirm that the peak area of (E )-cinnamic System suitability
acid obtained with 10 mL of this solution is equivalent to 3.5 Test for required detectability: When the procedure is run
to 6.5z of that obtained with 10 mL of the standard solu- with the sample solution under the above operating condi-
tion. tions, the S/N of the signal of around d 6.20 ppm is not less
2) (E )-Cinnamic acid for assay 2 (Purity value by quan- than 100.
titative NMR) System performance: When the procedure is run with the
Unity of peak—Conduct this procedure without exposure sample solution under the above operating conditions, the
to light, using light-resistant vessels. Dissolve 1 mg of (E )- signal of around d 6.20 ppm is no overlapped with any obvi-
cinnamic acid for assay 2 in 50 mL of the mobile phase, and ous signal of foreign substance.
use this solution as the sample solution. Perform the test System repeatability: When the test is repeated 6 times
with 10 mL of the sample solution as directed under Liquid with the sample solution under the above operating condi-
Chromatography <2.01> according to the following conditions, the relative standard deviation of the ratio of the reso-
tions, and compare the absorption spectra of at least 3 points nance intensity, A, to that of the internal reference is not
including the top of (E )-cinnamic acid peak and around the more than 1.0z.
two middle peak heights of before and after the top: no
(E )-Cinnamic acid for thin-layer chromatography
difference in form is observed between their spectra.
C9H8O2 White, crystals or crystalline powder, having a
Operating conditions
characteristic aromatic odor. Freely soluble in methanol and
Column, column temperature, mobile phase, and flow
in ethanol (99.5), and practically insoluble in water.
rate: Proceed as directed in the operating conditions in the
Absorbance <2.24> E 11zcm (273 nm): 1307 – 1547 (5 mg
Assay (1) under Ryokeijutsukanto Extract.
dried with silica gel for 24 hours, methanol, 1000 mL).
Detector: A photodiode array detector (wavelength: 273
Melting point <2.60>: 132 – 1369C
nm, measuring range of spectrum: 220 – 400 nm).
Purity Related substances—Conduct this procedure
System suitability
without exposure to light, using light-resistant vessels. Dis-
System performance: Proceed as directed in the system
solve 10 mg of (E )-cinnamic acid for thin-layer chromatog-
suitability in the Assay (1) under Ryokeijutsukanto Extract.
raphy in 5 mL of methanol, and use this solution as the
Assay—Weigh accurately 5 mg of (E )-cinnamic acid for
sample solution. Pipet 1 mL of the sample solution, add
assay 2 and 1 mg of 1,4-BTMSB-d4 for nuclear magnetic
methanol to make exactly 100 mL, and use this solution as
resonance spectroscopy using an ultramicrobalance, dissolve
the standard solution. Proceed the test with 10 mL each of
both in 1 mL of deuterated chloroform for nuclear magnetic
the sample solution and standard solution as directed in the
resonance spectroscopy, and use this solution as the sample
Identification (1) under Ryokeijutsukanto Extract: the spot
solution. Transfer the sample solution into an NMR tube 5
other than the principal spot of around R f 0.5 obtained with
mm in outer diameter, measure 1H-NMR as directed under
the sample solution is not more intense than the spot ob-
Nuclear Magnetic Resonance Spectroscopy <2.21> and Crude
tained with the standard solution.
Drugs Test <5.01> according to the following conditions,
using 1,4-BTMSB-d4 for nuclear magnetic resonance spec- (E )-Cinnamic acid for component determination See
troscopy as the internal reference compound. Calculate the (E )-cinnamic acid for assay.
resonance intensity A (equivalent to 1 hydrogen) of the sig-
Cinobufagin for assay C26H34O6 A white crystalline
nal around d 6.20 ppm assuming the signal of the internal
powder. It is odorless.
reference compound as d 0 ppm.
Absorbance <2.24> E 11zcm (295 nm): 125 – 137 (10 mg,
Amount (z) of (E )-cinnamic acid (C9H8O2) methanol, 250 mL). Use the sample dried in a desiccator
＝ MS × I × P/(M × N ) × 0.6541 (silica gel) for 24 hours for the test.
Purity Related substances—Proceed with 40 mg of
M: Amount (mg) of (E )-cinnamic acid for assay 2 taken
cinobufagin for assay as directed in the Purity under bufalin
MS: Amount (mg) of 1,4-BTMSB-d4 for nuclear magnetic
for assay.
resonance spectroscopy taken
Content: not less than 98.0z. Assay—Weigh accurately
I: Signal resonance intensity A based on the signal reso-
about 10 mg of cinobufagin for assay, previously dried in a
nance intensity of 1,4-BTMSB-d4 for nuclear magnetic
desiccator (silica gel) for 24 hours, dissolve in methanol to
resonance spectroscopy as 18.000
make exactly 10 mL, and use this solution as the sample so-
N: Number of the hydrogen derived from A
lution. Perform the test with 20 mL of the sample solution as
P: Purity (z) of 1,4-BTMSB-d4 for nuclear magnetic
directed under Liquid Chromatography <2.01> according to
resonance spectroscopy

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
230 General Tests / 9.41 Reagents, Test Solutions JP XVII
the following conditions. Determine each peak area by the Clonazepam for assay C15H10ClN3O3 [Same as the
automatic integration method and calculate the amount of monograph Clonazepam]
cinobufagin by the area percentage method.
32D Clone3 cells A cloned cell line established by cultur-
Operating conditions
ing mouse bone marrow origin 32D cell line in the presence
Detector: An ultraviolet absorption photometer (wave-
of G-CSF.
length: 295 nm).
Column: A stainless steel column 4 to 6 mm in inside Clorazepate dipotassium for assay C16H10ClKN2O3.KOH
diameter and 15 to 30 cm in length, packed with octadecyl- [Same as the monograph Clorazepate Dipotassium. When
silanized silica gel for liquid chromatography (5 to 10 mm in dried it contains not less than 99.0z of clorazepate dipotas-
particle diameter). sium (C16H10ClKN2O3.KOH).]
Column temperature: A constant temperature of about
Clotrimazole C22H17ClN2 [Same as the namesake
409 C.
monograph]
Mobile phase: A mixture of water and acetonitrile (1:1).
Flow rate: Adjust so that the retention time of cinobufa- Cloxazolam C17H14Cl2N2O2 [Same as the namesake
gin is about 7 minutes. monograph]
Selection of column: Dissolve 10 mg each of cinobufagin
Cobalt (II) chloride-ethanol TS Dissolve 0.5 g of cobalt
for assay, bufalin for assay and resibufogenin for assay in
(II) chloride hexahydrate, previously dried at 1059C for 2
methanol to make 200 mL. Proceed with 20 mL of this solu-
hours, in ethanol (99.5) to make 100 mL.
tion under the above operating conditions. Use a column
giving elution of bufalin, cinobufagin and resibufogenin in Cobalt (II) chloride hexahydrate CoCl2.6H2O [K 8129,
this order, and clearly dividing each peak. Special class]
Detection sensitivity: Pipet 1 mL of the sample solution,
Cobalt (II) chloride TS Dissolve 2 g of cobalt (II) chlo-
add methanol to make exactly 100 mL, and use this solution
ride hexahydrate in 1 mL of hydrochloric acid and water to
as the standard solution (1). Pipet 1 mL of the standard solu-
make 100 mL (0.08 mol/L).
tion (1), add methanol to make exactly 20 mL, and use this
solution as the standard solution (2). Adjust the detection Cobalt (II) nitrate hexahydrate Co(NO3)2.6H2O
sensitivity so that the peak area of cinobufagin obtained [K 8552, Special class]
from 20 mL of the standard solution (2) can be measured by
Cobaltous chloride See cobalt (II) chloride hexahydrate.
the automatic integration method, and the peak height of
cinobufagin from 20 mL of the standard solution (1) is about Cobaltous nitrate See cobalt (II) nitrate hexahydrate.
20z of the full scale.
Codeine phosphate for assay See codeine phosphate hy-
Time span of measurement: About twice as long as the
drate for assay.
retention time of cinobufagin, beginning after the solvent
peak. Codeine phosphate hydrate for assay
C18H21NO3.H3PO4. 1/2 H2O [Same as the monograph
Cinobufagin for component determination See cinobu-
Codeine Phosphate Hydrate. It contains not less than 99.0z
fagin for assay.
of codeine phosphate (C18H21NO3.H3PO4), calculated on the
Cinoxacin for assay C12H10N2O5 [Same as the mono- anhydrous basis.]
graph Cinoxacin. When dried, it contains not less than
Collodion Clear, colorless, viscous liquid, having a
99.0z of cinoxacin (C12H10N2O5).]
diethyl ether-like odor.
Cisplatin Cl2H6N2Pt [Same as the namesake mono- pH <2.54>: 5.0–8.0
graph] Stir 5 g of collodion while warming, add 10 mL of water
gradually, and dry at 1109C after evaporating to dryness:
Citric acid See citric acid monohydrate.
mass of the residue is 0.250–0.275 g.
Citric acid-acetic acid TS To 1 g of citric acid monohy-
Concentrated chromotropic acid TS See chromotropic
drate add 90 mL of acetic anhydride and 10 mL of acetic
acid, concentrated.
acid (100), and dissolve under shaking.
Concentrated diazobenzenesulfonic acid TS See dia-
Citric acid-acetic anhydride TS To 1 g of citric acid
zobenzenesulfonic acid TS, concentrated.
monohydrate add 50 mL of acetic anhydride, and dissolve
by heating. Prepare before use. Congo red C32H22N6Na2O6S2 [K 8352, Special class]
Citric acid monohydrate C6H8O7.H2O [K 8283, or Congo red TS Dissolve 0.5 g of congo red in 100 mL of a
same as the monograph Citric Acid Hydrate] mixture of water and ethanol (95) (9:1).
Citric acid-phosphate-acetonitrile TS Dissolve 2.1 g of Coomassie brilliant blue G-250 C47H48N3NaO7S2 A
citric acid monohydrate, 13.4 g of dipotassium hydrogen deep violet powder. A solution in ethanol (99.5) (1 in
phosphate and 3.1 g of potassium dihydrogen phosphate in 100,000) exhibits an absorption maxima at a wavelength of
1000 mL of a mixture of water and acetonitrile (3:1). 608 nm.
0.01 mol/L Citric acid TS Dissolve 2.1 g of citric acid Coomassie brilliant blue R-250 C45H44N3NaO7S2 Deep
monohydrate in water to make 1000 mL. blue-purple powder. Odorless.
Content: not less than 50z.
1 mol/L Citric acid TS for buffer solution Dissolve
210.14 g of citric acid monohydrate in water to make 1000 Coomassie brilliant blue TS for interferon alfa Dissolve
mL. 20 mg of Coomassie brilliant blue G-250 in diluted perchlo-
ric acid (43 in 1000) to make 100 mL, and filter. Determine
Clofibrate C12H15ClO3 [Same as the namesake mono-
the absorbance of the filtrate at 465 nm as directed under
graph]
Ultraviolet-visible Spectrophotometry <2.24>, and add

Coomassie brilliant blue G-250 or diluted perchloric acid Each mL of 0.01 mol/L disodium dihydrogen
(43 in 1000) so that the absorbance is 1.3 – 1.5. ethylenediamine tetraacetate VS
＝ 0.9756 mg of Cu(OH)2
Coomassie staining TS Dissolve 125 mg of Coomassie
brilliant blue R-250 in 100 mL of a mixture of water, metha- Copper (II) nitrate trihydrate Cu(NO3)2.3H2O Blue,
nol and acetic acid (100) (5:4:1), and filter. crystals or crystalline powder. Very soluble in water, and
freely soluble in ethanol (99.5).
Copper Cu [K 8660, Special class]
Identification (1) A solution of copper (II) nitrate tri-
Copper (standard reagent) Cu In addition to JIS K hydrate (1 in 10) responds to the Qualitative Tests <1.09> (2)
8005 standard reagent for volumetric analysis, certified ref- for cupric salt.
erence material which can be used for volumetric analysis (2) A solution of copper (II) nitrate trihydrate (1 in 10)
may be used. responds to the Qualitative Tests <1.09> (1) for nitrate.
Purity (1) Iron—Weigh accurately 5.0 g of copper (II)
Copper (II) acetate monohydrate Cu(CH3COO)2.H2O
nitrate trihydrate, add 10 mL of a mixture of water and
Blue-green, crystals or crystalline powder.
nitric acid (2:1), add water to make exactly 100 mL, and use
Identification—(1) Dissolve 1 g of copper (II) acetate
this solution as the sample stock solution. Pipet 20 mL of the
monohydrate in 10 mL of diluted sulfuric acid (1 in 2), and
sample stock solution, add water to make exactly 100 mL,
heat: the odor of acetic acid is perceptible.
and use this solution as the sample solution. Separately,
(2) Dissolve 0.1 g of copper (II) acetate monohydrate in
pipet 20 mL of the sample stock solution, add exactly 3 mL
20 mL of water, and add 3 mL of ammonia solution (28): a
of Standard Iron Solution add water to make exactly 100
dark blue color is developed.
mL, and use this solution as the standard solution. Perform
Copper (II) acetate TS, strong Dissolve 13.3 g of copper the test with the sample solution and standard solution as di-
(II) acetate monohydrate in a mixture of 195 mL of water rected under Atomic Absorption Spectrophotometry <2.23>
and 5 mL of acetic acid. according to the following conditions, and determine the ab-
sorbances, AT and AS, of the sample solution and standard
Copper (II) chloride-acetone TS Dissolve 0.3 g of copper
solution: AT is not greater than (AS － AT) (not more than
(II) chloride dihydrate in acetone to make 10 mL.
0.003z).
Copper (II) chloride dihydrate CuCl2.2H2O [K 8145, Gas: Combustible gas—Acetylene.
Special class] Supporting gas—Air.
Lamp: Iron hollow-cathode lamp.
Copper (II) citrate TS Dissolve 25 g of copper (II) sulfate
Wavelength: 248.3 nm.
pentahydrate, 50 g of citric acid monohydrate and 144 g of
(2) Zinc—Use the sample solution in (1) as the sample
anhydrous sodium carbonate in water to make 1000 mL.
solution. Separately, pipet 20 mL of the sample stock solu-
Copper (II) disodium ethylenediamine tetraacetate tetra- tion in (1), add exactly 5 mL of a solution, prepared by add-
hydrate C10H12CuN2Na2O8.4H2O A blue powder. ing water to exactly 4 mL of Standard Zinc Solution to make
pH <2.54>: 7.0 – 9.0 exactly 10 mL, add water to make exactly 100 mL, and use
Purity Clarity and color of solution—Add 0.10 g of cop- this solution as the standard solution. Perform the test with
per (II) disodium ethylenediamine tetraacetate tetrahydrate the sample solution and standard solution as directed under
to 10 mL of freshly boiled and cooled water: the solution is Atomic Absorption Spectrophotometry <2.23> according to
blue in color and clear. the following conditions, and determine the absorbances, AT
Content: not less than 98.0z. Assay—Weigh accurately and AS, of the sample solution and standard solution: AT is
about 0.45 g of coppor (II) disodium ethylenediamine tetra- not greater than (AS － AT) (not more than 0.005z).
acetate tetrahydrate, and add water to make exactly 100 mL. Gas: Combustible gas—Acetylene.
Pipet 10 mL of this solution, adjust the pH of the mixture to Supporting gas—Air.
about 1.5 by adding 100 mL of water and dilute nitric acid, Lamp: Zinc hollow-cathode lamp.
then add 5 mL of a solution of 1,10-phenanthroline mono- Wavelength: 213.9 nm.
hydrate in methanol (1 in 20), and titrate <2.50> with 0.01 (3) Calcium—Use the sample solution in (1) as the sam-
mol/L bismuth nitrate VS until the color of the solution ple solution. Separately, pipet 20 mL of the sample stock so-
changes from yellow to red (indicator: 2 drops of xylenol lution in (1), add exactly 5 mL of a solution, prepared by
orange TS). adding water to exactly 1 mL of Standard Calcium Solution
to make exactly 10 mL, add water to make exactly 100 mL,
Each mL of 0.01 mol/L bismuth nitrate VS
and use this solution as the standard solution. Perform the
＝ 4.698 mg of C10H12CuN2Na2O8.4H2O
test with the sample solution and standard solution as di-
Copper (II) hydroxide Cu(OH)2 Light blue powder. rected under Atomic Absorption Spectrophotometry <2.23>
Practically insoluble in water. according to the following conditions, and determine the ab-
Content: not less than 95.0z as Cu(OH)2. Assay— sorbances, AT and AS, of the sample solution and standard
Weigh accurately about 0.6 g of Copper (II) hydroxide, and solution: AT is not greater than (AS – AT) (not more than
dissolve in 3 mL of hydrochloric acid and water to make ex- 0.005z).
actly 500 mL. Pipet 25 mL of this solution, add 75 mL of Gas: Combustible gas—Acetylene.
water, 10 mL of a solution of ammonium chloride (3 in 50), Supporting gas—Air or nitrous oxide.
3 mL of diluted ammonia solution (28) (1 in 10) and 0.05 g Lamp: Calcium hollow-cathode lamp.
of murexide-sodium chloride indicator, and titrate <2.50> Wavelength: 422.7 nm.
with 0.01 mol/L disodium dihydrogen ethylenediamine (4) Nickel—Use the sample solution in (1) as the sample
tetraacetate VS until the color of the liquid is changed from solution. Separately, pipet 20 mL of the sample stock solu-
yellow-green to red-purple. tion in (1), add exactly 4 mL of Standard Nickel Solution
and water to make exactly 100 mL, and use this solution as
the standard solution. Perform the test with the sample solu-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
232 General Tests / 9.41 Reagents, Test Solutions JP XVII
tion and standard solution as directed under Atomic Absorp- monograph]
tion Spectrophotometry <2.23> according to the following
Cottonseed oil A refined, nonvolatile fatty oil obtained
conditions, and determine the absorbances, AT and AS, of
from the seed of plants of Gossypium hirsutum Linn áe (Gos-
the sample solution and standard solution: AT is not greater
sypium) or of other similar species. A pale yellow, odorless,
than (AS － AT) (not more than 0.002z).
oily liquid. Miscible with chloroform, with diethyl ether, and
Gas: Combustible gas—Acetylene.
with hexane and with carbon disulfide. Slightly soluble in
Supporting gas—Air.
ethanol (95).
Lamp: Nickel hollow-cathode lamp.
Refractive index <2.45> n 20
D : 1.472 – 1.474
Wavelength: 232.0 nm.
Specific gravity <2.56> d 25
25: 0.915 – 0.921
Content—Not less than 77.0z and not more than 80.0z
Acid value <1.13>: not more than 0.5.
as Cu(NO3)2. Assay—Weigh accurately about 0.6 g of cop-
Saponification value <1.13>: 190 – 198
per (II) nitrate trihydrate, dissolve in water to make exactly
Iodine value <1.13>: 103 – 116
250 mL. Pipet 25 mL of this solution, add 75 mL of water, 6
mL of ammonium chloride solution (1 in 10), and 1 mL of a Cresol CH3C6H4(OH) [Same as the namesake mono-
mixture of water and ammonia solution (28) (10:1), and graph]
titrate <2.50> with 0.01 mol/L disodium dihydrogen ethyl-
m-Cresol CH3C6H4(OH) [K 8305, Special class]
enediamine tetraacetate VS until the color of the solution is
changed from green to red-purple (indicator: 50 mg of p-Cresol C7H 8O [K 8306, Special class]
murexide-sodium chloride indicator).
Cresol red C21H18O5S [K 8308, Special class]
Each mL of 0.01 mol/L disodium dihydrogen
Cresol red TS Dissolve 0.1 g of cresol red in 100 mL of
ethylenediamine tetraacetate VS
ethanol (95), and filter if necessary.
＝ 1.876 mg of Cu(NO3)2
Crystalline trypsin To trypsin obtained from bovine pan-
Copper (II) sulfate CuSO4 [K 8984, First class]
creas gland add an appropriate amount of trichloroacetic
Copper (II) sulfate pentahydrate CuSO4.5H2O acid to precipitate the trypsin, and recrystallize in ethanol
[K 8983, Special class] (95). White to yellowish white, crystals or powder. It is odor-
less. Freely soluble in water and in sodium tetraborate-calci-
Copper (II) sulfate-pyridine TS Dissolve 4 g of copper
um chloride buffer solution (pH 8.0).
(II) sulfate pentahydrate in 90 mL of water, then add 30 mL
Content: not less than 45 FIP Units of trypsin per mg.
of pyridine. Prepare before use.
Assay—(i) Sample solution: Weigh accurately an appropri-
Copper (II) sulfate TS Dissolve 12.5 g of copper (II) sul- ate amount of crystallized trypsin according to the labeled
fate pentahydrate in water to make 100 mL (0.5 mol/L). Units, dissolve in 0.001 mol/L hydrochloric acid TS to pre-
pare a solution containing 50 FIP Units per mL, and use this
Copper (II) sulfate TS, alkaline Dissolve 150 g of potas-
solution as the sample solution. Prepare before use, and
sium bicarbonate, 101.4 g of potassium carbonate and 6.93 g
preserve in ice.
of copper (II) sulfate pentahydrate in water to make 1000
(ii) Apparatus: Use a glass bottle as a reaction reservoir 20
mL.
mm in inside diameter and 50 mm in height, equipped with a
Coptisine chloride for thin-layer chromatography rubber stopper for attachment to a glass/silver-silver chlo-
C19H14NO4Cl Orange-red, crystals or crystalline powder. ride electrode for pH determination, nitrogen-induction tube
Slightly soluble in methanol, and very slightly soluble in and an exhaust port. Fix the reaction reservoir in a ther-
water and in ethanol (99.5). Melting point: about 2609C mostat, and keep the temperature at 25 ± 0.19 C by means
(with decomposition). of a precise thermoregulator.
Identification Determine the absorption spectrum of a (iii) Procedure: Pipet 1.0 mL of N-a-benzoyl-L-arginine ethyl
solution of coptisine chloride for thin-layer chromatography ester TS, transfer to the reaction reservoir, and add 9.0 mL
(1 in 100,000) as directed under Ultraviolet-visible Spectro- of sodium tetraborate-calcium chloride buffer solution (pH
photometry <2.24>: it exhibits maxima between 237 nm and 8.0). Allow to stand in the thermostat for 10 minutes to
241 nm, between 264 nm and 268 nm, between 354 nm and make the temperature of the contents reach to 25 ± 0.19C,
358 nm, and between 452 nm and 462 nm. adjust the pH of the solution to 8.00 by adding dropwise 0.1
Purity Related substances—Dissolve 1 mg of coptisine mol/L sodium hydroxide VS while stirring and passing a
chloride for thin-layer chromatography in 2 mL of metha- current of nitrogen, add exactly 0.05 mL of the sample solu-
nol, and use this solution as the sample solution. Pipet 1 mL tion previously allowed to stand at 25 ± 0.19C, then imme-
of the sample solution, add methanol to make exactly 20 diately add dropwise 0.1 mol/L sodium hydroxide VS by a
mL, and use this solution as the standard solution. Perform 50 mL-micropipet (minimum graduation of 1 mL) while stir-
the test with these solutions as directed under Thin-layer ring to keep the reaction solution at pH 8.00, and read the
Chromatography <2.03>. Spot 1 mL each of the sample solu- amount of 0.1 mol/L sodium hydroxide VS consumed and
tion and standard solution on a plate of silica gel for thin- the reaction time when the pH reached 8.00. Continue this
layer chromatography, develop the plate with a mixture of procedure up to 8 minutes. Separately, transfer 10 mL of so-
methanol, ammonium acetate solution (3 in 10) and acetic dium tetraborate-calcium chloride buffer solution (pH 8.0),
acid (100) (20:1:1) to a distance of about 10 cm, and air-dry and perform a blank determination in the same manner.
the plate. Examine under ultraviolet light (main wavelength: (iv) Calculation: Plot the amount of consumption (mL) of
365 nm): the spot other than the principal spot (R f value is 0.1 mol/L sodium hydroxide VS against the reaction time
about 0.4) obtained from the sample solution is not more in- (minutes), select linear reaction times, t1 and t2, designate the
tense than the spot obtained from the standard solution. corresponding consumption amount of 0.1 mol/L sodium
hydroxide VS as v1 and v2, respectively, and designate mmol
Corn oil [Same as the namesake monograph]
of sodium hydroxide consumed per minute as D (FIP Unit).
Cortisone acetate C23H30O6 [Same as the namesake

v2 － v1 1 of the sample solution

D ( mmol NaOH/min) ＝ ×f×
t2 － t1 10
Storage—Preserve in a cold place.
f: Factor of 0.1 mol/L sodium hydroxide VS
Crystal violet C25H30CIN3.9H2O [K 8294, Special
FIP Units per mL of crystalline trypsin class]
( D1 － D 0 ) × T Crystal violet TS Dissolve 0.1 g of crystal violet 10 mL
＝
L×M of acetic acid (100).
D1: mmol of sodium hydroxide consumed in 1 minute Culture medium for celmoleukin Take a specified
when the sample solution is used amount of RPMI-1640 powdered medium, add water to dis-
D0: mmol of sodium hydroxide consumed in 1 minute solve, and add N-2-hydroxyethylpiperidine-N?-2-ethansul-
when the solution for blank determination is used fonic acid as a buffering agent to a concentration of 0.025
M: Amount (mg) of crystalline trypsin taken mol/L. To 1000 mL of this solution add 0.1 g (potency) of
L: Amount (mL) of the sample solution put in the reac- streptomycin sulfate, 100,000 units of potassium benzyl-
tion reservoir penicillin, and 2 g of sodium hydrogen carbonate, adjust the
T: Total volume (mL) of the sample solution prepared by pH to 7.1 to 7.2 with sodium hydroxide TS, and then steri-
dissolving in 0.001 mol/L hydrochloric acid TS lize by filtration. To this solution add fetal calf serum heated
at 569C for 30 minutes to 20 volz.
One FIP Unit is an amount of enzyme which decomposes
1 mmol of N-a-benzoyl-L-arginine ethyl ester per minute Cu-PAN Prepare by mixing 1 g of 1-(2-pyridylazo)-2-
under the conditions described in the Assay. naphthol (free acid) with 11.1 g of coppor (II) disodium
Storage—Preserve in a cold place. ethylenediamine tetraacetate tetrahydrate. A grayish orange-
yellow, grayish red-brown or light grayish purple powder.
Crystalline trypsin for ulinastatin assay A proteolytic en-
Absorbance—Dissolve 0.50 g of Cu-PAN in diluted 1,4-
zyme prepared from bovine pancreas. White to light yellow
dioxane (1 in 2) to make exactly 50 mL. Pipet 1 mL of this
crystalline powder. Odorless. Sparingly soluble in water, and
solution, add methanol to make exactly 100 mL. Read the
dissolves in 0.001 mol/L hydrochloric acid TS.
absorbance of this solution at 470 nm as directed under Ul-
Content: not less than 3200 trypsin Units per mg. As-
traviolet-visible Spectrophotometry <2.24>, using water as
say—(i) Sample solution: Weigh accurately about 20 mg of
the blank solution: the absorbance is not less than 0.48.
crystalline trypsin for ulinastatin assay, and dissolve in 0.001
Purity Clarity and color of solution—Dissolve 0.50 g of
mol/L hydrochloric acid TS so that each mL of the solution
Cu-PAN in 50 mL of diluted 1,4-dioxane (1 in 2): the solu-
contains about 3000 trypsin Units. Dilute this solution with
tion is clear and yellow-brown.
0.001 mol/L hydrochloric acid TS so that each mL of the so-
lution contains about 40 trypsin Units, and use this solution Cu-PAN TS Dissolve 1 g of Cu-PAN in 100 mL of
as the sample solution. diluted 1,4-dioxane (1 in 2).
(ii) Diluent: Dissolve 4.54 g of potassium dihydrogen phos-
Cupferron C 6 H 9 N 3 O2 [K 8289, Special class]
phate in water to make exactly 500 mL (Solution I). Dissolve
4.73 g of anhydrous disodium hydrogen phosphate in water Cupferron TS Dissolve 6 g of cupferron in water to
to make exactly 500 mL (Solution II). To 80 mL of Solution make 100 mL. Prepare before use.
II add a suitable amount of Solution I to adjust to pH 7.6.
Cupric acetate See copper (II) acetate monohydrate.
(iii) Substrate solution: Dissolve 85.7 mg of N-a-benzoyl-L-
arginine ethyl ester hydrochloride in water to make exactly Cupric acetate TS, strong See copper (II) acetate
100 mL, and use this solution as the substrate stock solution. monohydrate TS, strong.
Pipet 10 mL of the stock solution, add the diluent to make
Cupric carbonate See cupric carbonate monohydrate.
exactly 100 mL, and use this solution as the substrate solu-
tion. The absorbance of the substrate solution determined at Cupric carbonate monohydrate CuCO3.Cu(OH)2.H2O
253 nm as directed under Ultraviolet-visible Spectropho- A blue to blue-green powder. It is insoluble in water, and
tometry <2.24> using water as the blank is between 0.575 and dissolves foamingly in dilute acid. It dissolves in ammonia
0.585. If the absorbance of the substrate solution is not in TS and shows a deep blue color.
this range, adjust with the diluent or the substrate stock solu- Purity (1) Chloride <1.03>: not more than 0.036z.
tion. (2) Sulfate <1.14>: not more than 0.120z.
(iv) Procedure: Pipet 3 mL of the substrate solution, previ- (3) Iron—Dissolve 5.0 g of cupric carbonate monohy-
ously warmed at 25 ± 0.19 C, into a 1-cm quartz cell, add ex- drate in excess ammonia TS and filter. Wash the residue with
actly 0.2 mL of the sample solution, and start the determina- ammonia TS, dissolve in dilute hydrochloric acid, add excess
tion of the absorbance change at 253 nm for 5 minutes at 25 ammonia TS and filter. Wash the residue with ammonia TS,
± 0.19C using a solution prepared by adding exactly 0.2 mL and dry to constant mass: the residue is not more than 10
of 0.001 mol/L hydrochloric acid TS to exactly 3 mL of the mg.
substrate solution as the blank. Determine the difference of
Cupric chloride See copper (II) chloride dihydrate.
the absorbance change per minute, A, when the difference
has been constant for at least 3 minutes. Cupric chloride-acetone TS See copper (II) chloride-
(v) Calculation: Trypsin Units per mg is obtained by use of acetone TS.
the following equation. One trypsin Unit is an amount of the
Cupric sulfate See copper (II) sulfate pentahydrate.
enzyme which gives 0.003 change in absorbance per minute
under the conditions described above. Cupric sulfate, anhydrous See copper (II) sulfate (anhy-
drous).
A
Trypsin Units per mg ＝
0.003 × M Cupric sulfate-pyridine TS See copper (II) sulfate-pyri-
dine TS.
M: Amount (mg) of the substance to be assayed in 0.2 mL

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
234 General Tests / 9.41 Reagents, Test Solutions JP XVII
Cupric sulfate solution, alkaline See copper (II) sulfate line powder. Slightly soluble in methanol, very slightly solu-
solution, alkaline. ble in ethanol (99.5), and practically insoluble in water.
Absorbance <2.24> E 11zcm (422 nm): 1460 – 1700 [dried for
Cupric sulfate TS See copper (II) sulfate TS.
24 hours in a desiccator (in vacuum, silica gel), 2.5 mg,
1 mol/L Cupriethylenediamine TS Put 100 g of copper methanol, 1000 mL.]
(II) hydroxide in a 1-L thick-walled bottle marked a 500-mL Melting point <2.60>: 180 – 1849C
line, and add water to make 500 mL. Connect the bottle with Purity Related substances—(1) Dissolve 4 mg of curcu-
a liquid introducing funnel, a nitrogen introducing glass tube min for assay in 2 mL of methanol, and use this solution as
and a gas removing glass tube. Adjust so that the lower end the sample solution. Pipet 1 mL of this solution, add metha-
of the nitrogen introducing tube is located at about 1.3 cm nol to make exactly 50 mL, and use this solution as the
above of the bottom of the bottle. Introduce the nitrogen for standard solution. Perform the test with these solutions as
about 3 hours to replacing the inside gas by adjusting the directed under Thin-layer chromatography <2.03>. Spot 5 mL
pressure (about 14 kPa) to get a mild bubbling. Then add each of the sample solution and standard solution on a plate
gradually 160 mL of ethylenediamine TS through the funnel of silica gel for thin-layer chromatography. Develop the
while introducing the nitrogen and cooling the bottle with plate with a mixture of dichloromethane and methanol (19:1)
the running water, and replace the funnel with a glass rod to to a distance of about 10 cm, and air-dry the plate. Examine
close tightly. After introducing the nitrogen for further 10 under ultraviolet light (main wavelength: 365 nm): the spots
minutes, replace the gas removing tube with a glass rod to other than the principal spot at the R f value of about 0.5 ob-
close tightly. Keep the inside pressure with the nitrogen to tained from the sample solution are not more intense than
about 14 kPa. After allowing the bottle to stand for about 16 the spot obtained from the standard solution.
hours while occasional shaking, filter the content if neces- (2) Dissolve 1.0 mg of curcumin for assay in 5 mL of
sary using a glass-filter under reducing pressure, and reserve methanol, and use this solution as the sample solution. Pipet
under nitrogen atmosphere. The concentration of copper (II) 1 mL of the sample solution, add methanol to make exactly
ion of this solution is about 1.3 mol/L. Determine the con- 50 mL, and use this solution as the standard solution. Per-
centration of ethylenediamine of this solution X (mol/L) and form the test with exactly 10 mL each of the sample solution
copper (II) ion Y (mol/L) by the following Assays, and and standard solution as directed under Liquid Chromatog-
adjust to that X is 1.96–2.04, Y is 0.98–1.02 and X/Y is raphy <2.01> according to the following conditions. Deter-
1.96–2.04 by adding water, copper (II) hydroxide or ethyl- mine each peak from both solutions by the automatic in-
enediamine TS, then determine X and Y again in the same tegration method: the total area of the peaks other than
manner, and use this solution as the test solution. curcumin obtained from the sample solution is not larger
Assay (1) Ethylenediamine—Pipet 1 mL (V1) of the so- than the peak area of curcumin obtained from the standard
lution to be assayed, add 60 mL of water, and titrate <2.50> solution.
with 0.1 mol/L hydrochloric acid VS (pH Determination Operating conditions
<2.54>; End point is about pH 8.4). Column, column temperature, mobile phase and flow
rate: Proceed as directed in the operating conditions in the
N1 a
X＝ Assay under Turmeric.
V1
Detector: A visible absorption photometer (wavelength:
X: Concentration of ethylenediamine (mol/L) 422 nm).
a: Volume of 0.1 mol/L hydrochloric acid VS consumed Time span of measurement: About 4 times as long as the
for the titration (mL) retention time of curcumin, beginning after the solvent peak.
N1: Concentration of 0.1 mol/L hydrochloric acid VS System suitability
(mol/L) System performance and system repeatability: Proceed as
directed in the system suitability in the Assay under
(2) Copper (II) ion—Pipet 2 mL (V2) of the solution to
Turmeric.
be assayed, add 20 mL of water, about 3 g of potassium
Test for required detectability: Pipet 1 mL of the standard
iodide and 50 mL of 2 mol/L sulfuric acid TS, shake for 5
solution, add methanol to make exactly 20 mL. Confirm
minutes, and titrate <2.50> the liberated iodine with 0.1
that the peak area of curcumin obtained from 10 mL of this
mol/L sodium thiosulfate VS. When the solution turns light
solution is equivalent to 3.5 to 6.5z of that of curcumin ob-
yellow at near the end point add 3 mL of starch TS and 10
tained from 10 mL of the standard solution.
mL of a solution of ammonium thiocyanate (1 in 5), and
then titrate until the blue color disappears. Curcumin for component determination See curcumin
for assay.
N2 b
Y＝
V2 Cyanoacetic acid C3H3NO2 White to light yellow crys-
tals. Very soluble in water.
Y: Concentration of copper (II) ion (mol/L)
Content: not less than 99z. Assay—Weigh accurately
b: Volume of 0.1 mol/L sodium thiosulfate VS consumed
about 300 mg of cyanoacetic acid, add 25 mL of water and
for the titration (mL)
25 mL of ethanol (95) to dissolve, and titrate <2.50> with 0.1
N2: Concentration of 0.1 mol/L sodium thiosulfate VS
mol/L sodium hydroxide VS (potentiometric titration). Per-
(mol/L)
form a blank determination, and make any necessary correc-
Curcumin C21H20O6 A reddish yellow crystalline pow- tion.
der.
Each mL of 0.1 mol/L sodium hydroxide VS
Melting point <2.60>: 180 – 1839 C
＝ 85.06 mg of C3H3NO2
Preserve in a light-resistant tight container.
Cyanocobalamin C63H88CoN14O14P [Same as the
Curcumin TS Dissolve 0.125 g of curcumin in acetic acid
namesake monograph]
(100) to make 100 mL. Prepare before use.
Cyanogen bromide TS To 100 mL of ice-cold water add
Curcumin for assay C21H20O6 Yellow to orange crystal-

1 mL of bromine, shake vigorously, and add ice-cold potas- times as long as the retention time of 1,1-cyclobutanedicar-
sium cyanide TS dropwise until the color of bromine just dis- boxylic acid, beginning after the solvent peak.
appears. Prepare this test solution in a draft chamber before Content: not less than 99.0z. Assay—Dissolve about 30
use. mg of 1,1-cyclobutanedicarboxylic acid, accurately weighed,
On handling this solution, be careful not to inhale its in 50 mL of water, and titrate <2.50> with 0.1 mol/L sodium
vapors, which are very toxic. hydroxide VS (potentiometric titration). Perform the blank
determination in the same manner, and make any necessary
1-Cyanoguanidine NH2C(NH)NHCN A white crystal-
correction.
line powder. Freely soluble in water.
Melting point <2.60>: 209 – 2129C Each mL of 0.1 mol/L sodium hydroxide VS
Loss on drying <2.41>: not more than 0.1z (1 g, 1059C, ＝ 7.207 mg of C6H8O4
3 hours).
Cyclohexane C6H12 [K 8464, Special class]
Nitrogen content <1.08>: 66.0 – 67.3z (after drying).
Cyclohexylamine C6H11NH2 A clear and colorless liq-
Cyanopropylmethylphenylsilicone for gas chromatogra-
uid, having a characteristic amine-like odor. Miscible with
phy Prepared for gas chromatography.
water, with N, N-dimethylformamide and with acetone.
6z Cyanopropylphenyl-94z dimethyl silicone polymer Purity Related substances—Use cyclohexylamine as the
for gas chromatography Prepared for gas chromatog- sample solution. Separately, pipet 1 mL of cyclohexylamine,
raphy. add hexane to make exactly 100 mL, and use this solution as
the standard solution. Perform the test as directed in Thin-
6z Cyanopropyl-6z phenyl-methyl silicone polymer for
layer Chromatography <2.03>. Spot 5 mL each of the sample
gas chromatography Prepaired for gas chromatography.
solution and standard solution on a plate of silica gel for
7z Cyanopropyl-7z phenylmethylsilicone polymer for thin-layer chromatography, develop the plate with a mixture
gas chromatography Prepared for gas chromatography. of ethyl acetate, methanol, ammonia water (28) and cyclo-
hexane (6:2:1:1) to a distance of about 10 cm, and air-dry
Cycloartenyl ferulate for thin-layer chromatography
the plate. Allow the plate to stand in iodine vapor: the spot
C40H58O4 A white to light brown, crystalline powder or
other than the principal spot obtained with the sample solu-
powder. Soluble in acetone, slightly soluble in acetonitrile,
tion is not more intense than the spot obtained with the
and practically insoluble in water and in methanol. Melting
standard solution.
point: about 1559 C.
Identification (1) Determine the absorption spectrum Cyclohexylmethanol C7H14O A liquid having slight
in heptane (1 in 50,000) as directed under Ultraviolet-visible camphor odor. Soluble in ethanol (99.5).
Spectrophotometry <2.24>: it exhibits maxima between 229 Refractive index <2.45> n 20
D : about 1.464
nm and 233 nm, between 289 nm and 293 nm, and between Bioling point <2.57>: about 1859C.
313 nm and 317 nm.
Cyclophosphamide hydrate for assay
(2) Determine the infrared absorption spectrum of cy-
C7H15Cl2N2O2P.H2O [Same as the monograph Cyclophos-
cloartenyl ferulate for thin-layer chromatography as directed
phamide Hydrate. It contains not less than 99.0z of cyclo-
in the potassium bromide disk method under Infrared Spec-
phosphamide hydrate (C7H15Cl2N2O2P.H2O).]
trophotometry <2.25>: it exhibits absorption at the wave
numbers of about 2940 cm－1, 1691 cm－1, 1511 cm－1 and Cyclosporine U C81H109N11O12 White powder.
1270 cm－1. Optical rotation <2.49> [a]20
D : about －1909C (0.1 g,
Purity Related substances—Dissolve 2.0 mg of cycloar- methonol, 20 mL 100 mm).
tenyl ferulate for thin-layer chromatography in 2 mL of ace-
L-Cysteicacid C3H7NO5S White powder.
tone, and use this solution as the sample solution. Pipet 1
Optical rotation <2.49> [a]20
D : ＋7.5 – ＋9.09(1.5 g, water,
mL of the sample solution, add methanol to make exactly
20 mL, 100 mm).
100 mL, and use this solution as the standard solution. Pro-
Melting point <2.60>: about 2609 C.
ceed the test with 5 mL each of the sample solution and
standard solution as directed in the Identification under L-Cysteine hydrochloride See L-cysteine hydrochloride
Brown Rice: the spot other than the principle spot, having monohydrate.
R f value of about 0.4, obtained from the sample solution is
L-Cysteine hydrochloride monohydrate
not more intense than the spot obtained from the standard
HSCH2CH(NH2)COOH.HCl.H2O [K 8470, Special class]
solution.
L-Cystine HOOCCH(NH2)CH2SSCH2CH(NH2)COOH
Cyclobutanecarboxylic acid C5H8O2 A clear and color-
[K 9048, L(－)-Cystine, Special class]
less liquid. Congealing point: －7.59C.
Cytochrome c An oxidase (molecular weight: 8000 –
1,1-Cyclobutanedicarboxylic acid C6H8O4 White crys-
13,000) derived from bovine cardiac muscle.
tals.
Melting point <2.60>: 159 – 1639 C Cytosine C4H5N3O White, crystalline powder or pow-
Purity Related substances—Dissolve 20 mg of 1,1-cyclo- der.
butanedicarboxylic acid in 100 mL of the mobile phase used Absorbance <2.24> E 11zcm (276 nm): not less than 800 (after
in the Purity (1) under Carboplatin, and use this solution as drying, 40 mg, 10,000 mL of 0.1 mol/L hydrochloric acid
the sample solution. Perform the test with 25 mL of the sam- TS).
ple solution as directed in the Purity (1) under Carboplatin.
Dacuronium bromide for thin-layer chromatography
Determine each peak area by the automatic integration
C33H58Br2N2O3 White crystalline powder. Very soluble in
method, and calculate their amounts by the area percentage
water, freely soluble in ethanol (95), and practically insolu-
method: the total amount of the peaks other than 1,1-cyclo-
ble in acetic anhydride. Hygroscopic.
butanedicarboxylic acid is not more than 2z. However, the
Identification—Determine the infrared absorption spec-
time span of measurement for this calculation is about 2

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
236 General Tests / 9.41 Reagents, Test Solutions JP XVII
trum of dacuronium bromide for thin-layer chromatography and add hexane for purity of crude drug to make exactly 100
according to the potassium bromide disk method under In- mL.
frared Spectrophotometry <2.25>: it exhibits the absorptions
o, p?-DDT (1,1,1-Trichloro-2-(2-chlorophenyl)-2-(4-chlo-
at the wave numbers at about 2940 cm－1, 1737 cm－1, 1630
rophenyl)ethane) C14H9Cl5
cm－1, 1373 cm－1, 1233 cm－1 and 1031 cm－1.
Melting point <2.60>: 73 – 759C
Purity Related substances—Dissolve 10 mg of dacuroni-
Purity Related substances—Proceed as directed in the
um bromide for thin-layer chromatography in 2 mL of
Purity of p, p?-DDD.
ethanol (95), and use this solution as the sample solution.
Pipet 1 mL of the sample solution, add ethanol (95) to make p , p?- DDT ( 1 , 1 , 1 - Trichloro - 2 , 2 - bis ( 4 - chlorophenyl)
exactly 100 mL, and use this solution as the standard solu- ethane) C14H9Cl5
tion. Perform the test with 10 mL each of the sample solution Melting point <2.60>: 108 – 1109C
and standard solution as directed in the Purity (2) Related Purity Related substances—Proceed as directed in the
substances under Pancuronium Bromide: the spots other Purity of p, p?-DDD using the following standard solution
than the principal spot from the sample solution do not show (1).
more intense color than the spot from the standard solution. Standard solution (1): Pipet 1 mL of the sample solution,
Water <2.48>: not more than 1.0z (1 g, volumetric titra- and add hexane for purity of crude drug to make exactly 100
tion, direct titration). mL.
Content: not less than 98.0z, calculated on the anhydrous
Decolorized fuchsin TS Add 1 g of fuchsin in 100 mL of
basis. Assay—Weigh accurately about 0.2 g of dacuronium
water, heat at about 509C, then cool with occasional shak-
bromide for thin-layer chromatography, dissolve in 50 mL
ing. After standing for 48 hours, mix and filter. To 4 mL of
of acetic anhydride by warming, and titrate <2.50> with 0.1
the filtration add 6 mL of hydrochloric acid and water to
mol/L perchloric acid VS (potentiometric titration). Per-
make 100 mL. Use after standing for at least 1 hour. Prepare
form a blank determination, and make any necessary correc-
before use.
tion.
n-Decyl trimethylammonium bromide C13H30NBr
Each mL of 0.1 mol/L perchloric acid VS
White powder. Melting point: about 2329 C (with decompo-
＝ 34.53 mg of C33H58Br2N2O3
sition).
p, p?-DDD (2,2-Bis(4-chlorophenyl)-1,1-dichloroethane) Content: not less than 99z. Assay—Weigh accurately
C14H10Cl4 about 0.5 g of n-decyl trimethylammonuim bromide, dis-
Melting point <2.60>: 108 – 1109 C solve in 50 mL of water, and titrate <2.50> with 0.1 mol/L
Purity Related substances—Dissolve 10 mg of p, p?-DDD silver nitrate VS (indicator: 1 mL of potassium chromate
in hexane for purity of crude drug to make exactly 100 mL, TS). Perform a blank determination in the same manner and
pipet 1 mL of this solution, add hexane for purity of crude make any necessary correction.
drug to make exactly 100 mL, and use this solution as the
Each mL of 0.1 mol/L silver nitrate VS
sample solution. Pipet 2 mL of the sample solution, add
＝ 28.03 mg of C13H30NBr
hexane for purity of crude drug to make exactly 100 mL, and
use this solution as the standard solution (1). Perform the 0.005 mol/L n-Decyl trimethylammonium bromide TS
test with exactly 1 mL each of the sample solution and stand- Dissolve 6.94 g of potassium dihydrogen phosphate, 3.22 g
ard solution (1) as directed under Gas Chromatography of disodium hydrogen phosphate dodecahydrate and 1.40 g
<2.02> according to the following conditions, and measure of n-decyl trimethylammonium bromide in water to make
each peak area from these solutions by the automatic in- 1000 mL.
tegration method: the total peak area other than p, p?-DDD
Defibrinated blood of rabbit Transfer 100 mL of blood
from the sample solution is not larger than the peak area of
obtained from rabbit to a flask, put in about 20 glass balls 8
p, p?-DDD from the standard solution (1).
mm in diameter, shake for 5 minutes gently, and filter
Operating conditions
through gauze. Prepare before use.
Proceed the operating conditions in the Purity 4.3. under
Crude Drugs Test <5.01> except detection sensitivity and time Dehydrated ethanol See ethanol (99.5).
span of measurement.
Dehydrated ether See diethyl ether, dehydrated.
Detection sensitivity: Pipet 1 mL of the standard solution
(1), add hexane for purity of crude drug to make exactly 20 Dehydrated pyridine See pyridine, dehydrated.
mL, and use this solution as the standard solution (2).
Dehydrocorydaline nitrate for assay C22H24N2O7 Yel-
Adjust the detection sensitivity so that the peak area of p, p?-
low, crystals or crystalline powder. It is sparingly soluble in
DDD obtained from 1 mL of the standard solution (2) can be
methanol, and slightly soluble in water and in ethanol (99.5).
measured by the automatic integration method, and the peak
Melting point: about 2409C (with decomposition).
height of p, p?-DDD from 1 mL of the standard solution (1) is
Absorbance <2.24> E 11zcm (333 nm): 577 – 642 (3 mg, water,
about 20z of the full scale.
500 mL). Use the sample dried in a desiccator (silica gel) for
Time span of measurement: About twice as long as the
not less than 1 hour for the test.
retention time of p, p?-DDD, beginning after the solvent
Purity (1) Related substances 1—Dissolve 5.0 mg of
peak.
dehydrocorydaline nitrate for assay in 1 mL of a mixture of
p , p?-DDE (2,2-Bis(4-chlorophenyl)-1,1-dichloroethy- water and methanol (1:1), and use this solution as the sample
lene) C14H8Cl4 solution. Pipet 0.5 mL of the sample solution, add a mixture
Melting point <2.60>: 88 – 909C of water and methanol (1:1) to make exactly 50 mL, and use
Purity Related substances—Proceed as directed in the this solution as the standard solution. Perform the test with
Purity of p,p?-DDD using the following standard solution these solutions as directed under Thin-layer Chromatogra-
(1). phy <2.03>. Spot 5 mL of the sample solution and standard
Standard solution (1): Pipet 1 mL of the sample solution, solution on a plate of silica gel for thin-layer chromatogra-

phy. Develop immediately with a mixture of methanol, a so- point: 166 – 1709C.
lution of ammonium acetate (3 in 10) and acetic acid (100) Identification Determine the absorption spectrum of a
(20:1:1) to a distance of about 10 cm, and air-dry the plate. solution of demethoxycurcumin in methanol (1 in 400,000)
Spray Dragendorff's TS on the plate, air-dry, and spray so- as directed under Ultraviolet-visible Spectrophotometry
dium nitrite TS: the spots other than the principal spot from <2.24>: it exhibits a maximum between 416 nm and 420 nm.
the sample solution are not more intense than the spot from Purity Related substances—(1) Dissolve 4 mg of de-
the standard solution. methoxycurcumin in 2 mL of methanol, and use this solu-
(2) Related substances 2—Dissolve 5.0 mg of dehydro- tion as the sample solution. Pipet 1 mL of this solution, add
corydaline nitrate for assay in 10 mL of the mobile phase, methanol to make exactly 20 mL, and use this solution as the
and use this solution as the sample solution. Pipet 1 mL of standard solution. Perform the test as directed under Thin-
the sample solution, add the mobile phase to make exactly layer Chromatography <2.03>. Spot 5 mL each of the sample
100 mL, and use this solution as the standard solution. Per- solution and standard solution on a plate of silica gel for
form the test with exactly 5 mL each of the sample solution thin-layer chromatography. Develop the plate with a mixture
and standard solution as directed under Liquid Chromatog- of dichloromethane and methanol (19:1) to a distance of
raphy <2.01> according to the following conditions, and about 10 cm, and air-dry the plate. Examine under ultravio-
determine each peak area from these solutions by the auto- let light (main wavelength: 365 nm): the spots other than the
matic integration method: the total area of peaks other than principal spot at the R f value of about 0.3 obtained from the
dehydrocorydaline from the sample solution is not larger sample solution are not more intense than the spot obtained
than the peak area of dehydrocorydaline from the standard from the standard solution.
solution. (2) Dissolve 1.0 mg of demethoxycurcumin in 5 mL of
Operating conditions methanol, and use this solution as the sample solution. Pipet
Column, column temperature, mobile phase, and flow 1 mL of the sample solution, add methanol to make exactly
rate: Proceed as directed in the operating conditions in the 20 mL, and use this solution as the standard solution. Per-
Assay under Corydalis Tuber. form the test with exactly 10 mL each of the sample solution
Detector: Ultraviolet absorption photometer (wavelength: and standard solution as directed under Liquid Chromatog-
230 nm). raphy <2.01> according to the following conditions. Deter-
Time span of measurement: About 3 times as long as the mine each peak area from both solutions by the automatic
retention time of dehydrocorydaline, beginning after the integration method: the total area of the peaks other than
peak of nitric acid. demethoxycurcumin obtained from the sample solution is
System suitability not larger than the peak area of demethoxycurcumin ob-
System performance and system repeatability: Proceed as tained from the standard solution.
directed in the system suitability in the Assay under Coryda- Operating conditions
lis Tuber. Column, column temperature, mobile phase and flow
Test for required detectability: To exactly 1 mL of the rate: Proceed as directed in the operating conditions in the
standard solution add the mobile phase to make exactly 20 Assay under Turmeric.
mL. Confirm that the peak area of dehydrocorydaline ob- Detector: A visible absorption photometer (wavelength:
tained from 5 mL of this solution is equivalent to 3.5 to 422 nm).
6.5z of that obtained from 5 mL of the standard solution. Time span of measurement: About 4 times as long as the
retention time of demethoxycurcumin, beginning after the
Dehydrocorydaline nitrate for component determination
solvent peak.
See dehydrocorydaline nitrate for assay.
System suitability
Dehydrocorydaline nitrate for thin-layer chromatography System performance and system repeatability: Proceed as
C22H24N2O7 Yellow, crystals or crystalline powder. Spar- directed in the system suitability in the Assay under
ingly soluble in methanol, and slightly soluble in water and Turmeric.
in ethanol (99.5). Melting point: about 2409C (with decom- Test for required detectability: Pipet 1 mL of the standard
position). solution, and add methanol to make exactly 20 mL. Confirm
Purity Related substances—Dissolve 5.0 mg of dehydro- that the peak area of demethoxycurcumin obtained from 10
corydaline nitrate for thin-layer chromatography in 1 mL of mL of this solution is equivalent to 3.5 to 6.5z of that of de-
a mixture of water and methanol (1:1), and use this solution methoxycurcumin obtained from 10 mL of the standard solu-
as the sample solution. Pipet 0.5 mL of the sample solution, tion.
add a mixture of water and methanol (1:1) to make exactly
N-Demethylerythromycin C36H65NO13 White to light
50 mL, and use this solution as the standard solution. Per-
yellowish white powder.
form the test with these solutions as directed under Thin-
layer Chromatography <2.03>. Spot 5 mL each of the sample N-Demethylroxithromycin C40H74N2O15 White pow-
solution and standard solution on a plate of silica gel for der.
thin-layer chromatography. Develop immediately with a Identification—Determine the infrared absorption spec-
mixture of methanol, a solution of ammonium acetate (3 in trum of a solution of the substance to be tested in chlo-
10) and acetic acid (100) (20:1:1) to a distance of about 10 roform (1 in 20) as directed in the solution method under In-
cm, and air-dry the plate. Examine under ultraviolet light frared Spectrophotometry <2.25> using a 0.1-mm cell made
(main wavelength: 365 nm) and then spray Dragendorff's TS of potassium bromide: it exhibits absorption at the wave
on the plate: the spot other than the principal spot obtained numbers of about 3600 cm－1, 3520 cm－1, 3450 cm－1, 3340
from the sample solution is not more intense than the spot cm－1, 1730 cm－1 and 1627 cm－1.
obtained from the standard solution in either case.
2?-Deoxyuridine for liquid chromatography C9H12N2O5
Demethoxycurcumin C20H18O5 Yellow to orange, crys- White crystalline powder.
talline powder or powder. Sparingly soluble in methanol and Melting point <2.60>: 162 – 1669C
in ethanol (99.5), and practically insoluble in water. Melting Purity—Dissolve 3.0 mg of 2?-deoxyuridine for liquid

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
238 General Tests / 9.41 Reagents, Test Solutions JP XVII
chromatography in diluted methanol (1 in 25) to make 50 Congealing point <2.42>: －2.0 – －5.59C
mL. Perform the test with 10 mL of this solution as directed Refractive index <2.45> n 20
D : 1.390 – 1.398
under Liquid Chromatography <2.01> according to the oper- Specific gravity <2.56> d 20
20: 0.98 – 1.00
ating conditions in the Purity under Idoxuridine Ophthalmic Boiling point <2.57>: 85 – 919C
Solution. Determine each peak area by the automatic in- Purity Clarity of solution—Dissolve 1.0 g of diacetyl in
tegration method to the range about twice the retention time 10 mL of water: the solution is clear.
of 2?-deoxyuridine, and calculate the amount of 2?-deoxyuri- Content: not less than 95.0z. Assay—Weigh accurately
dine by the area percentage method: it shows a purity of not about 0.4 g of diacetyl, add exactly 75 mL of hydroxylamine
less than 98.5z. TS, and heat on a water bath for 1 hour under a reflux con-
Content: not less than 98.5z. Assay—Weigh accurately denser. After cooling, titrate <2.50> the excess hydroxyla-
about 5 mg of 2?-deoxyuridine for liquid chromatography, mine with 0.5 mol/L hydrochloric acid VS until the color of
previously dried in vacuum at 609C for 3 hours, and dissolve the solution changes from blue to yellow-green through
in water to make exactly 250 mL. Pipet 10 mL of this solu- green (indicator: 3 drops of bromophenol blue TS). Perform
tion, dilute with water to make exactly 20 mL. Perform the a blank determination in the same manner.
test with this solution as directed under Ultraviolet-visible
Each mL of 0.5 mol/L hydrochloric acid VS
Spectrophotometry <2.24>, and determine absorbance A at
＝ 21.52 mg of C4H6O2
the maximum wavelength at about 262 nm.
Diacetyl TS Dissolve 1 mL of diacetyl in water to make
Amount (mg) of deoxyuridine (C9H12N2O5)
100 mL, and dilute 5 mL of this solution with water to make
A 100 mL. Prepare before use.
＝ × 5000
447
3,3?-Diaminobenzidine tetrahydrochloride C12H14N4.
Dermatan sulfate Dermatan sulfate is mucopolysaccha- 4HCl Occurs as white to yellow brown, needle-shaped crys-
ride purified from the skin and small intestines of pigs by tals, and is soluble in water.
alkaline extraction, followed by digestion with protease and
2,3-Diaminonaphthalene C10H10N2 Light yellow-brown,
fractionation by alcohol. When cellulose acetate membrane
crystals or powder. Slightly soluble in ethanol (95) and in
electrophoresis of dermatan sulfate is performed and the
diethyl ether, and practically insoluble in water.
membrane is stained in a toluidine blue O solution (1 in 200),
Melting point <2.60>: 193 – 1989C
a single band appears.
Sensitivity—Pipet separately 40 mL each of the selenium
Operation conditions of cellulose acetate membrane elec-
standard solution and diluted nitric acid (1 in 60) as the
trophoresis—
blank solution into beakers, and to these solutions add am-
Cellulose acetate membrane: 6 cm in width and 10 cm in
monia solution (28) to adjust the pH to between 1.8 and 2.2.
length.
Dissolve 0.2 g of hydroxylammonium chloride in each of
Mobile phase: Dissolve 52.85 g of calcium acetate mono-
these solutions under gentle shaking, add 5 mL of 2,3-
hydrate in water to make 1000 mL.
diaminonaphthalene TS, mix by shaking, and allow to stand
Run time: 3 hours (1.0 mA/cm).
for 100 minutes. Transfer these solutions to separators sepa-
Deuterated dimethylsulfoxide for nuclear magnetic reso- rately, rinse the beakers with 10 mL of water, add these rins-
nance spectroscopy (CD3)2SO Prepared for nuclear mag- ings to the separators, extract each with 5.0 mL of cyclo-
netic resonance spectroscopy. hexane by thorough shaking for 2 minutes, and centrifuge
the cyclohexane layers to remove moisture. When the absor-
Deuterated formic acid for nuclear magnetic resonance
bance at 378 nm of cyclohexane extract obtained from
spectroscopy DCOOD Prepared for nuclear magnetic
selenium standard solution is determined using the solution
resonance spectroscopy.
obtained from the blank solution as the reference solution as
Deuterated hydrochloric acid for nuclear magnetic reso- directed under Ultraviolet-visible Spectrophotometry <2.24>,
nance spectroscopy DCl Prepared for nuclear magnetic it is not less than 0.08.
resonance spectroscopy. Selenium standard solution—Weigh accurately 40 mg of
selenium, dissolve in 100 mL of diluted nitric acid (1 in 2), by
Deuterated methanol for nuclear magnetic resonance spec-
heating on water bath if necessary, and add water to make
troscopy CD3OD Prepared for nuclear magnetic reso-
exactly 1000 mL. Pipet 5 mL of this solution, and add water
nance spectroscopy.
to make exactly 200 mL. Pipet 2 mL of this solution, and
Deuterated NMR solvents Prepared for nuclear mag- add diluted nitric acid (1 in 60) to make exactly 50 mL. Pre-
netic resonance spectroscopy. For example: deuterated pare before use. This solution contains 0.04 mg of selenium
dimethylsulfoxide [(CD3)2SO], deuterated pyridine (C5D5N), (Se) per mL.
deuterochloroform (CDCl3), heavy water (D2O), etc.
2,4-Diaminophenol dihydrochloride C6H8N2O.2HCl
Deuterated pyridine for nuclear magnetic resonance spec- Pale yellow-brown to grayish yellow-green crystalline pow-
troscopy C5D5N Prepared for nuclear magnetic reso- der. Freely soluble in water, slightly soluble in ethanol (95),
nance spectroscopy. and practically insoluble in diethyl ether.
Purity Clarity of solution—Dissolve 1.0 g of 2,4-di-
Deuterochloroform for nuclear magnetic resonance spec-
aminophenol dihydrochloride in 20 mL of water: the solu-
troscopy CDCl3 Prepared for nuclear magnetic resonance
tion is clear or a slight turbidity is produced.
spectroscopy.
Loss on drying <2.41>: not more than 0.5z (1 g, 1059C,
Devarda's alloy [K 8653, For Nitrogen analysis] 3 hours).
Residue on ignition <2.44>: not more than 0.5z (1 g).
Diacetyl CH3COCOCH3 A yellow to yellow-green,
Content: not less than 98.0z. Assay—Weigh accurately
clear liquid, having a strong, pungent odor. Miscible with
about 0.2 g of 2,4-diaminophenol dihydrochloride, dissolve
ethanol (95) and with diethyl ether, and freely soluble in
in 50 mL of water, and titrate <2.50> with 0.1 mol/L silver
water.

nitrate VS (potentiometric titration). Perform a blank deter- of sulfaminic acid (1 in 20). Prepare before use.
mination, and make any necessary correction.
Diazo TS Weigh accurately 0.9 g of sulfanilic acid, add
Each mL of 0.1 mol/L silver nitrate VS 0.9 mL of hydrochloric acid and 20 mL of water, and dis-
＝ 9.853 mg of C6H8N2O.2HCl solve by heating. After cooling, filter, and dilute the filtrate
with water to make exactly 100 mL. Pipet 1.5 mL of this so-
2,4-Diaminophenol dihydrochloride TS Dissolve 1 g of
lution, cool in an ice bath, and add exactly 1 mL of sodium
2,4-diaminophenol dihydrochloride and 20 g of sodium bi-
nitrite solution (1 in 20) dropwise, while shaking. Cool in an
sulfite in 100 mL of water, and filter, if necessary.
ice bath for 10 minutes, add cold water to make exactly 50
2,4-Diaminophenol hydrochloride See 2,4-diaminophe- mL. Store in a cold place, and use within 8 hours.
nol dihydrochloride.
Dibasic ammonium phosphate See diammonium hydro-
2,4-Diaminophenol hydrochloride TS See 2,4-diamino- gen phosphate.
phenol dihydrochloride TS.
Dibasic potassium phosphate See dipotassium hydrogen
Diammonium hydrogen citrate C6H14N2O7 [K 8284, phosphate.
Special class]
Dibasic potassium phosphate-citric acid buffer solution
Diammonium hydrogen phosphate (NH4)2HPO4 (pH 5.3) See dipotassium hydrogen phosphate-citric acid
[K 9016, Special class] buffer solution (pH 5.3).
Diazepam for assay C16H13ClN2O [Same as the mono- 1 mol/L Dibasic potassium phosphate TS for buffer solu-
graph, Diazepam. When dried, it contains not less than tion See 1 mol/L dipotassium hydrogen phosphate TS for
99.0z of diazepam (C16H13ClN2O), and meets the additional buffer solution.
following requirement.]
Dibasic sodium ammonium phosphate See ammonium
Purity Related substance—Dissolve 50 mg of diazepam
sodium hydrogen phosphate tetrahydrate.
for assay in 10 mL of water, add methanol to make 100 mL,
and use this solution as the sample solution. Pipet 1 mL of Dibasic sodium phosphate See disodium hydrogen phos-
the sample solution, and add methanol to make exactly 100 phate dodecahydate.
mL. Pipet 2 mL of this solution, add methanol to make ex-
Dibasic sodium phosphate, anhydrous See disodium
actly 20 mL, and use this solution as the standard solution.
hydrogen phosphate.
Perform the test with exactly 10 mL each of the sample solu-
tion and standard solution as directed under Liquid Chroma- Dibasic sodium phosphate, anhydrous, for pH determina-
tography <2.01> according to the following conditions, and tion See disodium hydrogen phosphate for pH determina-
determine each peak area by the automatic integration tion.
method: the area of the peak other than diazepam from the
Dibasic sodium phosphate-citric acid buffer solution (pH
sample solution is not larger than the peak area of diazepam
4.5) See disodium hydrogen phosphate-citric acid buffer
from the standard solution.
solution (pH 4.5).
Operating conditions
Detector, column, column temperature, mobile phase, and Dibasic sodium phosphate-citric acid buffer solution (pH
flow rate: Proceed as directed in the operating conditions in 5.4) See disodium hydrogen phosphate-citric acid buffer
the Assay under Diazepam Tablets. solution (pH 5.4).
Time span of measurement: About 4.5 times as long as the
Dibasic sodium phosphate-citric acid buffer solution (pH
retention time of diazepam, beginning after the solvent peak.
6.0) See disodium hydrogen phosphate-citric acid buffer
System suitability
solution (pH 6.0).
System performance: When the procedure is run with 10
mL of the standard solution under the above operating con- Dibasic sodium phosphate TS See disodium hydrogen
ditions, the number of theoretical plates and the symmetry phosphate TS.
factor of the peak of diazepam are not less than 5000 and
0.05 mol/L Dibasic sodium phosphate TS See 0.05
not more than 1.5, respectively.
mol/L disodium hydrogen phosphate TS.
System repeatability: When the test is repeated 6 times
with 10 mL of the standard solution under the above operat- 0.5 mol/L Dibasic sodium phosphate TS See 0.5 mol/L
ing conditions, the relative standard deviation of the peak disodium hydrogen phosphate TS.
area of diazepam is not more than 2.0z.
Dibekacin sulfate C18H37N5O8.xH2SO4 [Same as the
Diazobenzenesulfonic acid TS Weigh 0.9 g of sulfanilic namesake monograph]
acid, previously dried at 1059C for 3 hours, dissolve it in 10
Dibenz[a,h]anthracene C22H14 Very pale yellow to
mL of dilute hydrochloric acid by heating, and add water to
green-yellow, crystalline powder or powder. Practically in-
make 100 mL. Pipet 3.0 mL of this solution, add 2.5 mL of
soluble in water, in methanol and in ethanol (99.5). Melting
sodium nitrite TS, and allow to stand for 5 minutes while
point: 265 – 2709C.
cooling with ice. Then add 5 mL of sodium nitrite TS and
Identification Perform the test with dibenz[a,h]anthra-
water to make 100 mL, and allow to stand in ice water for 15
cene as directed in the Purity: the mass spectrum of the main
minutes. Prepare before use.
peak shows a molecular ion peak (m/z 278) and a fragment
Diazobenzenesulfonic acid TS, concentrated Weigh 0.2 g ion peak (m/z 139).
of sulfanilic acid, previously dried at 1059C for 3 hours, dis- Purity Related substances—Dissolve 3.0 mg of
solve it in 20 mL of 1 mol/L hydrochloric acid TS by warm- dibenz[a,h]anthracene in methanol to make 100 mL, and use
ing. Cool this solution with ice, and add 2.2 mL of a solution this solution as the sample solution. Perform the test with 1
of sodium nitrite (1 in 25) dropwise under stirring. Allow to mL of the sample solution as directed under Gas Chromatog-
stand in ice water for 10 minutes, and add 1 mL of a solution raphy <2.02> according to the following conditions, and de-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
240 General Tests / 9.41 Reagents, Test Solutions JP XVII
termine each peak area by the automatic integration method. make exactly 20 mL, and use this solution as the sample so-
Calculate the amounts of these peaks by the area percentage lution. Separately, weigh accurately about 8 mg of acetic
method: the total amount of the peaks other than acid (100), add 25 mL of methanol, and add the solution
dibenz[a,h]anthracene is not more than 7.0z. containing 1.02 g of anhydrous disodium hydrogen phos-
Operating conditions phate and 6.80 g of potassium dihydrogen phosphate in 1000
Detector: A mass spectrophotometer (EI). mL of water to make exactly 50 mL. Pipet 5 mL of this solu-
Mass scan range: 15.00 – 300.00. tion, add a mixture of the solution containing 1.02 g of
Time of measurement: 12 – 30 minutes. disodium hydrogen phosphate, anhydrous and 6.80 g of
Column: A fused silica column 0.25 mm in inside diameter potassium dihydrogen phosphate in 1000 mL of water and
and 30 m in length, coated inside with 5z diphenyl-95z methanol (1:1) to make exactly 20 mL, and use this solution
dimethylpolysiloxane for gas chromatography in thickness as the control solution. Perform the test with exactly 20 mL
of 0.25 – 0.5 mm. each of the sample solution and control solution as directed
Column temperature: Inject at a constant temperature of under Liquid Chromatography <2.01> according to the fol-
about 459C, raise the temperature to 2409C at a rate of 409C lowing conditions, and determine each peak area by the
per minute, maintain at 2409C for 5 minutes, raise to 3009C automatic integration method. After making correction for
at a rate of 49C per minute, raise to 3209C at a rate of 109C the peak areas based on the valiance of the base-line and the
per minute, and maintain at 3209C for 3 minutes. peak of acetic acid on the chromatogram obtained with the
Injection port temperature: A constant temperature of sample solution, calculate the amount of N, N?-diben-
about 2509C. zylethylenediamine by the area percentage method.
Interface temperature: A constant temperature of about Operating conditions
3009C. Detector: An ultraviolet absorption photometer (wave-
Carrier gas: Helium. length: 220 nm).
Flow rate: Adjust so that the reaction time of the peak of Column: A stainless steel column 4.6 mm in inside diame-
dibenz[a,h]anthracene is about 27 minutes. ter and 25 cm in length, packed with octadecylsilanized silica
Split ratio: Splitless. gel for liquid chromatography (5 mm in particle diameter).
System suitability Column temperature: A constant temperature of about
Test for required detectability: Pipet 1 mL of the sample 409C.
solution, and add methanol to make exactly 10 mL. Confirm Mobile phase: A mixture of water, methanol and 0.25
that the peak area of dibenz[a,h]anthracene obtained from 1 mol/L potassium dihydrogen phosphate TS (pH 3.5)
mL of this solution is equivalent to 5 to 15z of that of (11:7:2).
dibenz[a,h]anthracene obtained from 1 mL of the standard Flow rate: Adjust so that the retention time of N, N?-
solution. dibenzylethylenediamine is about 4 minutes.
Time span of measurement: About 5 times as long as the
Dibenzyl C14H14 White crystals, freely soluble in
retention time of N, N?-dibenzylethylenediamine.
diethyl ether, soluble in methanol and in ethanol (95), and
System suitability
practically insoluble in water.
System performance: Dissolve an amount of benzyl-
Melting point <2.60> 50 – 549C
penicillin benzathine, equivalent to about 85,000 Units, in 25
Purity Related substances—Dissolve 32 mg of dibenzyl
mL of methanol, add a solution containing 1.02 g of anhy-
in methanol to make exactly 50 mL, and use this solution as
drous disodium hydrogen phosphate and 6.80 g of potassium
the sample solution. Perform the test with 20 mL of the
dihydrogen phosphate in 1000 mL of water to make exactly
sample solution as directed under Liquid Chromatography
50 mL. Pipet 5 mL of this solution, add a mixture of the so-
<2.01> according to the operating conditions in the Assay
lution containing 1.02 g of anhydrous disodium hydrogen
under Vinblastine Sulfate for Injection: any peak other than
phosphate and 6.80 g of potassium dihydrogen phosphate in
dibenzyl does not appear. Adjust the detection sensitivity so
1000 mL of water and methanol (1:1) to make exactly 20
that the peak height of dibenzyl obtained from 20 mL of the
mL. When the procedure is run with 20 mL of this solution
solution prepared by adding methanol to 10 mL of the sam-
under the above operating conditions, N, N?-diben-
ple solution to make 20 mL, is 3 to 5 cm, and the time span
zylethylenediamine and benzylpenicillin are eluted in this
of measurement is about 1.2 times as long as the retention
order with the resolution between these peaks being not less
time of dibenzyl after the solvent peak.
than 20.
N, N?-Dibenzylethylenediamine diacetate System repeatability: When the test is repeated 6 times
C16H20N2.2C2H4O2 A white to slightly pale yellow crystal- with 20 mL of the standard solution under the above operat-
line powder. ing conditions, the relative standard deviation of the peak
Identification—Determine the infrared absorption spec- area of N, N?-dibenzylethylenediamine is not more than
trum of the substance to be examined as directed in the po- 2.0z.
tassium bromide disk method under Infrared Spectropho-
2,6-Dibromo-N-chloro-1,4-benzoquinone monoimine
tometry <2.25>: it exhibits absorption at the wave numbers
C6H2Br2ClNO [K 8491, Special class]
of about 1530 cm－1, 1490 cm－1, 1460 cm－1, 1400 cm－1 and
1290 cm－1. 2,6-Dibromo-N-chloro-1,4-benzoquinone monoimine TS
Content: not less than 99.0z. Assay—Weigh accurately Dissolve 0.5 g of 2,6-dibromo-N-chloro-1,4-benzoquinone
about 25 mg of N, N?-dibenzylethylenediamine diacetate, monoimine in methanol to make 100 mL.
dissolve in 25 mL of methanol, and add a solution contain-
2,6-Dibromo-N-chloro-1,4-benzoquinone monoimine TS,
ing 1.02 g of disodium hydrogen phosphate, anhydrous and
dilute Dissolve 0.2 g of 2,6-dibromo-N-chloro-1,4-benzo-
6.80 g of potassium dihydrogen phosphate in 1000 mL of
quinone monoimine in methanol to make 100 mL.
water to make exactly 50 mL. Pipet 5 mL of this solution,
add a mixture of the solution containing 1.02 g of anhydrous 2,6-Dibromoquinone chlorimide See 2,6-dibromo-N-
disodium hydrogen phosphate and 6.80 g of potassium dihy- chloro-1, 4-benzoquinone monoimine.
drogen phosphate in 1000 mL of water and methanol (1:1) to

2,6-Dibromoquinone chlorimide TS See 2,6-dibromo-N- 2,6-Dichlorophenol-indophenol sodium See 2,6-

chloro-1, 4-benzoquinone monoimine TS. dichloroindophenol sodium dihydrate.
Dibucaine hydrochloride C20H29N3O2.HCl [Same as 2,6-Dichlorophenol-indophenol sodium TS See 2,6-
the namesake monograph] dichloroindophenol sodium TS.
Dibutylamine C8H19N Colorless, clear liquid. 2,6-Dichlorophenol-indophenol sodium TS for titration
Refractive index <2.45> n 20
D 1.415 – 1.419 See 2,6-dichloroindophenol sodium TS for titration.
Density <2.56> (209C): 0.756 – 0.761 g/mL
Diclofenac sodium C14H10Cl2NNaO2 [Same as the
Di-n-butyl ether (C4H9)2O Clear, colorless, water-non- namesake monograph]
miscible liquid.
Dicyclohexyl C12H22
Specific gravity <2.56> d 20
4 : 0.768 – 0.771
Specific gravity <2.56> d 20
20: about 0.864
Di-n-butyl phthalate C6H4(COOC4H9)2 Clear, colorless Boiling point <2.57>: about 2279C
liquid. Melting point <2.60>: about 49C
Purity Related substances—Dissolve 0.5 g of di-n-butyl
N, N?-Dicyclohexylcarbodiimide C13H22N2 Colorless or
phthalate in 50 mL of methanol, and use this solution as the
white, crystals or crystalline mass. Dissolves in ethanol (95),
sample solution. Perform the test with 10 mL of the sample
but decomposes in water to produce a white precipitate.
solution as directed in the Assay under Nicardipine Hydro-
Melting point <2.60>: 35 – 369C
chloride Injection, and determine the peak area by the
automatic integration method. Calculate the amount of di-n- N, N?-Dicyclohexylcarbodiimide-dehydrated ethanol TS
butyl phthalate by the area percentage method: the amount See N, N?-dicyclohexylcarbodiimide-ethanol TS.
of di-n-butyl phthalate is not less than 98.0z, and no peak
N, N?-Dicyclohexylcarbodiimide-ethanol TS Dissolve 6 g
appears at the same position as nicardipine. Adjust the de-
of N, N?-dicyclohexylcarbodiimide in ethanol (99.5) to make
tection sensitivity so that the peak height of di-n-butyl
100 mL.
phthalate obtained from 10 mL of the sample solution is 50
Storage—Preserve in tight containers, in a cold place.
to 100z of the full scale, and measure about 2 times as long
as the retention time of di-n-butyl phthalate, beginning after Dicyclohexyl phthalate C6H4(COOC6H11)2 A white,
the solvent peak. crystalline powder.
Melting point <2.60>: 63 – 669C
1,2-Dichlorobenzene C6H4Cl2 A colorless liquid.
Purity Clarity and color of solution—Dissolve 1.0 g of
Specific gravity <2.56> d 20
4 : 1.306
dicyclohexyl phthalate in 20 mL of ethanol (95): the solution
Boiling point <2.57>: 180 – 1819C
is clear and colorless.
1,2-Dichloroethane ClCH2CH2Cl [K 8465, Special
Dicyclohexylurea C6H11NHCONHC6H11 A white crys-
class]
talline powder, having no odor.
Dichlorofluorescein C20H10Cl2O5 Orange to red-brown Purity Related substances—Dissolve 50 mg of dicyclo-
powder. hexylurea in methanol to make 100 mL. Pipet 10 mL of this
Identification (1) Dissolve 0.1 g in 10 mL of sodium solution, and add methanol to make 100 mL. Pipet 20 mL of
hydroxide TS: the solution is an orange-red color, and red- this solution, add 10 mL of 0.5 mol/L sodium hydroxide TS,
orange precipitates appear by the addition of 10 mL of dilute shake, then add 5 mL of diluted hydrochloric acid (1 in 10),
hydrochloric acid. shake, and use this solution as the sample solution. Perform
(2) Dissolve 0.1 g in 10 mL of sodium hydroxide TS, and the test with 50 mL of the sample solution as directed under
add 40 mL of water: a green-yellow fluorescence is ex- Liquid Chromatography <2.01> according to the following
hibited. conditions, determine the area of each peak by the automatic
integration method, and calculate the amount by the area
Dichlorofluorescein TS Dissolve 0.1 g of dichloro-
percentage method: the total amount of the peaks other than
fluorescein in 60 mL of ethanol (95), add 2.5 mL of 0.1
dicyclohexylurea is not more than 3.0z.
mol/L sodium hydroxide VS, and dilute with water to make
Operating conditions
100 mL.
Detector, column, column temperature, mobile phase, and
2,6-Dichloroindophenol sodium dihydrate flow rate: Proceed as directed in the operating conditions in
C12H6Cl2NNaO2.2H2O [K 8469, Special class] the Purity (4) (ii) under Acetohexamide.
Time span of measurement: About 5 times as long as the
2,6-Dichloroindophenol sodium TS Add 0.1 g of 2,6-
retention time of dicyclohexylurea, beginning after the sol-
dichloroindophenol sodium dihydrate to 100 mL of water,
vent peak.
warm, and filter. Use within 3 days.
System suitability
2,6-Dichloroindophenol sodium TS for titration See the System performance, and system repeatability: Proceed as
monograph Ascorbic Acid Powder. directed in the system suitability in the Purity (4) (ii) under
Acetohexamide.
2,6-Dichloroindophenol sodium-sodium acetate TS Mix
Test for required detectability: To exactly 5 mL of the
equal volumes of 2,6-dichloroindophenol sodium dihydrate
standard solution add water to make exactly 200 mL. Con-
solution (1 in 20) and acetic acid-sodium acetate TS (pH
firm that the peak area of dicyclohexylurea obtained with 50
7.0). Prepare before use.
mL of this solution is equivalent to 1.8 to 3.3z of that ob-
Dichloromethane CH2Cl2 [K 8161, Special class] tained with 50 mL of the standard solution.
2,6-Dichlorophenol C6H4Cl2O White to purplish white Diethanolamine C4H11NO2 Colorless viscous liquid.
crystals. Melting point <2.60>: 27 – 309C
Melting point <2.60>: 65 – 679C Water <2.48>: less than 0.1z.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
242 General Tests / 9.41 Reagents, Test Solutions JP XVII
Diethanolamine hydrochloride See 2,2?-iminodiethanol Operating conditions
hydrochloride. Proceed the operating conditions in the 4. Purity 4.3.
under the Crude Drugs Test <5.01> except detection sensi-
Diethylamine (C2H5)2NH A clear, colorless liquid, hav-
tivity and time span of measurement.
ing an amine-like odor. Miscible with water and with ethanol
Detection sensitivity: Pipet 1 mL of the standard solution
(95). The solution in water is alkaline, and readily absorbs
(1), add hexane for purity of crude drug to make exactly 20
carbon dioxide in air.
mL, and use this solution as the standard solution (2).
Specific gravity <2.56> d 10
4 : 0.702 – 0.708
Adjust the detection sensitivity so that the peak area of g-
Distilling range <2.57>: 54 – 589C; not less than 96 volz.
BHC obtained from 1 mL of the standard solution (2) can be
Content: not less than 99.0z. Assay—Weigh accurately
measured by the automatic integration method, and the peak
about 1.5 g of diethylamine in a flask containing exactly 30
height of g-BHC from 1 mL of the standard solution (1) is
mL of 0.5 mol/L sulfuric acid VS, and titrate <2.50> the
about 20z of the full scale.
excess of sulfuric acid with 1 mol/L sodium hydroxide VS
Time span of measurement: About three times as long as
(indicator: 2 drops of methyl red TS). Perform a blank
the retention time of g-BHC, beginning after the peak of sol-
determination.
vent.
Each mL of 0.5 mol/L sulfuric acid VS
N, N-Diethyl-N?-1-naphthylethylenediamine oxalate
＝ 73.14 mg of (C2H5)2NH
C18H24N2O4 A white crystalline powder.
Diethylene glycol HO(CH2CH2O)2H Colorless and Identification—Determine the infrared absorption spec-
odorless liquid. Miscible with water and with ethanol (95). trum as directed in the potassium bromide disk method
Specific gravity <2.56> d 20
20: 1.118 – 1.120 under Infrared Spectrophotometry <2.25>: it exhibits absorp-
tion at the wave numbers of about 3340 cm－1, 2940 cm－1,
Diethylene glycol adipinate for gas chromatography Pre-
1581 cm－1, 1536 cm－1, 1412 cm－1, 789 cm－1, 774 cm－1 and
pared for gas chromatography.
721 cm－1.
Diethylene glycol dimethyl ether (CH3OCH2CH2)2O Purity Clarity of solution—To 0.1 g add 20 mL of
Clear and colorless liquid, miscible with water. water, and dissolve by warming: the solution is clear.
Specific gravity <2.56> d 20
4 : 0.940 – 0.950
N, N-Diethyl-N?-1-naphthylethylenediamine oxalate-ace-
Distilling range <2.57>: 158 – 1609C, not less than 95
tone TS Dissolve 1 g of N, N-diethyl-N?-1-naphthylethy-
volz.
lenediamine oxalate in 100 mL of a mixture of acetone and
Diethylene glycol monoethyl ether [2-(2-ethoxyethoxy) water (1:1). Prepare before use.
ethanol] C2H5(OCH2CH2)2OH Clear, colorless liquid, of
N, N-Diethyl-N?-1-naphthylethylenediamine oxalate TS
which boiling point is about 2039C. Miscible with water.
Dissolve 1 g of N, N-diethyl-N?-1-naphthylethylenediamine
Refractive index <2.45> n 20
D : 1.425 – 1.429
oxalate in water to make 1000 mL.
Specific gravity <2.56> d 20
20: 0.990 – 0.995
Acid (as CH3COOH): less than 0.01z. Diethyl phthalate C6H4(COOC2H5)2 A colorless, clear
liquid.
Diethylene glycol monoethyl ether for water determination
Refractive index <2.45> n 20
D : 1.500 – 1.505
See Water Determination <2.48>.
Diethyl terephthalate C6H4(COOC2H5)2 White to pale
Diethylene glycol succinate ester for gas chromatography
brownish white, crystalline or mass.
Prepared for gas chromatography.
Melting point <2.60>: 44 – 469C
Diethylene glycol succinate polyester for gas chromatogra- Content: not less than 99z. Assay—Dissolve 0.1 g of
phy Prepared for gas chromatography. diethyl terephthalate in 10 mL of methanol. Perform the test
with 2 mL of this solution as directed under Gas Chromatog-
Diethyl ether C2H5OC2H5 [K 8103, Special class]
raphy <2.02> according to the following conditions, and de-
Diethyl ether, dehydrated C2H5OC2H5 [K 8103, Spe- termine the area of each peak by the automatic integration
cial class. The water content is not more than 0.01z.] method.
Diethyl ether for purity of crude drug C2H5OC2H5 peak area of diethyl terephthalate
Content (z) ＝ × 100
[K 8103, Special class] Use diethyl ether meeting the fol- total of all peak areas
lowing additional specification. Evaporate 300.0 mL of
Operating conditions
diethyl ether for purity of crude drug in vacuum at a temper-
Detector: A hydrogen flame-ionization detector.
ature not higher than 409C, add the diethyl ether to make
Column: A glass tube 4 mm in inside diameter and 2 m in
exactly 1 mL, and use this solution as the sample solution.
length, packed with siliceous earth for gas chromatography,
Separately, dissolve 2.0 mg of g-BHC in hexane for purity of
177- to 250- mm in particle diameter, coated with methylsili-
crude drug to make exactly 100 mL. Pipet 1 mL of this solu-
cone polymer for gas chromatography at the ratio of 10z.
tion, and add hexane for purity of crude drug to make ex-
Column temperature: A constant temperature of about
actly 100 mL. Pipet 2 mL of this solution, add hexane for
2009C.
purity of crude drug to make exactly 100 mL, and use this
Carrier gas: Helium.
solution as the standard solution (1). Perform the test with
Flow rate: Adjust so that the retention time of diethyl
exactly 1 mL each of the sample solution and standard solu-
terephthalate is between 6 and 7 minutes.
tion (1) as directed under Gas Chromatography <2.02> ac-
Time span of measurement: About 5 times as long as the
cording to the following conditions, and determine each
retention time of diethyl terephthalate, beginning after the
peak area by the automatic integration method: the total
solvent peak.
area of peaks other than the solvent peak from the sample
solution is not larger than the peak area of g-BHC from the Difenidol hydrochloride C21H27NO.HCl [Same as the
standard solution (1). namesake monograph]

4,4?-Difluorobenzophenone C13H8F2O A white crystal- Melting point <2.60>: about 1259

C
line powder.
2,7-Dihydroxynaphthalene C10H6(OH)2
Melting point <2.60>: 106 – 1099
C
Purity: not less than 97.0z.
Digitonin C56H92O29 White to whitish, crystals or crys-
2,7-Dihydroxynaphthalene TS Dissolve 0.10 g of 2,7-di-
talline powder.
hydroxynaphthalene in 1000 mL of sulfuric acid, and allow
Optical rotation <2.49> [a]20
D : －47 – －509(2 g dried at
to stand until the yellow color initially developed disappears.
1059C for 2 hours, diluted acetic acid (100) (3 in 4), 50 mL,
If the solution is blackened notably, prepare freshly.
100 mm).
Sensitivity—Dissolve 0.5 g of digitonin in 20 mL of Diisopropylamine [(CH3)2CH]2NH Colorless, clear liq-
ethanol (95) by warming, and add ethanol (95) to make 50 uid, having an amine-like odor. Miscible with water and with
mL. To 0.5 mL of this solution add 10 mL of a solution of ethanol (95). The solution in water is alkaline.
cholesterol in ethanol (95) (1 in 5000), cool to 109C, and Refractive index <2.45> n 20
D : 1.391 – 1.394
allow to stand for 30 minutes while vigorous shaking occa- Specific gravity <2.56> d 20
20: 0.715 – 0.722
sionally: A precipitate is produced.
Diltiazem hydrochloride C22H26N2O4S.HCl [Same as
Digoxin C41H64O14 [Same as the namesake monograph] the namesake monograph]
Dihydrocodeine phosphate for assay C18H23NO3.H3PO4 Diltiazem hydrochloride for assay C22H26N2O4S.HCl
[Same as the monograph Dihydrocodeine Phosphate. It con- [Same as the monograph Diltiazem Hydrochloride.
tains not less than 99.0z of dihydrocodeine phosphate However, when dried, it contains not less than 99.0z of
(C18H23NO3.H3PO4), calculated on the dried basis.] diltiazem hydrochloride (C22H26N2O4S.HCl).]
Dihydroergocristine mesilate for thin-layer chromatogra- Dilute acetic acid See acetic acid, dilute.
phy C35H41N5O5.CH4O3S A pale yellowish white powder.
Dilute bismuth subnitrate-potassium iodide TS for spray-
Freely soluble in methanol, in ethanol (95) and in chlo-
ing Dissolve 10 g of L-tartaric acid in 50 mL of water, and
roform, sparingly soluble in water. Melting point: about
add 5 mL of bismuth subnitrate TS.
1909C (with decomposition).
Purity Related substances—Dissolve 6 mg of dihydro- Dilute bromophenol blue TS See bromophenol blue TS,
ergocristine mesilate for thin-layer chromatography in exact dilute.
100 mL of a mixture of chloroform and methanol (9:1), and
Dilute p-dimethylaminobenzaldehyde-ferric chloride TS
perform the test with 5 mL of this solution as directed in the
See 4-dimethylaminobenzaldehyde-iron (III) chloride TS,
Purity (3) under Dihydroergotoxine Mesilate: any spot other
dilute.
than the principal spot at the R f value around 0.4 does not
appear. Dilute 4-dimethylaminobenzaldehyde-iron (III) chloride
TS See 4-dimethylaminobenzaldehyde-iron (III) chloride
1-[(2R,5S )-2,5-Dihydro-5-(hydroxymethyl)-2-furyl] thy-
TS, dilute.
mine for thin-layer chromatography C10H12N2O4 Occurs
as a white powder. Diluted ethanol See ethanol, diluted.
Purity—Dissolve 0.1 g of 1-[(2R,5S)-2,5-dihydro-5-
Dilute ethanol See ethanol, dilute.
(hydroxymethyl)-2-furyl]thymine for thin-layer chromatog-
raphy in 100 mL of methanol and perform the test as di- Dilute ferric ammonium sulfate TS See ammonium iron
rected in the Purity (2) under Zidovudine: spots other than (III) sulfate TS, dilute.
the principal spot with an R f value of about 0.23 are not ob-
Dilute ferric chloride TS See iron (III) chloride TS,
served.
dilute.
3,4-Dihydro-6-hydroxy-2(1H)-quinolinone C9H9NO2 A
Dilute formaldehyde TS See formaldehyde TS, dilute.
white to light brown, powder or granule. Melting point:
about 2409C (with decomposition). Dilute Giemsa's TS See Giemsa's TS, dilute.
Identification—Determine the infrared absorption spec-
Dilute hydrochloric acid See hydrochloric acid, dilute.
trum as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>: it exhibits absorp- Dilute hydrogen peroxide TS See hydrogen peroxide TS,
tion at the wave numbers of about 3210 cm－1, 1649 cm－1, dilute.
1502 cm－1, 1252 cm－1 and 816 cm－1.
Dilute iodine TS See iodine TS, dilute.
2,4-Dihydroxybenzoic acid C7H6O4 White to pale
Dilute iron-phenol TS See iron-phenol TS, dilute.
brown powder.
Purity Clarity of solution—Dissolve 1.0 g of 2,4-di- Dilute lead subacetate TS See lead subacetate TS, dilute.
hydroxybenzoic acid in 20 mL of ethanol (95): the solution is
Dilute methyl red TS See methyl red TS, dilute.
clear.
Content: not less than 95z. Assay—Weigh accurately Dilute nitric acid See nitric acid, dilute.
about 1 g of 2,4-dihydroxybenzoic acid, dissolve in 50 mL of
Dilute potassium hydroxide-ethanol TS See potassium
ethanol (95) and 50 mL of water, and titrate <2.50> with 0.1
hydroxide-ethanol TS, dilute.
mol/L sodium hydroxide VS.
Dilute sodium hydroxide TS See sodium hydroxide TS,
Each mL of 0.1 mol/L sodium hydroxide VS
dilute.
＝ 15.41 mg of C7H6O4
Dilute sodium pentacyanonitrosylferrate (III)-potassium
1,3-Dihydroxynaphthalene C10H6(OH)2 Crystals or pur-
hexacyanoferrate (III) TS See sodium pentacyanonitro-
ple-brown powder. Freely soluble in water and in ethanol
sylferrate (III)-potassium hexacyanoferrate (III) TS, dilute.
(95).

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
244 General Tests / 9.41 Reagents, Test Solutions JP XVII
Dilute sulfuric acid See sulfuric acid, dilute. nation in the same manner.
Dilute thymol blue TS See thymol blue TS, dilute. Each mL of 0.5 mol/L sulfuric acid VS
＝ 45.08 mg of (CH3)2NH
Dilute vanadium pentoxide TS See vanadium (V) oxide
TS, dilute. 2,6-Dimethylaniline C8H11N A clear liquid. Soluble in
ethanol (95), and sparingly soluble in water. Specific gravity
Dilution fluid for particle counter A fluid used for blood
d 20
20: about 0.98.
dilution.
4-Dimethylaminoantipyrine C13H17N3O Colorless or
Dimedon C8H12O2 White to pale yellow crystalline
white crystals, or a white crystalline powder.
powder.
Purity Related substances—Proceed the test with 5 mL of
Melting point <2.60>: 145 – 1499
C
a solution of 4-dimethylaminoantipyrine (1 in 2000) as di-
Dimenhydrinate for assay C17H21NO.C7H7ClN4O2 rected in the Assay under Cefpiramide Sodium, determine
[Same as the monograph Dimenhydrinate. When dried, each peak area in a range of about 2 times as long as the
it contains not less than 53.8z and not more than 54.9z retention time of 4-dimethylaminoantipyrine, beginning
of diphenhydramine (C17H21NO) and not less than 45.2z after the solvent peak by the automatic integration method,
and not more than 46.1z of 8-chlorotheophylline and calculate the total amount of the peaks other than 4-
(C7H7ClN4O2).] dimethylaminoantipyrine by the area percentage method:
not more than 1.0z.
Dimethoxymethane C3H8O2 Colorless, clear and vola-
tile liquid. Miscible with methanol, with ethanol (95) and (Dimethylamino)azobenzenesulfonyl chloride
with diethyl ether. C14H14ClN3O2S Prepared for amino acid analysis or
biochemistry.
N, N-Dimethylacetamide CH3CON(CH3)2 Clear and col-
orless liquid. p-Dimethylaminobenzaldehyde See 4-dimethylamino-
Specific gravity <2.56> d: 0.938 – 0.945 (Method 3). benzaldehyde.
Boiling point <2.57>: 163 – 1659 C
4-Dimethylaminobenzaldehyde (CH3)2NC6H4CHO
Purity—Perform the test with 3 mL of N, N-dimethyl-
[K 8496, p-Dimethylaminobenzaldechyde, Special class]
acetamide as directed under Gas Chromatography <2.02>
according to the following conditions, determine each peak p-Dimethylaminobenzaldehyde-ferric chloride TS See 4-
area by the automatic integration method, and calculate the dimethylaminobenzaldehyde-iron (III) chloride TS.
amount of N, N-dimethylacetamide by the area percentage
p-Dimethylaminobenzaldehyde-ferric chloride TS, dilute
method: not less than 98.0z.
See 4-dimethylaminobenzaldehyde-iron (III) chloride TS,
Operating conditions
dilute.
Detector: A hydrogen flame-ionization detector.
Column: A fused silica column 0.25 mm in inside diameter p-Dimethylaminobenzaldehyde-hydrochloric acid TS See
and 30 m in length, coated the inside surface 0.5 mm in thick- 4-dimethylaminobenzaldehyde-hydrochloric acid TS.
ness with polyethylene glycol 20 M for gas chromatography.
4-Dimethylaminobenzaldehyde-hydrochloric acid TS
Column temperature: The sample is injected at a constant
Dissolve 1.0 g of 4-dimethylaminobenzaldehyde in 50 mL of
temperature of about 709 C, keep this temperature for 1
hydrochloric acid while cooling, and add 50 mL of ethanol
minute, then raise to 2009 C in a rate of 109C per minute,
(95).
and keep 2009C for 3 minutes.
Carrier gas: Helium. 4-Dimethylaminobenzaldehyde-hydrochloric acid-acetic
Flow rate (linear velocity): About 30 cm per second. acid TS Dissolve 8 g of 4-dimethylaminobenzaldehyde in
Time span of measurement: About 2 times as long as the 50 mL of a mixture of acetic acid (100) and hydrochloric
retention time of N, N-dimethylacetamide. acid (19:1). Prepare before use.
System suitability
4-Dimethylaminobenzaldehyde-iron (III) chloride TS
Test for required detectability: To exactly 1.0 g of N, N-
Dissolve 0.125 g of 4-dimethylaminobenzaldehyde in a cold
dimethylacetamide add acetone to make exactly 100 mL.
mixture of 65 mL of sulfuric acid and 35 mL of water, then
Pipet 5 mL of this solution, and add acetone to make exactly
add 0.05 mL of iron (III) chloride TS. Use within 7 days.
50 mL. Confirm that the peak area of N, N-dimethyl-
acetamide obtained from 3 mL of this solution is equivalent 4-Dimethylaminobenzaldehyde-iron (III) chloride TS,
to 40 to 60z of the full-scale. dilute To 80 mL of water add carefully 100 mL of 4-
System repeatability: When the test is repeated with 3 mL dimethylaminobenzaldehyde-iron (III) chloride TS and 0.15
of N, N-dimethylacetamide under the above operating condi- mL of iron (III) chloride TS, while cooling with ice.
tions, the relative standard deviation of the peak area of
p-Dimethylaminobenzaldehyde TS See 4-dimethylamino-
N, N-dimethylacetamide is not more than 2.0z.
benzaldehyde TS.
Water <2.48>: not more than 0.2z (0.1 g, Coulometric
titration). 4-Dimethylaminobenzaldehyde TS Dissolve 10 g of 4-
dimethylaminobenzaldehyde in a cold mixture of 90 mL of
Dimethylamine (CH3)2NH Colorless, clear liquid, hav-
sulfuric acid and 10 mL of water. Prepare before use.
ing amine-like, characteristic odor. It is miscible with water
and with ethanol (99.5). It is alkaline. p-Dimethylaminobenzaldehyde TS for spraying See 4-
Specific gravity <2.56> d 20
20: 0.85 – 0.93 dimethylaminobenzaldehyde TS for spraying.
Content: 38.0 – 45.0z. Assay—Weigh accurately about
4-Dimethylaminobenzaldehyde TS for spraying
1 g of dimethylamine, transfer to a flask containing exactly
Dissolve 1.0 g of 4-dimethylaminobenzaldehyde in 20 mL of
20 mL of 0.5 mol/L sulfuric acid VS, and titrate <2.50> the
dilute sulfuric acid. Prepare before use.
excess sulfuric acid with 1 mol/L sodium hydroxide VS (in-
dicator: 2 drops of methyl red TS). Perform a blank determi- p-Dimethylaminobenzylidene rhodanine See 4-dimethyl-

aminobenzylidene rhodanine. in ethanol (95) to make 100 mL.

4-Dimethylaminobenzylidene rhodanine C12H12N2OS2 Dimethyl malonate C5H8O4 Clear, colorless or pale yel-
[K 8495, Special class] low liquid.
Specific gravity <2.56> d 20
4 : 1.152 – 1.162
p-Dimethylaminobenzylidene rhodanine TS See 4-
Water <2.48>: not more than 0.3z.
dimethylaminobenzylidene rhodanine TS.
Residue on ignition <2.44>: not more than 0.1z.
4-Dimethylaminobenzylidene rhodanine TS Dissolve 20
N, N-Dimethyl-n-octylamine C10H23N Colorless liquid.
mg of 4-dimethylaminobenzylidene rhodanine in acetone to
Refractive index <2.45> n 20
D : 1.424
make 100 mL.
N, N-Dimethyl-p-phenylenediammonium dichloride
p-Dimethylaminocinnamaldehyde See 4-dimethylamino-
H2NC6H4N(CH3)2.2HCl [K 8193, N, N-Dimethyl-p-phenyl
cinnamaldehyde.
enediammonium dichloride, Special class]
4-Dimethylaminocinnamaldehyde C11H13NO Orange,
N, N-Dimethyl-p-phenylenediammonium hydrochloride
crystals or crystalline powder, having a characteristic odor.
See N, N-dimethyl-p-phenylenediamine dichloride.
Freely soluble in dilute hydrochloric acid, sparingly soluble
in ethanol (95) and in diethyl ether, and practically insoluble Dimethyl phthalate C10H10O4 A colorless, clear liquid,
in water. having a slight aroma.
Melting point <2.60>: 140 – 1429 C Refractive index <2.45> n 20
D : 1.513 – 1.517
Purity Clarity of solution—Dissolve 0.20 g of 4- Specific gravity <2.56> d 20
20: 1.191 – 1.196
dimethylaminocinnamaldehyde in 20 mL of ethanol (95): the
Dimethylpolysiloxane for gas chromatography Prepared
solution is clear.
for gas chromatography.
Loss on drying <2.41>: not more than 0.5z (1 g, 1059C,
2 hours). Dimethylsulfoxide (CH3)2SO [K 9702, Special class]
Residue on ignition <2.44>: not more than 0.1z (1 g).
Dimethylsulfoxide for ultraviolet-visible spectrophotome-
Nitrogen content <1.08>: 7.8 – 8.1z (1059C, 2 hours, after
try (CH3)2SO Colorless crystals or clear colorless liquid,
drying).
having a characteristic odor. It is highly hygroscopic.
p-Dimethylaminocinnamaldehyde TS See 4-dimethyl Congealing point <2.42>: not less than 18.39C.
aminocinnamaldehyde TS. Purity—Read absorbance of dimethylsulfoxide for ultra-
violet-visible spectrophotometry, immediately after saturat-
4-Dimethylaminocinnamaldehyde TS Before use, add 1
ing with nitrogen, using water as the blank as directed under
mL of acetic acid (100) to 10 mL of a solution of 4-
Ultraviolet-visible Spectrophotometry <2.24>: its value is not
dimethylaminocinnamaldehyde in ethanol (95) (1 in 2000).
more than 0.20 at 270 nm, not more than 0.09 at 275 nm,
Dimethylaminophenol (CH3)2NC6H4OH Dark purple, not more than 0.06 at 280 nm, and not more than 0.015 at
crystals or crystalline mass. 300 nm. It exhibits no characteristic absorption between 260
Melting point <2.60>: 859C nm and 350 nm.
Water <2.48>: not more than 0.1z.
Dimethylaniline See N, N-dimethylaniline.
2,6-Dimethyl-4-(2-nitrosophenyl)3,5-pyridinedicarboxylic
N,N-Dimethylaniline C6H5N(CH3)2 Colorless or light
acid dimethyl ester for thin-layer chromatography
yellow liquid, having a characteristic odor.
C17H16N2O5 Irradiate xenon light at 50,000 lx of illumina-
Specific gravity <2.56> d 20
20: 0.955 – 0.960
tion for 8 hours to a methanol solution of nifedipine (1 in
Distilling range <2.57>: 192 – 1959C, not less than 95
100), and evaporate the methanol on a water bath.
volz.
Recrystallize the residue 4 times from 1-propanol, and dry in
Dimethylformamide See N, N-dimethylformamide. a desiccator (in vacuum, phosphorus pentoxide). Pale blue
crystals. Very soluble in chloroform, freely soluble in ace-
N,N-Dimethylformamide HCON(CH3)2 [K 8500, Spe-
tone, and practically insoluble in water.
cial class]
Melting point <2.60>: 93 – 959C
N,N-Dimethylformamide for liquid chromatography Content: not less than 99.0z. Assay—Weigh accurately
HCON(CH3)2 [K 8500, N, N-Dimethylformamide, Special about 0.4 g of 2,6-dimethyl-4-(2-nitrosophenyl)-3,5-
class] Read absorbance as directed under Ultraviolet-visible pyridinedicarboxylic acid dimethyl ester for thin-layer chro-
Spectrophotometry <2.24> (in a 1-cm cell, using water as the matography, dissolve in 70 mL of acetic acid (100), and
blank): the absorbance is not more than 0.60 at 270 nm, not titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio-
more than 0.15 at 280 nm, and not more than 0.05 at 300 metric titration). Perform a blank determination in the same
nm. manner.
Dimethylglyoxime C4H8N2O2 [K 8498, Special class] Each mL of 0.1 mol/L perchloric acid VS
＝ 32.83 mg of C17H16N2O5
Dimethylglyoxime-thiosemicarbazide TS Solution A:
Dissolve 0.5 g of dimethylglyoxime in hydrochloric acid to 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium
make 100 mL. Prepare before use. Solution B: Dissolve 0.1 g bromide C18H16BrN5S Yellow crystals. Melting point:
of thiosemicarbazide in 50 mL of water with the acid of about 1959C (with decomposion).
warming if necessary, and add diluted hydrochloric acid
3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium
(1 in 2) to make 100 mL. Prepare before use.
bromide TS Dissolve 5 g of 3-(4,5-dimethylthiazole-2-yl)-
Mix 10 mL each of solution A and solution B, add diluted
2,5-diphenyl-2H-tetrazolium bromide in phosphate-buffered
hydrochloric acid (1 in 2) to make 100 mL, and allow the
sodium chloride TS to make 1000 mL.
mixture to stand for 1 hour. Use within 24 hours.
Dimorpholamine for assay (C20H38N4O4) [Same as the
Dimethylglyoxime TS Dissolve 1 g of dimethylglyoxime

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
246 General Tests / 9.41 Reagents, Test Solutions JP XVII
monograph Dimorpholamine. When dried, it contains not 1,4-Dioxane C 4 H 8 O2 [K 8461, Special class]
less than 99.0z of dimorpholamine (C20H38N4O4).]
Diphenhydramine C17H21NO [Same as the namesake
2,2?-dinaphthylether C20H14O White crystals. monograph]
Melting point <2.60>: 102 – 1079
C
Diphenhydramine tannate [Same as the namesake mono-
m-Dinitrobenzene See 1,3-dinitrobenzene. graph]
1,2-Dinitrobenzene C6H4(NO2)2 Occurs as yellowish Diphenyl C12H10 White, crystals or crystalline powder,
white to brownish yellow, crystals or a crystalline powder. having a characteristic odor. Freely soluble in acetone and in
Identification—Determine the infrared absorption spec- diethyl ether, soluble in ethanol (95), and practically insolu-
trum of 1,2-dinitrobenzene as directed in the paste method ble in water.
under Infrared Spectrophotometry <2.25>: it exhibits absorp- Melting point <2.60>: 68 – 729C
tion at the wave numbers of about 3100 cm－1, 1585 Purity—Dissolve 0.1 g of diphenyl in 5 mL of acetone and
cm－1, 1526 cm－1, 1352 cm－1, and 793 cm－1. use this solution as the sample solution. Perform the test
Melting point <2.60>: 116 – 1199C with 2 mL of the sample solution as directed under Gas Chro-
matography <2.02> according to the following conditions.
1,3-Dinitrobenzene C6H4(NO2)2 Light yellow to red-
Measure each peak area by the automatic integration method
dish-yellow, crystals or crystalline powder.
and calculate the amount of diphenyl by the area percentage
Melting point <2.60>: 88 – 929C.
method: it shows the purity of not less than 98.0z.
Preserve in a light-resistant tight container.
Operating conditions
m-Dinitrobenzene TS See 1,3-dinitrobenzene TS. Detector: A hydrogen flame-ionization detector.
Column: A glass tube about 3 mm in inside diameter and
1,3-Dinitrobenzene TS Dissolve 1 g of 1,3-dinitroben-
about 2 m in length, packed with 150 to 180 mm mesh
zene in 100 mL of ethanol (95). Prepare before use.
siliceous earth for gas chromatography coated with 10z of
m-Dinitrobenzene TS, alkaline See 1,3-dinitrobenzene polyethylene glycol 20 M for thin-layer chromatography.
TS, alkaline. Column temperature: A constant temperature of about
1809C.
1,3-Dinitrobenzene TS, alkaline Mix 1 mL of tetra-
Carrier gas: Nitrogen.
methylammonium hydroxide and 140 mL of ethanol (99.5),
Flow rate: Adjust so that the retention time of diphenyl is
titrate a part of the mixture with 0.01 mol/L hydrochloric
about 8 minutes.
acid VS, and dilute the remainder with ethanol (99.5) to give
Detection sensitivity: Adjust the detection sensitivity so
a 0.008 mol/L solution. Before use, mix 40 mL of this solu-
that the peak height of diphenyl obtained from 2 mL of the
tion with 60 mL of a solution of 1,3-dinitrobenzene in ben-
slution prepared by adding acetone to 1.0 mL of the sample
zene (1 in 20).
solution to make 100 mL, is 5 to 15z of the full scale.
2,4-Dinitrochlorobenzene See 1-chloro-2, 4-dinitroben- Time span of measurement: About 3 times as long as the
zene. retention time of diphenyl, beginning after the solvent peak.
2,4-Dinitrofluorobenzene See 1-fluoro-2, 4-dinitroben- Diphenylamine (C6H5)2NH [K 8487, Special class]
zene.
Diphenylamine-acetic acid TS Dissolve 1.5 g of
2,4-Dinitrophenol C6H3OH(NO2)2 Yellow, crystals or diphenylamine in 1.5 mL of sulfuric acid and acetic acid
crystalline powder. (100) to make 100 mL.
Melting point <2.60>: 110 – 1149
C
Diphenylamine-acetic acid (100) TS See diphenilamine-
2,4-Dinitrophenol TS Dissolve 0.5 g of 2,4-dinitrophenol acetic acid TS.
in 100 mL of ethanol (95).
Diphenylamine TS Dissolve 1 g of diphenylamine in 100
2,4-Dinitrophenylhydrazine (NO2)2C6H3NHNH2 mL of sulfuric acid. Use the colorless solution.
[K 8480, Special class]
9,10-Diphenylanthracene C26H18 Yellow crystalline
2,4-Dinitrophenylhydrazine-diethylene glycol dimethyl powder. Soluble in diethyl ether, and practically insoluble in
ether TS Dissolve 3 g of 2,4-dinitrophenylhydrazine in 100 water.
mL of diethylene glycol dimethyl ether while heating, cool, Melting point <2.60>: about 2489 C
and filter if necessary.
1,4-Diphenylbenzene C18H14 White scaly crystals, hav-
2,4-Dinitrophenylhydrazine-ethanol TS Dissolve 1.5 g of ing a slight aromatic odor. It is freely soluble in ethanol
2,4-dinitrophenylhydrazine in a cold mixture of 10 mL of (99.5), and slightly soluble in water.
sulfuric acid and 10 mL of water, then add a mixture of 1 Identification—Determine the infrared absorption spec-
volume of aldehyde-free ethanol and 3 volumes of water to trum as directed in the potassium bromide disk method
make 100 mL, and filter if necessary. under Infrared Spectrophotometry <2.25>: it exhibits absorp-
tion at the wave numbers of about 3050 cm－1, 3020 cm－1,
2,4-Dinitrophenylhydrazine TS Dissolve 1.5 g of 2,4-
1585 cm－1, 1565 cm－1, 1476 cm－1, 1450 cm－1, 995 cm－1,
dinitrophenylhydrazine in a cold mixture of 10 mL of sulfu-
834 cm－1, 740 cm－1 and 680 cm－1.
ric acid and 10 mL of water, then add water to make 100
mL, and filter if necessary. Diphenylcarbazide See 1,5-diphenilcarbonohydrazide.
Dinonyl phthalate C6H4(COOC9H19)2 Colorless to pale Diphenylcarbazide TS See 1,5-diphenilcarbonohydrazide
yellow, clear liquid. TS.
Specific gravity <2.56> d 20
20: 0.967 – 0.987
Diphenylcarbazone C6H5N2CON2H2C6H5 A yellowish-
Acid value <1.13>: not more than 2.
red crystalline powder.
Dioxane See 1,4-dioxane. Identification—Determine the infrared absorption spec-

trum of diphenylcarbazone as directed in the potassium bro- Content: not less than 99.0z. Assay—Dissolve about
mide disk method as directed under Infrared Spectropho- 0.2 g of 1,1-diphenyl-4-pyperidino-1-butene hydrochloride
tometry <2.25>: it exhibits absorption at the wave numbers of for thin-layer chromatography, previously weighed accu-
about 1708 cm－1, 1602 cm－1, 1497 cm－1, 1124 cm－1, 986 rately, in 20 mL of acetic acid (100), add 20 mL of acetic an-
cm－1, 748 cm－1 and 692 cm－1. hydride, and titrate <2.50> with 0.05 mol/L perchloric acid
Preserve in a light-resistant tight container. VS (potentiometric titration). Perform a blank determina-
tion, and make any necessary correction.
Diphenylcarbazone TS Dissolve 1 g of diphenylcarba-
zone in ethanol (95) to make 1000 mL. Each mL of 0.05 mol/L perchloric acid VS
＝ 16.39 mg of C21H25N.HCl
1,5-Diphenylcarbonohydrazide C13H14N4O [K 8488,
Special class] Dipicolinic acid C7H5NO4 White powder.
Identification—Determine the infrared absorption spec-
1,5-Diphenylcarbonohydrazide TS Dissolve 0.2 g of 1,5-
trum as directed in the potassium bromide disk method
diphenylcarbonohydrazide in 100 mL of a mixture of
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
ethanol (95) and acetic acid (100) (9:1).
tion at the wave numbers of about 2630 cm－1, 1701 cm－1,
5z Diphenyl-95z dimethylpolysiloxane for gas chroma- 1576 cm－1, 1416 cm－1, 1300 cm－1 and 1267 cm－1.
tography Prepared for gas chromatography. Purity Clarity and color of solution—Dissolve by warm-
ing 0.5 g of dipicolinic acid in 20 mL of ethanol (99.5), and
Diphenyl ether C12H10O Colorless crystals, having a
cool: a clear, colorless liquid.
geranium-like aroma. Dissolves in alcohol (95) and in diethyl
Content: Not less than 98.0z. Assay—Weigh accurately
ether, and practically insoluble in water.
about 0.1 g, add 25 mL of ethanol (99.5), dissolve by warn-
Specific gravity <2.56> d 20
20: 1.072 – 1.075
ing, cool, then titrate <2.50> with 0.1 mol/L sodium hydrox-
Boiling point <2.57>: 254 – 2599C
ide VS (indicator: 2 drops of phenolphthalein TS). Perform
Melting point <2.60>: 289C
a blank determination in the same manner and make any
Diphenyl imidazole C15H12N2 White, crystals or crys- necessary correction.
talline powder, freely soluble in acetic acid (100), and spar-
Each mL of 0.1 mol/L sodium hydroxide VS
ingly soluble in methanol.
＝ 8.356 mg of C7H5O4N
Melting point <2.60>: 234 – 2389 C
Loss on drying <2.41>: not more than 0.5z (0.5 g, 1059C, Dipotassium hydrogen phosphate K2HPO4 [K 9017,
3 hours). Special class]
Content: not less than 99.0z. Assay—Dissolve about
Dipotassium hydrogen phosphate-citric acid buffer solu-
0.3 g of diphenyl imidazole, previously dried and weighed
tion (pH 5.3) Mix 100 mL of 1 mol/L dipotassium hydro-
accurately, in 70 mL of acetic acid (100), and titrate <2.50>
gen phosphate TS for buffer solution and 38 mL of 1 mol/L
with 0.1 mol/L perchloric acid VS (indicator: 2 drops of
citric acid TS for buffer solution, and add water to make 200
crystal vioret TS).
mL.
Each mL of 0.1 mol/L perchloric acid VS
1 mol/L Dipotassium hydrogen phosphate TS for buffer
＝ 22.03 mg of C15H12N2
solution Dissolve 174.18 g of dipotassium hydrogen phos-
Diphenyl phthalate C6H4(COOC6H5)2 White crystalline phate in water to make 1000 mL.
powder.
Dipotassium tetraborate tetrahydrate K2B4O7.4H2O
Melting point <2.60>: 71 – 769C
White, crystalline powder or powder. Slightly soluble in
Purity Related substances—Dissolve 60 mg of diphenyl
ethanol (99.5).
phthalate in 50 mL of chloroform and use this solution as
the sample solution. Proceed with 10 mL of the sample solu- Diprophylline C10H14N4O4 A white, powder or grain.
tion as directed in the Assay under Tolnaftate Solution: any Freely soluble in water, and slightly soluble in ethanol (95).
peak other than the principal peak at the retention time of Identification—Determine the infrared absorption spec-
about 8 minutes and the peak of the solvent does not appear. trum of the substance to be examined, previously dried at
Adjust the detection sensitivity so that the peak height of 1059C for 4 hours, as directed in the potassium bromide disk
diphenyl phthalate obtained from 10 mL of the sample solu- method under Infrared Spectrophotometry <2.25>: it exhibits
tion is 50 to 100z of the full scale, and the time span of absorption at the wave numbers of about 3460 cm－1, 3330
measurement is about twice as long as the retention time of cm－1, 1651 cm－1, 1242 cm－1, 1059 cm－1 and 1035 cm－1.
diphenyl phthalate, beginning after the solvent peak.
a,a?-Dipyridyl See 2,2?-bipyridyl.
1,1-Diphenyl-4-pyperidino-1-butene hydrochloride for
Disodium chromotropate dihydrate
thin-layer chromatography C21H25N.HCl To 1 g of
C10H6Na2O8S2.2H2O [K 8316, Special class] Preserve in
diphenidole hydrochloride add 30 mL of 1 mol/L hydrochlo-
light-resistant containers.
ric acid TS, and heat under a reflux condenser for 1 hour.
After cooling, extract twice with 30 mL-portions of chlo- Disodium dihydrogen ethylenediamine tetraacetate di-
roform, combine the chloroform extracts, wash twice with hydrate C10H14N2Na2O8.2H2O [K 8107, Special class]
10 mL portions of water, and evaporate chloroform under
0.4 mol/L Disodium dihydrogen ethylenediamine tetra-
reduced pressure. Recrystallize the residue from a mixture of
acetate TS (pH 8.5) Dissolve 148.9 g of disodium dihydro-
diethyl ether and ethanol (95) (3:1), and dry in a desiccator
gen ethylenediamine tetraacetate dihydrate in about 800 mL
(in vaccum, silica gel) for 2 hours. White crystals or crystal-
of water, adjust to pH 8.5 with 8 mol/L sodium hydroxide
line powder.
TS, and add water to make 1000 mL.
Absorbance <2.24> E 11zcm (250 nm): 386 – 446 (10 mg,
water, 1000 mL). 0.04 mol/L Disodium dihydrogen ethylenediamine tetra-
Melting point <2.60>: 176 – 1809 C acetate TS Dissolve 14.890 g of disodium dihydrogen ethyl-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
248 General Tests / 9.41 Reagents, Test Solutions JP XVII
enediamine tetraacetate dihydrate in water to make 1000 acid monohydrate in water to make 1000 mL.
mL.
Disodium hydrogen phosphate-citric acid buffer solution
0.1 mol/L Disodium dihydrogen ethylenediamine tetra- (pH 7.5) To 1000 mL of 0.05 mol/L disodium hydrogen
acetate TS Dissolve 37.2 g of disodium dihydrogen ethyl- phosphate TS add a solution prepared by dissolving 5.25 g of
enediamine tetraacetate dihydrate in water to make 1000 citric acid monohydrate in water to make 1000 mL to adjust
mL. the pH to 7.5.
Disodium ethylenediaminetetraacetate See disodium di- Disodium hydrogen phosphate-citric acid buffer solution
hydrogen ethylenediamine tetraacetate dihydrate. (pH 8.2) Dissolve 20.7 g of anhydrous disodium hydrogen
phosphate, 7.38 g of citric acid monohydrate, and 0.535 g of
Disodium ethylenediaminetetraacetate copper See cop-
sodium dihydrogen phosphate dihydrate in 400 mL of water,
por (II) disodium ethylenediamine tetraacetate tetrahydrate.
adjust to pH 8.2 with a solution of sodium hydroxide (1 in
0.1 mol/L Disodium ethylenediaminetetraacetate TS 2), and add water to make 500 mL.
See 0.1 mol/L disodium dihydrogen ethylenediamine tetra-
Disodium hydrogen phosphate-citric acid buffer solution
acetate TS.
for penicillium origin b-galactosidase (pH 4.5) Dissolve
Disodium hydrogen phosphate, anhydrous Na2HPO4 71.6 g of disodium hydrogen phosphate dodecahydrate in
[K 9020, Special class] water to make 1000 mL, and adjust the pH to 4.5 with a so-
lution prepared by dissolving 21.0 g of citric acid monohy-
Disodium hydrogen phosphate-citric acid buffer solution
drate in water to make 1000 mL (volume ratio: about 44:56).
(pH 3.0) Dissolve 35.8 g of disodium hydrogen phosphate
dodecahydrate in water to make 500 mL. To this solution Disodium hydrogen phosphate dodecahydrate
add a solution of citric acid monohydrate (21 in 1000) to Na2HPO4.12H2O [K 9019, Special class]
adjust the pH to 3.0.
Disodium hydrogen phosphate for pH determination
Disodium hydrogen phosphate-citric acid buffer solution Na2HPO4 [K 9020, for pH determination]
(pH 4.5) Dissolve 21.02 g of citric acid monohydrate in
Disodium hydrogen phosphate TS Dissolve 12 g of diso-
water to make 1000 mL, and adjust the pH to 4.5 with a so-
dium hydrogen phosphate dodecahydrate in water to make
lution prepared by dissolving 35.82 g of disodium hydrogen-
100 mL (0.3 mol/L).
phophate 12-water in water to make 1000 mL.
0.05 mol/L Disodium hydrogen phosphate TS Dissolve
Disodium hydrogen phosphate-citric acid buffer solution
7.098 g of anhydrous disodium hydrogen phosphate in water
(pH 5.0) Dissolve 7.1 g of anhydrous disodium hydrogen
to make 1000 mL.
phosphate in water to make 1000 mL, and adjust to pH 5.0
with a solution prepared by dissolving 5.25 g of citric acid 0.5 mol/L Disodium hydrogen phosphate TS Dissolve
monohydrate in water to make 1000 mL. 70.982 g of anhydrous disodium hydrogen phosphate in
water to make 1000 mL.
Disodium hydrogen phosphate-citric acid buffer solution
(pH 5.4) Dissolve 1.05 g of citric acid monohydrate and Disodium 1-nitroso-2-naphthol-3,6-disulfonate
2.92 g of disodium hydrogen phosphate dodecahydrate in C10H5NNa2O8S2 Yellow, crystals or crystalline powder.
200 mL of water, and adjust to pH 5.4 with phosphoric acid Identification—Determine the infrared absorption spec-
or sodium hydroxide TS, if necessary. trum as directed in the potassium bromide disk method as di-
rected under Infrared Spectrophotometry <2.25>: it exhibits
Disodium hydrogen phosphate-citric acid buffer solution
absorption at the wave numbers of about 3400 cm－1, 1639
(pH 5.5) To 1000 mL of 0.05 mol/L disodium hydrogen
cm－1, 1451 cm－1, 1270 cm－1, 1231 cm－1, 1173 cm－1, 1049
phosphate TS add an amount of a solution, prepared by dis-
cm－1, 848 cm－1 and 662 cm－1.
solving 5.25 g of citric acid monohydrate in water to make
Preserve in a light-resistant tight container.
1000 mL, to adjust to pH 5.5.
Dissolved acetylene C2H 2 [K 1902]
Disodium hydrogen phosphate-citric acid buffer solution
(pH 6.0) Dissolve 71.6 g of disodium hydrogen phosphate Distigmine bromide for assay C22H32Br2N4O4 [Same as
dodecahydrate in water to make 1000 mL. To this solution the monograph Distigmine Bromide. It contains not less
add a solution, prepared by dissolving 21.0 g of citric acid than 99.0z of distigmine bromide (C22H32Br2N4O4), calcu-
monohydrate in water to make 1000 mL, until the pH lated on the anhydrous basis.]
becomes 6.0 (ratio of volume: about 63:37).
Distilled water for injection [Use the water prescribed by
0.05 mol/L Disodium hydrogen phosphate-citric acid the monographs of Water for Injection or Sterile Water for
buffer solution (pH 6.0) To 1000 mL of 0.05 mol/L disodi- Injection in Containers. Prepared by distillation. It is not
um hydrogen phosphate TS add a solution prepared by dis- necessary to check the conformity to all the specification
solving 5.25 g of citric acid monohydrate in water to make items of the monograph, if it is confirmed that the water to
1000 mL to adjust pH 6.0. be used is suitable for the purpose of relevant test.]
Disodium hydrogen phosphate-citric acid buffer solution 2,6-Di-tert-butylcresol [(CH3)3C]2C6H2(CH3)OH A
(pH 6.8) To 1000 mL of 0.05 mol/L disodium hydrogen white crystalline powder. Freely soluble in ethanol (95).
phosphate TS add a solution prepared by dissolving 5.25 g of Melting point <2.60>: 69 – 719C
citric acid monohydrate in water to make 1000 mL to adjust Residue on ignition <2.44>: not more than 0.05z.
the pH to 6.8.
2,6-Di-tert-butylcresol TS Dissolve 0.1 g of 2,6-di-tert-
Disodium hydrogen phosphate-citric acid buffer solution butylcresol in ethanol (95) to make 10 mL.
(pH 7.2) Dissolve 7.1 g of anhydrous disodium hydrogen
2,6-Di-tert-butyl-p-cresol See 2,6-di-tert-butylcresol.
phosphate in water to make 1000 mL. Adjust this solution to
pH 7.2 with a solution prepared by dissolving 5.3 g of citric 2,6-Di-tert-butyl-p-cresol TS See 2,6-di-tert-butylcresol

TS. fate and 50 mg of proteinase K, and stir gently at 50 ± 19C

for 2 hours. Add an equal volume of phenol-saturated TE
1,3-Di (4-pyridyl) propane C13H14N2 A pale yellow
buffer solution, stir gently at room temperature for 2 hours,
powder.
and centrifuge. After removing the lower layer, repeat the
Melting point <2.60>: 61 – 629C
same operation. Collect the upper layer, and dialyze for 10
Water <2.48>: less than 0.1z.
hours against dialysis buffer B, then change the external so-
1,1?-[3,3?-Dithiobis(2-methyl-1-oxopropyl)]-L-diproline lution to dialysis buffer C, and dialyze for 24 hours. Collect
C18H28N2O6S2 White, crystals or crystalline powder. Spar- the inner solution, add 0.1 volume of acetic acid-sodium ace-
ingly soluble in methanol, and practically insoluble in water. tate buffer solution (pH 5.2) and 2.2 volume of ethanol
Identification—Determine the infrared absorption spec- (99.5), and stir gently. Collect the DNA precipitated by
trum of 1,1?-[3,3?-dithiobis(2-methyl-1-oxopropyl)]-L-dipro- winding on a glass rod, wash with diluted ethanol (7 in 10),
line according to potassium bromide disk method under In- dry in vacuum, dissolve the residue in 4 mL of TE buffer so-
frared Spectrophotometry <2.25>: it exhibits absorption at lution, and use this solution as the standard DNA. Dilute
the wave numbers of about 2960 cm－1, 1750 cm－1, 1720 with water so that each mL contains exactly 40 ng of DNA
cm－1, 1600 cm－1, 1480 cm－1, 1450 cm－1 and 1185 cm－1. following the specific absorbance of double-stranded DNA,
Purity Related substances—Dissolve 0.10 g of 1,1?-[3,3?- E 11zcm (260 nm), is 200.
dithiobis (2-methyl-1-oxopropyl)]-L-diproline in exactly 10
Docetaxel hydrate C43H53NO14.3H2O [Same as the
mL of methanol. Perform the test with this solution as di-
namesake monograph]
rected in the Purity (3) under Captopril: any spot other than
the principal spot at the R f value of about 0.2 does not ap- Dopamine hydrochloride for assay C8H11NO2.HCl
pear. [Same as the monograph Dopamine hydrochloride. When
Content: not less than 99.0z. Assay—Weigh accurately dried, it contains not less than 99.0z of dopamine hydro-
about 0.3 g of 1,1?-[3,3?-dithiobis (2-methyl-1-oxopropyl)]-L- chloride (C8H11NO2.HCl).]
diproline, dissolve in 20 mL of methanol, add 50 mL of
Doxepin hydrochloride C19H21NO.HCl White, crystals
water, and titrate <2.50> with 0.1 mol/L sodium hydroxide
or crystalline powder. Melting point: 185 – 1919C.
VS until the color of the solution changes from yellow
through bluish green to blue (indicator: 3 drops of bromo- Doxifluridine C9H11FN2O5 [Same as the namesake
thymol blue TS). Perform a blank determination in the same monograph]
manner, and make any necessary correction.
Doxifluridine for assay C9H11FN2O5 [Same as the
Each mL of 0.1 mol/L sodium hydroxide VS monograph Doxifluridine. When dried, it contains not less
＝ 21.63 mg of C18H28N2O6S2 than 99.5z of doxifluridine (C9H11FN2O5).]
Dithiodiglycolic acid C4H6O4S2 Prepared for amino Doxorubicin hydrochloride C27H29NO11.HCl [Same as
acid analysis or biochemistry. the namesake monograph]
Dithiodipropionic acid C6H10O4S2 Prepared for amino Dragendorff's TS Dissolve 0.85 g of bismuth subnitrate
acid analysis or biochemistry. in 10 mL of acetic acid (100) and 40 mL of water with
vigorous shaking (solution A). Dissolve 8 g of potassium
Dithiothreitol C4H10O2S2 Crystals.
iodide in 20 mL of water (solution B). Immediately before
Melting point <2.60>: about 429C
use, mix equal volumes of solution A, solution B and acetic
Dithizone C6H5NHNHCSN:NC6H5 [K 8490, Special acid (100). Store solution A and solution B in light-resistant
class] containers.
Dithizone solution for extraction Dissolve 30 mg of Dragendorff's TS for spraying Add 20 mL of diluted
dithizone in 1000 mL of chloroform, and add 5 mL of acetic acid (31) (1 in 5) to 4 mL of a mixture of equal
ethanol (95), and store. Before use, shake a suitable volume volumes of solution A and solution B of Dragendorff's TS.
of the solution with one-half of its volume of diluted nitric Prepare before use.
acid (1 in 100), and use the chloroform layer after discarding
Dried human normal plasma powder Freeze-dried nor-
the water layer.
mal plasma obtained from healthy human.
Dithizone TS Dissolve 25 mg of dithizone in ethanol (95)
Dried sodium carbonate Na2CO3 [Same as the name-
to make 100 mL. Prepare before use.
sake monograph]
DNA standard stock solution for interferon alfa
Droxidopa for assay C9H11NO5 [Same as the mono-
(NAMALWA) To 1 × 109 Namalwa cells add 0.1 mL of
graph Droxidopa. When dried, it contains not less than
proteinase K solution and 20 mL of N-lauroyl sarcosine so-
99.5z of droxidopa (C9H11NO5).]
dium TS, lyse the cells by gentle stirring at 50 ± 19 C for 3
hours, add 20 mL of water-saturated phenol, and stir gently DSS-d6 for nuclear magnetic resonance spectroscopy
at room temperature for 3 hours. Add 10 mL of a mixture of C6H9D6NaO3SSi Sodium 3-(trimethylsilyl)-1-propanesul-
chloroform and 3-methyl-l-butanol (24:1), centrifuge, and fonate-d6 that the traceability to the International System of
discard the lower layer. Add 20 mL of water-saturated Units has been secured.
phenol to the upper layer, stir gently at room temperature
Dydrogesterone for assay C21H28O2 [Same as the
for 2 hours, and centrifuge. Collect the lower layer, dialyze
monograph Dydrogesterone. When dried, it contains not less
for 24 hours against dialysis buffer A, add ribonuclease A
than 99.0z of dydrogesterone (C21H28O2).]
and ribonuclease T1 so that each mL of the inner solution
obtained contains 25 mg of ribonuclease A and 25 units of Eagle's minimum essential medium Dissolve 6.80 g of
ribonuclease T1, and stir gently at 37 ± 19C for 3 hours. sodium chloride, 400 mg of potassium chloride, 115 mg of
Add sodium lauryl sulfate solution (1 in 10) and proteinase K anhydrous sodium dihydrogen phosphate, 93.5 mg (as anhy-
solution so that each mL contains 5 mg of sodium lauryl sul- drous) of magnesium sulfate, 200 mg (as anhydrous) of cal-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
250 General Tests / 9.41 Reagents, Test Solutions JP XVII
cium chloride, 1.00 g of glucose, 126 mg of L-arginine hy- with sialic acid is bound to end by a-2, 6 linkage.
drochloride, 73.0 mg of L-lysine hydrochloride, 31.4 mg of
Elderberry lectin TS Dilute biotin-labeled elderberry lec-
L-cysteine hydrochloride monohydrate, 36.0 mg of L-tyro-
tin with 0.01 mol/L tris buffer solution-sodium chloride TS
sine, 42.0 mg of L-histidine hydrochloride monohydrate,
(pH 7.4) so that the concentration is 10 mg/mL. Prepare be-
52.0 mg of L-isoleucine, 52.0 mg of L-leucine, 15.0 mg of
fore use.
methionine, 32.0 mg of phenylalanine, 48.0 mg of L-threo-
nine, 10.0 mg of L-tryptophan, 46.0 mg of L-valine, 75.0 mg Eleutheroside B for liquid chromatography C17H24O9
of succinic acid, 100 mg of sodium succinate hexahydrate, A white crystalline powder. Sparingly soluble in methanol,
1.8 mg of choline bitartrate, 1.0 mg of folic acid, 2.0 mg of slightly soluble in water, and very slightly soluble in ethanol
myoinositol, 1.0 mg of nicotinamide, 1.0 mg of calcium D- (99.5). Melting point: 190 – 1949 C‚
pantothenate, 1.0 mg of pyridoxal hydrochloride, 0.1 mg of Identification—Determine the absorption spectrum of a
riboflavin, 1.0 mg of thiamine chloride hydrochloride, 20 mg solution of eleutheroside B for liquid chromatography in
of biotin and 6.0 mg of phenol red in 1000 mL of water, heat methanol (1 in 200,000) as directed under Ultraviolet-visible
in an autoclave at 1219C for 15 minutes and cool to room Spectrophotometry <2.24>: it exhibits a maximum between
temperature, then add separately sterilized 22 mL of 10z so- 261 nm and 265 nm.
dium hydrogen carbonate TS and 10 mL of glutamine TS. Purity Related substances—Dissolve 1.0 mg of eleu-
theroside B for liquid chromatography in 10 mL of the mo-
Eagle's minimum essential medium containing bovine
bile phase, and use this solution as the sample solution. Pipet
serum To Eagle's minimum essential medium add an
1 mL of the sample solution, add the mobile phase to make
adequate amount of bovine serum.
exactly 50 mL, and use this solution as the standard solution.
Ebastine for assay C32H39NO2 [Same as the monograph Perform the test with exactly 10 mL each of the sample solu-
Ebastine. When dried, it contains not less than 99.5z of tion and standard solution as directed under Liquid Chroma-
ebastine (C32H39NO2).] tography <2.01> according to the following conditions. De-
termine each peak area by the automatic integration method:
Ecabet sodium hydrate for assay C20H27NaO5S.5H2O
the total area of the peaks other than eleutheroside B ob-
[Same as the monograph Ecabet Sodium Hydrate. It con-
tained with the sample solution is not larger than the peak
tains not less than 99.5z of ecabet sodium (C20H27NaO5S),
area of eleutheroside B obtained with the standard solution.
calculated on the anhydrous basis.]
Operating conditions
E. coli protein Process E. coli cells (E. coli N4830/ Detector, column, column temperature, mobile phase, and
pTB281) retaining a plasmid deficient in the celmoleukin flow rate: Proceed as directed in the operating conditions in
gene according to the celmoleukin purification process in the the Identification under Eleutherococcus Senticosus Rhi-
following order; (i) extraction, (ii) butylated vinyl polymer zome.
hydrophobic chromatography, (iii) carboxymethylated vinyl Time span of measurement: About 3 times as long as the
polymer ion-exchange column chromatography, and (iv) sul- retention time of eleutheroside B, beginning after the solvent
fopropyl-polymer ion-exchange chromatography, and dur- peak.
ing process (iv) collect the fractions corresponding to the cel- System suitability
moleukin elution position. Dialyze the fractions obtained in System performance: Proceed as directed in the system
(iv) against 0.01 mol/L acetate buffer solution (pH 5.0), and suitability in the Identification under Eleutherococcus
take the dialysis solution as E.coli protein. Senticosus Rhizome.
Description—Clear and colorless solution. Test for required detectability: To exactly 1 mL of the
Identification—When the absorption spectrum is deter- standard solution add the mobile phase to make exactly 20
mined as directed under Ultraviolet-visible Spectrophoto- mL. Confirm that the peak area of eleutheroside B obtained
metry <2.24>, an absorption maximum is observed in the with 10 mL of this solution is equivalent to 3.5 to 6.5z of
region of 278 nm. that obtained with 10 mL of the standard solution.
Protein content: When determining the protein content
EMB plate medium Melt eosin methylene blue agar me-
using the Assay (1) Total protein content under Celmoleukin
dium by heating, and cool to about 509C. Transfer about 20
(Genetical Recombination), the protein content per mL is 0.1
mL of this medium to a Petri dish, and solidify horizontally.
to 0.5 mg.
Place the dish with the cover slightly opened in the incubator
E. coli protein stock solution A solution obtained by cul- to evaporate the inner vapor and water on the plate.
turing a bacteria that contains a plasmid lacking the teceleu-
Emedastine fumarate for assay C17H26N4O.2C4H4O4
kin gene but is otherwise exactly identical to the teceleukin-
[Same as the monograph Emedastine Fumarate. When dried
producing E. coli strain in every function except teceleukin
it contains not less than 99.5z of emedastine fumarate
production, and then purified using a purification technique
(C17H26N4O.2C4H4O4).]
that is more simple than that for teceleukin. Determine the
amount of protein by Brodford method using bovine serum Emetine hydrochloride for assay C29H40N2O4.2HCl A
albumin as the standard substance. Store shielded from light white or light-yellow crystalline powder. Soluble in water.
at －709C. Absorbance <2.24> E 11zcm (283 nm): 116 – 127 (10 mg,
diluted methanol (1 in 2), 400 mL). [after drying in a desicca-
Edaravone for assay C10H10N2O [Same as the mono-
tor (in vacuum, phosphorus (V) oxide, 509C) for 5 hours].
graph Edaravone. When dried, it contains not less than
Melting point <2.60>: about 2509C [with decomposition,
99.5z of edaravone (C10H10N2O).]
after drying in a desiccator (in vacuum, phosphorus (V)
Egg albumin for gel filtration molecular mass marker oxide, 509C) for 5 hours].
Obtained from chicken egg white, for gel filtration chroma- Purity Related substances—Dissolve 10 mg of emetine
tography. hydrochloride for assay in 10 mL of the mobile phase, and
use this solution as the sample solution. Pipet 1 mL of the
Elderberry lectin It is a lectin derived from the Japan or
sample solution, add the mobile phase to make exactly 100
Western elderberry, specifically recognizes a sugar chain

mL, and use this solution as the standard solution. Perform not more than 20 minutes, and cool quickly by immersing in
the test with exactly 10 mL each of the sample solution and cold water, or sterilize fractionally on each of three succes-
standard solution as directed under Liquid Chromatography sive days for 30 minutes at 1009C.
<2.01> according to the following conditions. Determine the
Ephedrine hydrochloride C10H15NO.HCl [Same as the
peak areas from both solutions by the automatic integration
namesake monograph]
method: the total area of peaks other than emetine from the
sample solution is not larger than the peak of emetine from Ephedrine hydrochloride for assay See ephedrine hydro-
the standard solution. chloride.
Operating conditions
Ephedrine hydrochloride for assay of crude drugs
Detector, column, column temperature, mobile phase, and
C10H15NO.HCl Ephedrine hydrochloride for assay or the
flow rate: Proceed as directed in the operating conditions in
substance that complies with the following requirements.
the Assay under Ipecac.
White, crystals or crystalline powder. Freely soluble in
Time span of measurement: About 3 times as long as the
water, and soluble in ethanol (95).
retention time of emetine.
Identification—Determine the infrared absorption spec-
System suitability
trum of ephedrine hydrochloride for assay of crude drugs,
System performance and System repeatability: Proceed as
previously dried, as directed in the potassium chloride disk
directed in the system suitability in the Assay under Ipecac.
method under Infrared Spectrophotometry <2.25>, and com-
Test for required detectability: Pipet 1 mL of the standard
pare the spectrum with the Reference Spectrum of Ephedrine
solution, add the mobile phase to make exactly 20 mL. Con-
Hydrochloride: both spectra exhibit similar intensities of ab-
firm that the peak area of emetine obtained with 10 mL of
sorption at the same wave numbers.
this solution is equivalent to 3.5 to 6.5z of that obtained
Optical rotation <2.49> [a]20D : －33.0 – －36.09(after dry-
with 10 mL of the standard solution.
ing, 0.1 g, water, 2 mL, 100 mm).
Emorfazone for assay C11H17N3O3 [Same as the mono- Melting point <2.60>: 218 – 2229C
graph Emorfazone. When dried, it contains not less than Purity Related substances—Dissolve 10 mg of ephedrine
99.0z of emorfazone (C11H17N3O3).] hydrochloride for assay of crude drugs in 10 mL of the mo-
bile phase, and use this solution as the sample solution. Pipet
Enalapril maleate C20H28N2O5.C4H4O4 [Same as the
1 mL of the sample solution, add the mobile phase to make
namesake monograph]
exactly 100 mL, and use this solution as the standard solu-
Endo's medium Melt 1000 mL of the ordinary agar me- tion. Perform the test with exactly 10 mL each of the sample
dium by heating in a water bath, and adjust the pH to be- solution and standard solution as directed under Liquid
tween 7.5 and 7.8. Add 10 g of lactose monohydrate previ- Chromatography <2.01> according to the following condi-
ously dissolved in a small quantity of water, mix thoroughly, tions. Determine each peak area by the automatic integration
and add 1 mL of fuchsin-ethanol TS. After cooling to about method: the total area of the peaks other than ephedrine ob-
509 C, add dropwise a freshly prepared solution of sodium tained from the sample solution is not larger than the peak
sulfite heptahydrate (1 in 10) until a light red color develops area of ephedrine obtained from the standard solution.
owing to reducing fuchsin, requiring about 10 to 15 mL of a Operating conditions
solution of sodium sulfite heptahydrate (1 in 10). Dispense Detector, column, column temperature, mobile phase, and
the mixture, and sterilize fractionally on each of three suc- flow rate: Proceed as directed in the operating conditions in
cessive days for 15 minutes at 1009C. the Assay under Ephedra Herb.
Time span of measurement: About 3 times as long as the
Endo's plate medium Melt Endo's medium by heating,
retention time of ephedrine, beginning after the solvent
and cool to about 509C. Transfer about 20 mL of this me-
peak.
dium to a Petri dish, and solidify horizontally. Place the dis-
System suitability
hes with the cover slightly opened in the incubator to evapo-
System performance and system repeatability: Proceed as
rate the inner vapor and water on the surface of the agar.
directed in the system suitability in the Assay under Ephedra
Enflurane C3H2ClF5O [Same as the namesake mono- Herb.
graph] Test for required detectability: Pipet 1 mL of the standard
solution, and add the mobile phase to make exactly 20 mL.
Enzyme TS The supernatant liquid is obtained as
Confirm that the peak area of ephedrine obtained with 10 mL
follows: To 0.3 g of an enzyme preparation potent in amylo-
of this solution is equivalent to 3.5 to 6.5z of that obtained
lytic and phosphorolytic activities, obtained from Aspergil-
with 10 mL of the standard solution.
lus oryzae, add 10 mL of water and 0.5 mL of 0.1 mol/L hy-
Loss on drying <2.41>: Not more than 0.5z (0.1 g, 1059C,
drochloric acid TS, mix vigorously for a few minutes, and
3 hours).
centrifuge. Prepare before use.
6-Epidoxycycline hydrochloride C22H24N2O8.HCl
Eosin See eosin Y.
Yellow to dark yellow, crystals or crystalline powder.
Eosin Y C20H6Br4Na2O5 Red, masses or powder. Purity Related substances—Dissolve 20 mg of 6-epiox-
Identification—To 10 mL of a solution (1 in 1000) add 1 ycycline hydrochloride in 25 mL of 0.01 mol/L hydrochloric
drop of hydrochloric acid: yellow-red precipitates appear. acid TS, and use this solution as the sample solution. Pro-
ceed the test with 20 mL of the sample solution as directed in
Eosin methylene blue agar medium Dissolve by boiling
the Purity (2) under Doxycycline Hydrochloride Hydrate,
10 g of casein peptone, 2 g of dipotassium hydrogen-
determine each peak area by the automatic integration
phosphate and 25 to 30 g of agar in about 900 mL of water.
method, and calculate the amounts of them by the area per-
To this mixture add 10 g of lactose monohydrate, 20 mL of a
centage method: the total area of the peaks other than 6-
solution of eosin Y(1 in 50), 13 mL of a solution of methy-
epidoxycycline is not more than 10z.
lene blue (1 in 200) and warm water to make 1000 mL. Mix
thoroughly, dispense, sterilize by autoclaving at 1219C for 4-Epioxytetracycline C22H24N2O9 Green-brown to bro-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
252 General Tests / 9.41 Reagents, Test Solutions JP XVII
wn powder. Ethanol (99.5) C2H5OH [K 8101, Special class]
Purity Related substances—Dissolve 20 mg of 4-epiox-
Ethanol (99.5) for liquid chromatography C2H5OH A
ytetracycline in 25 mL of 0.01 mol/L hydrochloric acid TS,
clear, colorless liquid, miscible with water.
and use this solution as the sample solution. Proceed the test
Purity Ultraviolet absorbing substance—Perform the
with 20 mL of the sample solution as directed in the Purity
test as directed under Ultraviolet-visible Spectrophotometry
(2) under Oxytetracycline Hydrochloride, determine each
<2.24> using water as the blank: the absorbances at 210 nm,
peak area by the automatic integration method, and calcu-
at 220 nm, at 230 nm, at 240 nm, at 254 nm and at 260 nm
late the amounts of them by the area percentage method: the
are not more than 0.70, 0.40, 0.20, 0.10, 0.02 and 0.01,
total amount of the peaks other than 4-epioxytetracycline is
respectively.
not more than 10z.
Ethanol, aldehyde-free Transfer 1000 mL of ethanol (95)
Eriochrome black T C20H12N3NaO7S [K 8736, Special
to a glass-stoppered bottle, add the solution prepared by dis-
class]
solving 2.5 g of lead (II) acetate trihydrate in 5 mL of water,
Eriochrome black T-sodium chloride indicator Mix 0.1 g and mix thoroughly. In a separate container, dissolve 5 g of
of eriochrome black T and 10 g of sodium chloride, and potassium hydroxide in 25 mL of warm ethanol (95), cool,
triturate until the mixture becomes homogeneous. and add this solution gently, without stirring, to the first so-
lution. After 1 hour, shake this mixture vigorously, allow to
Eriochrome black T TS Dissolve 0.3 g of eriochrome
stand overnight, decant the supernatant liquid, and distil the
black T and 2 g of hydroxylammonium chloride in methanol
ethanol.
to make 50 mL. Use within 1 week. Preserve in light-
resistant containers. Ethanol, dehydrated See ethanol (99.5).
Erythromycin B C37H67NO12 White to light yellowish Ethanol, dilute To 1 volume of ethanol (95) add 1
white powder. volume of water.
Purity Related substances—Dissolve 10 mg of
Ethanol, diluted Prepare by diluting ethanol (99.5).
erythromycin B in 1 mL of methanol, add a mixture of phos-
phate buffer solution (pH 7.0) and methanol (15:1) to make Ethanol for alcohol number determination See Alcohol
5 mL, and use this solution as the sample solution. Pipet 1 Number Determination <1.01>.
mL of the sample solution, add a mixture of phosphate
Ethanol for disinfection [Same as the namesake mono-
buffer solution (pH 7.0) and methanol (15:1) to make ex-
graph]
actly 20 mL, and use this solution as the standard solution.
Proceed with exactly 100 mL each of the sample solution and Ethanol for gas chromatography Use ethanol prepared
standard solution as directed in the Purity (3) under by distilling ethanol (99.5) with iron (II) sulfate heptahy-
Erythromycin, and determine each peak area from the solu- drate. Preserve in containers, in which the air has been dis-
tions by the automatic integration method: the total of areas placed with nitrogen, in a dark, cold place.
of the peaks other than erythromycin B from the sample so-
Ethanol-free chloroform See chloroform, ethanol-free.
lution is not more than the peak area of erythromycin B
from the standard solution. Ethanol-isotonic sodium chloride solution To 1 volume
of ethanol (95) add 19 volumes of isotonic sodium chloride
Erythromycin C C36H65NO13 White to light yellowish
solution.
white powder.
Purity Related substances—Dissolve 10 mg of Ethanol, methanol-free See ethanol (95), methanol-free.
erythromycin C in 1 mL of methanol, add a mixture of phos-
Ethanol, neutralized To a suitable quantity of ethanol
phate buffer solution (pH 7.0) and methanol (15:1) to make
(95) add 2 to 3 drops of phenolphthalein TS, then add 0.01
5 mL, and use this solution as the sample solution. Pipet 1
mol/L or 0.1 mol/L sodium hydroxide VS until a light red
mL of the sample solution, add a mixture of phosphate
color develops. Prepare before use.
buffer solution (pH 7.0) and methanol (15:1) to make ex-
actly 20 mL, and use this solution as the standard solution. Ethenzamide C9H11NO2 [Same as the namesake mono-
Proceed with exactly 100 mL each of the sample solution and graph].
standard solution as directed in the Purity (3) under
Ether See diethyl ether.
Erythromycin, and determine each peak area from the solu-
tions by the automatic integration method: the total of areas Ether, anesthetic C2H5OC2H5 [Same as the namesake
of the peaks other than erythromycin C from the sample so- monograph]
lution is not more than the peak area of erythromycin C
Ether, dehydrated See diethyl ether, dehydrated.
from the standard solution.
Ether for purity of crude drug See diethyl ether for
Essential oil Same as the essential oil under the mono-
purity of crude drug.
graph.
Ethinylestradiol C20H24O2 [Same as the namesake
Etacrynic acid for assay C13H12Cl2O4 [Same as the
monograph]
monograph Etacrynic acid. When dried, it contains not less
than 99.0z of etacrynic acid (C13H12Cl2O4).] 4?-Ethoxyacetophenone C2H5OC6H4COCH3 White
crystals.
Ethanol See ethanol (95).
Melting point <2.60>: 37 – 399C
Ethanol (95) C2H5OH [K 8102, Special class]
3-Ethoxy-4-hydroxybenzaldehyde C9H10O3 White to
Ethanol (95), methanol-free Perform the test for metha- pale yellowish white crystalline. Freely soluble in ethanol
nol, by using this methanol-free ethanol (95) in place of the (95), and slightly soluble in water.
standard solution, as directed in Methanol Test <1.12>: it is Melting point <2.60>: 76 – 789C
practically colorless. Content: not less than 98.0z. Assay—Weigh accurately

about 0.3 g of 3-ethoxy-4-hydroxybenzaldehyde, previously peak area from these solutions by the automatic integration
dried in a desiccator (phosphorous (V) oxide) for 4 hours, method: the total peak areas other than ethyl n-caprylate
dissolve in 50 mL of N, N-dimethylacetamide, and titrate from the sample solution is not larger than the peak area of
<2.50> with 0.1 mol/L sodium methoxide VS (indicator: ethyl n-caprylate from the standard solution (1).
thymol blue TS). Operating conditions
Proceed the operating conditions in the Assay under Men-
Each mL of 0.1 mol/L sodium methoxide VS
tha Oil except detection sensitivity and time span of meas-
＝ 16.62 mg of C9H10O3
urement.
p-Ethoxyphenol See 4-ethoxyphenol. Detection sensitivity: Pipet 1 mL of the standard solution
(1), add dichloromethane to make exactly 20 mL, and use
4-Ethoxyphenol C8H10O2 White to light yellow-brown,
this solution as the standard solution (2). Adjust the detec-
crystals or crystalline powder. Freely soluble in ethanol (95),
tion sensitivity so that the peak area of ethyl n-caprylate ob-
and very slightly soluble in water.
tained from 5 mL of the standard solution (2) can be meas-
Melting point <2.60>: 62 – 689C
ured by the automatic integration method, and the peak
Purity—Dissolve 0.5 g of 4-Ethoxyphenol in 5 mL of
height of ethyl n-caprylate from 5 mL of the standard solu-
ethanol (95), and use this solution as the sample solution.
tion (1) is about 20z of the full scale.
Perform the test with 1 mL of the sample solution as directed
Time span of measurement: 3 times as long as the reten-
under Gas Chromatography <2.02> according to the follow-
tion time of ethyl n-caprylate, beginning after the solvent
ing conditions. Measure each peak area by the automatic in-
peak.
tegration method and calculate the amount of substance
other than 4-ethoxyphenol by the area percentage method: it Ethyl carbamate H2NCOOC2H5 White, crystals or pow-
is not more than 2.0z. der.
Operating conditions Melting point <2.60>: 48 – 509C
Detector: Thermal conductivity detector. Purity Clarity of solution—Dissolve 5 g of ethyl carba-
Column: A glass column about 3 mm in inside diameter mate in 20 mL of water: the solution is clear.
and about 2 m in length, packed with 180- to 250-mm
Ethyl cyanoacetate NCCH2COOC2H5 Colorless or
siliceous earth for gas chromatography coated with methyl-
light yellow, clear liquid, having an aromatic odor. Specific
silicone porymer for gas chromatography.
gravity d 20
20: about 1.08.
Column temperature: A constant temperature of about
Identification—To 0.5 mL of a solution in ethanol (99.5)
1509C.
(1 in 10,000) add a mixture of 1 mL of a solution of quinhy-
Carrier gas: Herium.
drone in diluted ethanol (99.5) (1 in 2) (1 in 20,000) and 1
Flow rate: Adjust so that the retention time of 4-ethox-
drop of ammonia solution (28): a light blue color develops.
yphenol is about 5 minutes.
Detection sensitivity: Adjust the detection sensitivity so Ethylenediamine C2H 8N 2 [Same as the namesake
that the peak height of 4-ethoxyphenol obtained from 1 mL monograph]
of the sample solution is not less than 50z of the full scale.
Ethylenediamine TS Dissolve 70 g of ethylenediamine in
Time span of measurement: 3 times as long as the reten-
30 g of water.
tion time of 4-ethoxyphenol, beginning after the solvent
peak. Ethylene glycol HOCH2CH2OH [K 8105, Special
class]
Ethyl acetate CH3COOC2H5 [K 8361, Special class]
Ethylene glycol for water determination Distil ethylene
Ethylamine hydrochloride C2H5NH2.HCl White to
glycol, and collect the fraction distilling between 1959
C and
light yellowish brown, crystals or crystalline powder, having
1989C. The water content is not more than 1.0 mg per mL.
a deliquescency.
Ethylene oxide A colorless flammable gas. Use ethylene
Ethyl aminobenzoate C9H11NO2 [Same as the name-
oxide from a metal cylinder.
sake monograph]
Boiling point <2.57>: 9 – 129C
Ethylbenzene C6H5C2H5 A colorless liquid. Freely
Ethyl formate HCOOC2H5 A clear and colorless liq-
soluble in ethanol (99.5) and in acetone, and practically
uid. Miscible with ethanol (95) and with acetone, and soluble
insoluble in water.
in water.
Specific gravity <2.56> d 20
4 : 0.862 – 0.872
Identification—Determine the infrared absorption spec-
Boiling point <2.57>: about 1359C
trum of ethyl formate as directed in the liquid film method
Ethyl benzoate C6H5COOC2H5 Clear, colorless liquid. under Infrared Spectrophotometry <2.25>: it exhibits absorp-
Refractive index <2.45> n 20
D : 1.502 – 1.507 tion at the wave numbers of about 2980 cm－1, 2930 cm－1,
Specific gravity <2.56> d 20
20: 1.045 – 1.053 1718 cm－1, 1470 cm－1, 1449 cm－1, 1387 cm－1, 1302 cm－1,
1181 cm－1, 1004 cm－1, 840 cm－1 and 747 cm－1.
Ethyl n-caprylate C10H20O2 Clear and colorless to
Purity—(1) Perform the test with 1 mL of ethyl formate
almost colorless liquid.
as directed under Gas Chromatography <2.02> according to
Specific gravity <2.56> d 20
20: 0.864 – 0.871
the following conditions, determine each peak area by the
Purity Related substances—Dissolve 0.10 g of ethyl n-
automatic integration method, and calculate the amount of
caprylate in 10 mL of dichloromethane and use this solution
ethyl formate by the area percentage method: not less than
as the sample solution. Pipet 1 mL of the sample solution,
97.0z.
add dichloromethane to make exactly 100 mL, and use this
Operating conditions
solution as the standard solution (1). Perform the test with
Detector: A thermal conductivity detector.
exactly 5 mL each of the sample solution and standard solu-
Column: A fused silica column 0.25 mm in inside diameter
tion (1) as directed under Gas Chromatography <2.02> ac-
and 30 m in length, coated the inside surface with a layer
cording to the following conditions, and determine each
about 0.25 mm thick of polyethylene glycol 20M for gas

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
254 General Tests / 9.41 Reagents, Test Solutions JP XVII
chromatography. the peaks of 2-ethyl-2-phenylmalondiamide and the solvent
Column temperature: Maintain at 509C for 1 minute after does not appear. Adjust the detection sensitivity so that the
injecting sample, then rise to 1509C at the rate of 109C per peak height of 2-ethyl-2-phenylmalondiamide obtained from
minute, and maintain at 1509C for 1 minute. 2 mL of the sample solution is about 80z of the full scale,
Carrier gas: Helium. and the time span of measurement is about twice as long as
Flow rate: 41 cm per second. the retention time of 2-ethyl-2-phenylmalondiamide, begin-
Split ratio: 1:110. ning after the solvent peak.
Time span of measurement: About 5 times as long as the
Ethyl propionate CH3CH2COOC2H5 Colorless, clear
retention time of ethyl formate.
liquid.
(2) Acid (as formic acid) Dissolve 0.5 g of potassium
Specific gravity <2.56> d 20
4 : 0.890 – 0.892
iodate and 5 g of potassium iodide in 50 mL of water, and
add 2 g of ethyl formate. After allowing to stand for 10 Etidronate disodium for assay C2H6Na2O7P2 [Same as
minutes, add 2 drops of starch TS and 1.30 mL of 0.1 mol/L the monograph Etidronate Disodium. When dried, it
sodium thiosulfate VS: the solution is colorless (not more contains not less than 99.0z of etidronate disodium
than 0.3z). (C2H6Na2O7P2).]
Water <2.48>: not more than 0.5z (1 g, coulometric titra-
Etilefrine hydrochloride C10H15NO2.HCl [Same as the
tion).
namesake monograph]
2-Ethylhexyl parahydroxybenzoate C15H22O3 Pale yel-
Etilefrine hydrochloride for assay C10H15NO2.HCl
low, clear viscous liquid. Miscible with methanol (99.5).
[Same as the monograph Etilefrine Hydrochloride. When
Practically insoluble in water.
dried, it contains not less than 99.0z of etilefrine hydro-
Content: not less than 98.0z. Assay—Weigh accurately
chloride (C10H15NO2.HCl).]
about 1 g of 2-ethylhexyl parahydroxybenzoate, add exactly
20 mL of 1 mol/L sodium hydroxide VS, heat at about 709 C Etizolam for assay C17H15ClN4S [Same as the mono-
for 1 hour, and immediately cool in ice. Titrate <2.50> the graph Etizolam. When dried, it contains not less than 99.0z
excess sodium hydroxide with 0.5 mol/L sulfuric acid VS up of etizolam (C17H15ClN4S).]
to the second equivalent point (potentiometric titration).
Factor IIa A lyophilized factor IIa purified from human
Perform a blank determination.
plasma. A white to pale yellowish powder. It contains not
Each mL of 1 mol/L sodium hydroxide VS less than 2000 IU per mg of protein.
＝ 250.3 mg of C15H22O3
Factor Xa It is prepared from lyophilization of Factor
Ethyl iodide See iodoethane. Xa which has been prepared from bovine plasma. White or
pale yellow, masses or powder.
N-Ethylmaleimide C6H7NO2 White crystals, having a
Purity Clarity and color of solution—Dissolve 71
pungent, characteristic odor. Freely soluble in ethanol (95),
nkats-2222 of it in 10 mL water; the solution is clear and color-
and slightly soluble in water.
less or pale yellow.
Melting point <2.60>: 43 – 469C
Content: not less than 75z and not more than 125z of
Purity Clarity and color of solution—Dissolve 1 g of N-
the label.
ethylmaleimide in 20 mL of ethanol (95): the solution is clear
and colorless. Factor Xa TS Dissolve 71 nkats-2222 of factor Xa in 10
Content: not less than 99.0z. Assay—Dissolve about mL of water.
0.1 g of N-ethylmaleimide, accurately weighed, in 20 mL of
Famotidine for assay C8H15N7O2S3 [Same as the mono-
ethanol (95), add exactly 20 mL of 0.1 mol/L sodium hy-
graph Famotidine. When dried, it contains not less than
droxide VS, and titrate <2.50> with 0.1 mol/L hydrochloric
99.0z of famotidine (C8H15N7O2S3), and when proceed as
acid VS (indicator: 2 drops of phenolphthalein TS). Perform
directed in the Purity (3), the total related substance is not
a blank determination.
more than 0.4z.]
Each mL of 0.1 mol/L sodium hydroxide VS
Fatty acid methyl esters mixture TS Dissolve 0.50 g of a
＝ 12.51 mg of C6H7NO2
mixture of methyl myristate for gas chromatography, methyl
N-Ethylmorpholine C6H13NO A colorless to yellow- palmitate for gas chromatography, methyl palmitoleate for
brown liquid. gas chromatography, methyl stearate for gas chromatogra-
Refractive index <2.45> n20D : 1.439 – 1.443 phy, methyl oleate for gas chromatography, methyl linoleate
Specific gravity <2.56> d 20
4 : 0.908 – 0.916 for gas chromatography and methyl linolenate for gas chro-
matography, corresponding to the composition of Polysor-
Ethyl parahydroxybenzoate HOC6H4COOC2H5 [Same
bate 80, in heptane to make 50.0 mL.
as the namesake monograph]
Fatty oil Same as the fatty oil under the monograph.
2-Ethyl-2-phenylmalondiamide C11H14O2N2 White,
odorless crystals. Soluble in ethanol (95), and very slightly FBS-IMDM Dissolve an amount of the powder for 1 L
soluble in water. Melting point: about 1209 C (with decom- of Iscove's modified Dulbecco's powder medium, 0.1 g of
position). kanamycin sulfate (not less than 600 mg potency/mg), 3.0 g
Purity Related substances—To 5.0 mg of 2-ethyl-2- of sodium hydrogen carbonate and 36 mL of 2-mercapto
phenylmalondiamide add 4 mL of pyridine and 1 mL of bis- ethanol solution (1 in 10) in water to make 1000 mL, and
trimethylsilylacetamide, shake thoroughly, and heat at sterilize by filtration. To this solution add fetal bovine se-
1009C for 5 minutes. After cooling, add pyridine to make rum, previously heated at 569C for 30 minutes, so that the
exactly 10 mL, and use this solution as the sample solution. concentration of the serum is 10 volz.
Perform the test with 2 mL of the sample solution as directed
Fehling's TS The copper solution—Dissolve 34.66 g of
under Gas Chromatography <2.02> according to the condi-
copper (II) sulfate pentahydrate in water to make 500 mL.
tions in the Purity (3) under Primidone: any peak other than

Keep this solution in a glass-stoppered bottles in well-filled. Ferrous tartrate TS See iron (II) tartrate TS.
The alkaline tartrate solution—Dissolve 173 g of potas-
Ferrous thiocyanate TS See iron (II) thiocyanate TS.
sium sodium tartrate tetrahydrate and 50 g of sodium hy-
droxide in water to make 500 mL. Preserve this solution in a Ferrous trisodium pentacyanoamine TS See iron (II)
polyethylene container. trisodium pentacyanoamine TS.
Before use, mix equal volumes of both solutions.
(E )-Ferulic acid C10H10O4 White to light yellow, crys-
Fehling's TS for amylolytic activity test The copper solu- tals or crystalline powder. Freely soluble in methanol, solu-
tion—Dissolve 34.660 g of copper (II) sulfate pentahydrate, ble in ethanol (99.5), and practically insoluble in water.
accurately weighed, in water to make exactly 500 mL. Melting point: 173 – 1769C.
Preserve this solution in well-filled, glass-stoppered bottles. Identification—Determine the absorption spectrum of a
The alkaline tartrate solution—Dissolve 173 g of potas- solution in methanol (1 in 200,000) as directed under Ultravi-
sium sodium tartrate tetrahydrate and 50 g of sodium hy- olet-visible Spectrophotometry <2.24>: it exhibits maxima be-
droxide in water to make exactly 500 mL. Preserve this solu- tween 215 nm and 219 nm, between 231 nm and 235 nm, and
tion in polyethylene containers. between 318 nm and 322 nm.
Before use, mix exactly equal volumes of both solutions. Purity Related substances—Conduct this procedure
without exposure to light, using light-resistant vessels. Dis-
Felbinac for assay C14H12O2 [Same as the monograph
solve 1 mg of (E )-ferulic acid in 1 mL of methanol, and use
Felbinac. It, when dried, contains not less than 99.0z of fel-
this solution as the sample solution. Perform the test with
binac (C14H12O2).]
the sample solution as directed under Thin-layer Chromatog-
Ferric ammonium citrate See ammonium iron (III) raphy <2.03>. Spot 2 mL of the sample solution on a plate of
citrate. silica gel for thin-layer chromatography. Develop the plate
with a mixture of ethyl acetate, acetone and water (20:12:3)
Ferric ammonium sulfate See ammonium iron (III)
to a distance of about 7 cm, and air-dry the plate. Spray
sulfate dodecahydrate.
evenly dilute sulfuric acid on the plate, and heat at 1059C for
Ferric ammonium sulfate TS See ammonium iron (III) 5 minutes. Examine under ultraviolet light (main wave-
sulfate TS. length: 365 nm): no spot appears other than the principle
spot at the R f value of about 0.6.
Ferric ammonium sulfate TS, dilute See ammonium iron
(III) sulfate TS, dilute. (E)-Ferulic acid for assay C10H10O4 Use (E )-ferulic
acid. It meets the following additional requirements.
Ferric chloride See iron (III) chloride hexahydrate.
Absorbance <2.24> E 11zcm (320 nm): 878 – 969 (5 mg,
Ferric chloride-acetic acid TS See iron (III) chloride- methanol, 1000 mL).
acetic acid TS. Purity Related substances—Conduct this procedure
without exposure to light, using light-resistant vessels. Dis-
Ferric chloride-iodine TS See iron (III) chloride-iodine
solve 5 mg of (E )-ferulic acid for assay in 10 mL of a mix-
TS.
ture of water and methanol (1:1), and use this solution as the
Ferric chloride-methanol TS See iron (III) chloride- sample solution. Pipet 1 mL of the sample solution, add a
methanol TS. mixture of water and methanol (1:1) to make exactly 100
mL, and use this solution as the standard solution. Perform
Ferric chloride-pyridine TS, anhydrous See iron (III)
the test with exactly 10 mL each of the sample solution and
chloride-pyridin TS, anhydrous.
standard solution as directed under Liquid Chromatography
Ferric chloride TS See iron (III) chloride TS. <2.01> according to the following conditions, and determine
the peak areas of each peak by the automatic integration
Ferric chloride TS, acidic See iron (III) chloride TS,
method: the total area of the peaks other than (E )-ferulic
acidic.
acid obtained from the sample solution is not larger than the
Ferric chloride TS, dilute See iron (III) chloride TS, peak area of (E )-ferulic acid obtained from the standard so-
dilute. lution.
Operating conditions
Ferric nitrate See iron (III) nitrate enneahydrate.
Detector, column, column temperature, mobile phase, and
Ferric nitrate TS See iron (III) nitrate TS. flow rate: Proceed as directed in the operating conditions in
the Assay (1) under Tokishakuyakusan Extract.
Ferric perchlorate See iron (III) perchlorate hexahy-
Time span of measurement: About 6 times as long as the
drate.
retention time of (E )-ferulic acid, beginning after the solvent
Ferric perchlorate-dehydrated ethanol TS See iron (III) peak.
perchlorate-ethanol TS. System suitability
Test for required detectability: To exactly 1 mL of the
Ferric salicylate TS See Iron salicylate TS
standard solution add a mixture of water and methanol (1:1)
Ferric sulfate See iron (III) sulfate n-hydrate. to make exactly 20 mL. Confirm that the peak area of (E )-
ferulic acid obtained with 10 mL of this solution is equivalent
Ferric sulfate TS See iron (III) sulfate TS.
to 3.5 to 6.5z of that obtained with 10 mL of the standard
Ferrous ammonium sulfate See ammonium iron (II) solution.
sulfate hexahydrate. System performance: When the procedure is run with 10
mL of the standard solution under the above operating con-
Ferrous sulfate See iron (II) sulfate heptahydrate.
ditions, the number of theoretical plates and the symmetry
Ferrous sulfate TS See iron (II) sulfate TS. factor of the peak of (E )-ferulic acid are not less than 5000
and not more than 1.5, respectively.
Ferrous sulfide See iron (II) sulfide.
System repeatability: When the test is repeated 6 times

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
256 General Tests / 9.41 Reagents, Test Solutions JP XVII
with 10 mL of the standard solution under the above operat- 1114 cm－1 and 849 cm－1.
ing conditions, the relative standard deviation of the peak
Fluorescein sodium C20H10Na2O5 [Same as the name-
area of (E )-ferulic acid is not more than 1.5z.
sake monograph].
Fetal calf serum Serum obtained from fetal calves. Inter-
Fluorescein sodium TS Dissolve 0.2 g of fluorescein so-
leukin-2 dependent cell growth suppression substance is re-
dium in water to make 100 mL.
moved by heat at 569C for 30 min before use.
Fluorescence TS Mix 400 mL of sodium dithionite solu-
Fibrinogen Fibrinogen is prepared from human or bo-
tion containing 6.27 g in 200 mL of water, 210 mL of 2-
vine blood by fractional precipitation with ethanol or ammo-
mercaptoethanol, 321 mL of acetic acid (100), 400 mL of 1,2-
nium sulfate. It may contain citrate, oxalate and sodium
diamino-4,5-methylenedioxybenzene solution containing
chloride. A white amorphous solid. Add 1 mL of isotonic so-
31.1 mg in 1.0 mL of water and 2669 mL of water. Prepare
dium chloride solution to 10 mg of fibrinogen. It, when
before use.
warmed to 379C, dissolves with a slight turbidity, and clots
on the subsequent addition of 1 unit of thrombin. 4-Fluorobenzoic acid C7H5FO2 White, crystals or crys-
talline powder.
1st Fluid for disintegration test See 1st fluid for dissolu-
Identification—Determine the infrared absorption spec-
tion test.
trum as directed in the potassium bromide disk method
1st Fluid for dissolution test Dissolve 2.0 g of sodium under Infrared Spectrophotometry <2.25>: it exhibits absorp-
chloride in 7.0 mL of hydrochloric acid and water to make tion at the wave numbers of about 1684 cm－1, 1606 cm－1
1000 mL. It is clear and colorless, and has a pH of about and 1231 cm－1.
1.2. Melting point <2.60>: 182 – 1889C
Fixed oil Same as the vegetable oils under the mono- 1-Fluoro-2,4-dinitrobenzene C6H3(NO2)2F Light yel-
graph. C.
low, liquid or crystalline masses. Melting point: about 259
Identification—Determine the infrared absorption spec-
FL cell Established cell strain derived from normal hu-
trum as directed in the liquid film method as directed under
man amnion. Subculture the cells in Eagle's minimum essen-
Infrared Spectrophotometry <2.25>: it exhibits absorption at
tial medium containing bovine serum.
the wave numbers of about 3110 cm－1, 1617 cm－1, 1538
Flecainide acetate C17H20F6N2O3.C2H4O2 [Same as the cm－1, 1345 cm－1, 1262 cm－1 and 743 cm－1.
namesake monograph] Preserve in a light-resistant tight container.
Flecainide acetate for assay C17H20F6N2O3.C2H4O2 Fluorogenic substrate TS A solution containing ox-
[Same as the monograph Flecainide Acetate. When dried, idation-reduction indicator.
it contains not less than 99.0z of flecainide acetate
7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazole C6H2FN3O3
(C17H20F6N2O3.C2H4O2). Additionally, when perform the
Prepared for amino acid analysis or biochemistry.
test as directed in the Purity (3), the sample solution does not
show the spot corresponding to the spot obtained from the Fluoroquinolonic acid for thin-layer chromatography
standard solution, and when perform the test as directed in C13H9ClFNO3 A white to light brown powder.
the Purity (4), the total area of the peaks other than Purity Perform the test with 8 mL of a solution of
flecainide is not larger than the peak area of flecainide from fluoroquinolonic acid for thin-layer chromatography in
the standard solution.] acetonitrile (1 in 1250) as directed under Liquid Chromatog-
raphy <2.01> according to the following conditions, deter-
Flopropione C9H10O4 [Same as the namesake mono-
mine each peak area by the automatic integration method,
graph]
and calculate the amount of them by the area percentage
Flopropione for assay C9H10O4 [Same as the mono- method: the amount of the peak of fluoroquinolonic acid is
graph Flopropione. It contains not less than 99.0z of not less than 98.0z.
flopropione (C9H10O4), calculated on the anhydrous basis.] Operating conditions
Detector: An ultraviolet absorption photometer (wave-
Fluconazole for assay C13H12F2N6O [Same as the
length: 263 nm).
monograph Fluconazole]
Column: A stainless steel column 4 mm in inside diameter
Fluid thioglycolate medium See the Sterility Test <4.06>. and 12.5 cm in length, packed with octadecylsilanized silica
gel for liquid chromatography (5 mm in particle diameter).
Fluocinolone acetonide C24H30F2O6 [Same as the name-
Column temperature: A constant temperature of about
sake monograph]
409C.
9-Fluorenylmethyl chloroformate C15H11ClO2 White, Mobile phase A: Diluted phosphoric acid (1 in 500).
crystals or crystalline powder. Mobile phase B: Methanol.
Melting point <2.60>: 60 – 639C Flow of mobile phase: Control the gradient by mixing the
mobile phases A and B as directed in the following table.
9-Fluorenylmethyl chloroformate C15H11ClO2
Prepared for amino acid analysis or biochemistry.
Time after injection Mobile phase A Mobile phase B
Fluorescamine C17H10O4 A white powder. of sample (min) (volz) (volz)
Fluorescein C20H12O5 An yellowish red powder.
0 – 5.5 60 → 55 40 → 45
Identification—Determine the infrared absorption spec-
5.5 – 14 55 → 25 45 → 75
trum as directed in the potassium bromide disk method
14 – 15 25 → 15 75 → 85
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
tion at the wave numbers of about 1597 cm－1, 1466 cm－1,
1389 cm－1, 1317 cm－1, 1264 cm－1, 1247 cm－1, 1213 cm－1, Flow rate: 1.5 mL per minute (retention time of fluoro-

quinolonic acid: about 8 min). 2-Formylbenzoic acid CHOC6H4COOH White crys-

Time span of measurement: For 15 minutes after injec- tals. Melting point: 97 – 999C.
tion, beginning after the solvent peak. Content: not less than 99.0z. Assay—Weigh accurately
System suitability about 0.3 g of 2-formylbenzoic acid, previously dried (in
System performance: When perform the test with 8 mL of vacuum, phosphorus (V) oxide, 3 hours), dissolve in 50 mL
a solution of fluoroquinolonic acid for thin-layer chro- of freshly boiled and cooled water, and titrate <2.50> with
matography in acetonitrile (1 in 1250) according to the above 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of
operating conditions, the number of theoretical plates and phenol red TS).
the symmetry factor of the peak of fluoroquinolonic acid are
Each mL of 0.1 mol/L sodium hydroxide VS
not less than 10,000 and not more than 1.5, respectively.
＝ 15.01 mg of C8H6O3
Flurazepam for assay C21H23ClFN3O [Same as the
Forsythia fruit [Same as the namesake monograph.]
monograph Flurazepam. When drid, it contains not less than
99.3zof flurazepan (C21H23CIFN3O).] Freund's complete adjuvant A suspension of 5 mg of
mycobacteria of Corynebacterium butyricum, killed by
Flutoprazepam for assay C19H16ClFN2O [Same as the
heating, in 10 mL of a mixture of mineral oil and aricel A
monograph Flutoprazepam. When dried, it contains not less
(17:3).
than 99.5z of flutoprazepam (C19H16ClFN2O).]
Fructose C6H12O6 [Same as the namesake monograph]
Folic acid C19H19N7O6 [Same as the namesake mono-
graph] Fructose for thin-layer chromatography C6H12O6
Colorless to white, crystals or crystalline powder. Very solu-
Folin's TS Place 20 g of sodium tungstate (VI) dihy-
ble in water, and sparingly soluble in ethanol (99.5). It is
drate, 5 g of disodium molybdate (VI) dihydrate and about
deliquescence with the atmospheric moisture.
140 mL of water in a 300-mL volumetric flask, add 10 mL of
Optical rotation <2.49> [a]20 D : －88 – －949(1 g, diluted
diluted phosphoric acid (17 in 20) and 20 mL of hydrochloric
ammonia solution (28) (1 in 1000), 100 mL, 100 mm. Previ-
acid, and boil gently using a reflux condenser with ground-
ously, dried over silica gel as the desiccant for 3 hours).
glass joints for 10 hours. To the mixture add 30 g of lithium
Purity Related substances—Dissolve 2 mg of Fructose
sulfate monohydrate and 10 mL of water, and then add a
for thin-layer chromatography in 1 mL of a mixture of water
very small quantity of bromine to change the deep green
and methanol (1:1), and use this solution as the sample solu-
color of the solution to yellow. Remove the excess bromine
tion. Perform the test with the sample solution as directed
by boiling for 15 minutes without a condenser, and cool.
under Thin-layer chromatography <2.03>. Spot 2 mL of the
Add water to make 200 mL, and filter through a glass filter.
sample solution on a plate of silica gel for thin-layer chroma-
Store it free from dust. Use this solution as the stock solu-
tography. Develop the plate with a mixture of 2-propanol,
tion, and dilute with water to the directed concentration
water and methanol (3:2:2) to a distance of about 7 cm, and
before use.
air-dry the plate. Spray evenly 1,3-naphthalenediol TS to the
Folin's TS, dilute Titrate <2.50> Folin's TS with 0.1 plate, and heat at 1059 C for 10 minutes: any spot other than
mol/L sodium hydroxide VS (indicator: phenolphthalein the principle spot with an Rf value of about 0.6 does not
TS), and determine the acid concentration. Prepare by add- appear.
ing water to Folin's TS so the acid concentration is 1 mol/L.
Fuchsin A lustrous, green, crystalline powder or mass,
Formaldehyde solution HCHO [K 8872, Special class] slightly soluble in water and in ethanol (95).
Loss on drying <2.41>: 17.5 – 20.0z (1 g, 1059C, 4 hours).
Formaldehyde solution-sulfuric acid TS Add 1 drop of
Residue on ignition <2.44>: not more than 0.1z (1 g).
formaldehyde solution to 1 mL of sulfuric acid. Prepare be-
fore use. Fuchsin-ethanol TS Dissolve 11 g of fuchsin in 100 mL
of ethanol (95).
Formaldehyde solution TS To 0.5 mL of formaldehyde
solution add water to make 100 mL. Fuchsin-sulfurous acid TS Dissolve 0.2 g of fuchsin in
120 mL of hot water, and allow the solution to cool. Add a
Formaldehyde TS, dilute Dilute formaldehyde solution
solution prepared by dissolving 2 g of anhydrous sodium sul-
to 10 times its volume with water.
fite in 20 mL of water, then add 2 mL of hydrochloric acid
Formalin See formaldehyde solution. and water to make 200 mL, and allow to stand for at least 1
hour. Prepare before use.
Formalin TS See formaldehyde solution TS.
Fudosteine for assay C6H13NO3S [Same as the mono-
Formalin-sulfuric acid TS See formaldehyde solution-
graph Fudosteine]
sulfuric acid TS.
Fumaric acid for thin-layer chromatography C4H4O4
Formamide HCONH2 [K 8873, Special class]
White, crystalline powder, odorless, and has a characteristic
Formamide for water determination HCONH2 acid taste.
[K 8873, Special class; water content per g of formamide for Purity—Perform the test as directed in the Identification
water determination should be not more than 1 mg.] (5) under Clemastine Fumarate: any spot other than the
principal spot at the R f value of about 0.8 does not appear.
Formazin opalescence standard solution To 15 mL of
formazin stock suspension add water to make 1000 mL. Use Fuming nitric acid See nitric acid, fuming.
within 24 hours after preparation. Shake thoroughly before
Fuming sulfuric acid See sulfuric acid, fuming.
use.
Furfural C5H4O2 A clear, colorless liquid.
Formic acid HCOOH [K 8264, Special class, specific
Specific gravity <2.56> d 20
20: 1.160 – 1.165
gravity: not less than 1.21].
Distilling range <2.57>: 160 – 1639C, not less than 95

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
258 General Tests / 9.41 Reagents, Test Solutions JP XVII
volz. Geniposide for assay C17H24O10 Use geniposide for
thin-layer chromatography meeting the following additional
D-Galactosamine hydrochloride C6H13NO5.HCl White
specifications, 1) Geniposide for assay 1 or 2) Geniposide for
powder. Melting point: about 1809C (with decomposition).
assay 2 (Purity value by quantitative NMR). The former is
Optical rotation <2.49> [a]20
D : ＋90 – ＋979 (1 g, water,
used after drying (in vacuum, phosphorus (V) oxide, 24
100 mL, 100 mm).
hours), and the latter is corrected its content based on the
Galactose See D-galactose. amount (z) obtained in the Assay.
1) Geniposide for assay 1
D-Galactose C6H12O6 White, crystals, granules or pow-
Absorbance <2.24> E 11zcm (240 nm): 249 – 269 [10 mg
der.
dried in a desiccator (in vacuum, phosphorus (V) oxide) for
Identification—Determine the infrared absorption spec-
24 hours, diluted methanol (1 in 2), 500 mL].
trum as directed in the potassium bromide disk method
Purity Related substances—Dissolve 5 mg of geniposide
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
for assay in 50 mL of diluted methanol (1 in 2), and use this
tion at the wave numbers of about 3390 cm－1, 3210 cm－1,
solution as the sample solution. Pipet 1 mL of the sample so-
3140 cm－1, 1151 cm－1, 1068 cm－1, 956 cm－1, 836 cm－1, 765
lution, add diluted methanol (1 in 2) to make exactly 100
cm－1 and 660 cm－1.
mL, and use this solution as the standard solution. Perform
Optical rotation <2.49> [a]20 D : ＋79 – ＋829 (desiccator
the test with exactly 10 mL each of the sample solution and
(silica gel), 2.5 g after drying for 18 hours, diluted ammonia
standard solution as directed under Liquid Chromatography
solution (28) (1 in 300), 25 mL, 100 mm).
<2.01> according to the following conditions, and determine
Gallic acid See gallic acid monohydrate. each peak area by the automatic integration method: the
total area of the peaks other than geniposide obtained from
Gallic acid monohydrate C6H2(OH)3COOH.H2O
the sample solution is not larger than the peak area of
White to pale yellowish white, crystals or powder.
geniposide obtained from the standard solution.
Melting point <2.60>: about 2609C (with decomposition).
Operating conditions
Gelatin [Same as the namesake monograph] Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
Gelatin, acid-treated [Same as the monograph Gelatin.
the Assay under Gardenia Fruit.
Its isoelectric point is at pH between 7.0 and 9.0]
Time span of measurement: About 3 times as long as the
Gelatin peptone See peptone, gelatin. retention time of geniposide, beginning after the solvent
peak.
Gelatin-phosphate buffer solution Dissolve 13.6 g of po-
System suitability
tassium dihydrogen phosphate, 15.6 g of sodium dihydrogen
System performance and system repeatability: Proceed as
phosphate dihydrate and 1.0 g of sodium azide in water to
directed in the system suitability in the Assay under Garde-
make 1000 mL, adjust the pH to 3.0 with diluted phosphoric
nia Fruit.
acid (1 in 75) (solution A). Dissolve 5.0 g of acid-treated
Test for required detectability: Pipet 1 mL of the standard
gelatin in 400 mL of the solution A by warming, after cool-
solution, add diluted methanol (1 in 2) to make exactly 20
ing, adjust the pH to 3.0 with diluted phosphoric acid (1 in
mL. Confirm that the peak area of geniposide obtained with
75), and add the solution-A to make 1000 mL.
10 mL of this solution is equivalent to 3.5 to 6.5z of that
Gelatin-phosphate buffer solution (pH 7.0) Dissolve obtained with 10 mL of the standard solution.
1.15 g of sodium dihydrogen phosphate dihydrate, 5.96 g of 2) Geniposide for assay 2 (Purity value by quantitative
disodium hydrogen phosphate dodecahydrate and 5.4 g of NMR)
sodium chloride in 500 mL of water. Dissolve 1.2 g of gelatin Unity of peak—Dissolve 5 mg of geniposide for assay 2 in
to this solution by heating, and after cooling add water to 50 mL of diluted methanol (1 in 2). To 1 mL of this solution
make 600 mL. add diluted methanol (1 in 2) to make 100 mL, and use this
solution as the sample solution. Perform the test with 10 mL
Gelatin-phosphate buffer solution (pH 7.4) To 50 mL of
of the sample solution as directed under Liquid Chromatog-
0.2 mol/L potassium dihydrogen phosphate TS for buffer
raphy <2.01> according to the following conditions, and
solution add 39.50 mL of 0.2 mol/L sodium hydroxide TS
compare the absorption spectra of at least 3 points including
and 50 mL of water. Dissolve 0.2 g of gelatin to this solution
the top of geniposide peak and around the two middle peak
by heating, then after cooling adjust to pH 7.4 with 0.2
heights of before and after the top: no difference in form is
mol/L sodium hydroxide TS, and add water to make 200
observed between their spectra.
mL.
Operating conditions
Gelatin-tris buffer solution Dissolve 6.06 g of 2-amino- Column, column temperature, mobile phase, and flow
2-hydroxymethyl-1,3-propanediol and 2.22 g of sodium rate: Proceed as directed in the operating conditions in the
chloride in 700 mL of water. Separately, dissolve 10 g of Assay under Gardenia Fruit.
acid-treated gelatin in 200 mL of water by warming. After Detector: A photodiode array detector (wavelength: 240
cooling, mix these solutions, and adjust the pH to 8.8 with nm, measuring range of spectrum: 220 – 400 nm).
dilute hydrochloric acid, and add water to make 1000 mL. System suitability
System performance: Proceed as directed in the system
Gelatin-tris buffer solution (pH 8.0) Dissolve 40 g of 2-
suitability in the Assay under Gardenia Fruit.
amino-2-hydroxymethyl-1,3-propanediol and 5.4 g of so-
Assay—Weigh accurately 10 mg of geniposide for assay 2
dium chloride in 500 mL of water. Add 1.2 g of gelatin to
and 1 mg of 1,4-BTMSB-d4 for nuclear magnetic resonance
dissolve by heating, adjust to pH 8.0 with dilute hydrochlo-
spectroscopy using an ultramicrobalance, dissolve in 1 mL of
ric acid after cooling, and add water to make 600 mL.
deuterated methanol for nuclear magnetic resonance spec-
Gelatin TS Dissolve 1 g of gelatin in 50 mL of water by troscopy, and use this solution as the sample solution.
gentle heating, and filter if necessary. Prepare before use. Transfer the sample solution into an NMR tube 5 mm in
outer diameter, measure 1H-NMR as directed under Nuclear

Magnetic Resonance Spectroscopy <2.21> and Crude Drugs rected in the Identification (2) under Gardenia Fruit: no spot
Test <5.01> according to the following conditions, using 1,4- other than the principal spot at an R f value of about 0.3 is
BTMSB-d4 for nuclear magnetic resonance spectroscopy as observed.
the internal reference compound. Calculate the resonance in-
Gentamicin B C19H38N4O10 White to pale yellowish
tensities, A1 (equivalent to 1 hydrogen) and A2 (equivalent
white powder. Very soluble in water, and practically insolu-
to 1 hydrogen), of the signals around d 3.93 ppm and d 4.06
ble in ethanol (95).
ppm assuming the signal of the internal reference compound
Content: not less than 80.0z. Assay—Dissolve a suita-
as d 0 ppm.
ble amount of gentamicin B in 0.05 mol/L sulfuric acid TS
Amount (z) of geniposide (C17H24O10) to make the solution containing 0.1 mg of gentamicin B per
＝ MS × I × P/(M × N ) × 1.7147 mL, and use this solution as the sample solution. Perform
the test with 5 mL of the sample solution as directed under
M: Amount (mg) of geniposide for assay 2 taken
Liquid Chromatography <2.01> according to the following
MS: Amount (mg) of 1,4-BTMSB-d4 for nuclear magnetic
conditions, and measure each peak area by the automatic
resonance spectroscopy taken
integration method. Calculate the amount of gentamicin B
I: Sum of the signal resonance intensities, A1 and A2,
by the area percentage method.
based on the signal resonance intensity of
Operating conditions
1,4-BTMSB-d4 for nuclear magnetic resonance spectros-
Apparatus, detector, column, column temperature, reac-
copy as 18.000
tion coil, mobile phase, reagent, reaction temperature, flow
N: Sum of number of the hydrogen derived from A1 and
rate of the mobile phase, and flow rate of the reagent: Pro-
A2
ceed the operating conditions in the Assay under Isepamicin
P: Purity (z) of 1,4-BTMSB-d4 for nuclear magnetic
Sulfate.
resonance spectroscopy
Time span of measurement: About 3 times as long as the
Operating conditions retention time of gentamicin B.
Apparatus: An apparatus of nuclear magnetic resonance System suitability
spectrum measurement having 1H resonance frequency of Proceed the system suitability in the Assay under Isepami-
not less than 400 MHz. cin Sulfate.
Target nucleus: 1H.
Gentiopicroside for thin-layer chromatography C16H20O9
Digital resolution: 0.25 or lower.
A white powder. Freely soluble in water and in methanol,
Measuring spectrum range: 20 ppm or upper, including
and practically insoluble in diethyl ether. Melting point:
between －5 ppm and 15 ppm.
about 1109C (with decomposition).
Spinning: off.
Purity Related substances—Dissolve 10 mg of gen-
Pulse angle: 909.
13C decoupling: on. tiopicroside for thin-layer chromatography in 2 mL of meth-
anol, and use this solution as the sample solution. Pipet 1
Delay time: Repeating pulse waiting time not less than 60
mL of the sample solution, and methanol to make exactly
seconds.
100 mL, and use this solution as the standard solution. Per-
Integrating times: 8 or more times.
form the test with 10 mL each of the sample solution and
Dummy scanning: 2 or more times.
standard solution as directed in the Identification (2) under
Measuring temperature: A constant temperature between
Gentian: the spots other than the principal spot at the R f
209 C and 309C.
value of about 0.4 from the sample solution are not more in-
System suitability
tense than the spot from the standard solution.
Test for required detectability: When the procedure is run
with the sample solution under the above operating condi- Giemsa's TS Dissolve 3 g of azure II-eosin Y and 0.8 g
tions, the S/N of the two signals of around d 3.93 ppm and d of azure II in 250 g of glycerin by warming to 609C. After
4.06 ppm is not less than 100. cooling, add 250 g of methanol, and mix well. Allow to
System performance: When the procedure is run with the stand for 24 hours, and filter. Store in tightly stoppered
sample solution under the above operating conditions, the bottles.
two signals of around d 3.93 ppm and d 4.06 ppm are not Azure II-eosin Y is prepared by coupling eosin Y to azure
overlapped with any signal of obvious foreign substance, II. Azure II is the mixture of equal quantities of methylene
and the ratios of the resonance intensities, A1/A2, of each azure (azure I), prepared by oxidizing methylene blue, and
signal around d 3.93 ppm and d 4.06 ppm are between 0.99 methylene blue.
and 1.01, respectively.
Giemsa's TS, dilute Dilute Giemsa's TS to about 50
System repeatability: When the test is repeated 6 times
times its volume with a solution prepared by dissolving
with the sample solution under the above operating condi-
4.54 g of potassium dihydrogen phosphate and 4.75 g of
tions, the relative standard deviation of the ratio of the reso-
anhydrous disodium hydrogen phosphate in water to make
nance intensity, A1 or A2, to that of the internal reference is
1000 mL, and filter with a filter paper. Prepare before use.
not more than 1.0z.
[6]-Gingerol for assay C17H26O4 [6]-Gingerol for thin-
Geniposide for component determination See geniposide
layer chromatography. However, it meets the following re-
for assay.
quirements:
Geniposide for thin-layer chromatography C17H24O10 Absorbance <2.24> E 11zcm (281 nm): 101 – 112 [7 mg,
White, crystals or crystalline powder. Freely soluble in water ethanol (99.5), 200 mL].
and in methanol, and soluble in ethanol (99.5). Melting Purity Related substances—Dissolve 5 mg of [6]-gingerol
point: about 1609 C. for assay in 5 mL of methanol, and use this as the sample so-
Purity Related substances—Dissolve 1.0 mg of genipo- lution. Pipet 1 mL of the sample solution, add methanol to
side for thin-layer chromatography in exactly 1 mL of meth- make exactly 50 mL, and use this as the standard solution.
anol, and perform the test with 20 mL of this solution as di- Perform the test with exactly 10 mL each of the sample solu-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
260 General Tests / 9.41 Reagents, Test Solutions JP XVII
tion and standard solution as directed under Liquid Chroma- <2.01> according to the conditions directed in the Assay (2)
tography <2.01> according to the following conditions, and under Ginseng until ginsenoside Rc is eluted: the total area
determine each peak area by the automatic integration of the peaks other than ginsenoside Rc and solvent peak is
method: the total area of the peaks other than [6]-gingerol not larger than 1/10 times the total peak area excluding the
from the sample solution is not larger than the peak area of peak area of the solvent.
[6]-gingerol from the standard solution.
Ginsenoside Re C48H82O18 A white crystalline powder.
Operating conditions
It is odorless.
Detector, column, column temperature, mobile phase, and
Purity—Dissolve 1.0 mg of ginsenoside Re in diluted
flow rate: Proceed as directed in the operating conditions in
methanol (3 in 5) to make 10 mL. Perform the test with 10
the Assay (3) under Hangekobokuto Extract.
mL of this solution as directed under Liquid Chroma-
Time span of measurement: About 6 times as long as the
tography <2.01> according to the conditions directed in the
retention time of [6]-gingerol.
Assay (1) under Ginseng until ginsenoside Re is eluted: the
System suitability
total area of the peaks other than ginsenoside Re and solvent
System performance and system repeatability: Proceed as
peak is not larger than 1/10 times the total peak area exclud-
directed in the system suitability in the Assay (3) under
ing the peak area of the solvent.
Hangekobokuto Extract.
Test for required detectability: Pipet 1 mL of the standard Ginsenoside Rg1 for thin-layer chromatography
solution, and add methanol to make exactly 20 mL. Confirm C42H72O14 A white, powder or crystalline powder. Very
that the peak area of [6]-gingerol obtained with 10 mL of this soluble in methanol and in ethanol (99.5), and soluble in
solution is equivalent to 3.5 to 6.5z of that obtained with 10 water. It is hygroscopic.
mL of the standard solution. Identification Determine the infrared absorption spec-
trum as directed in the potassium bromide disk method
[6]-Gingerol for component determination See [6]-gin-
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
gerol for assay.
tion at the wave numbers of about 3390 cm－1, 1642 cm－1,
[6]-Gingerol for thin-layer chromatography C17H26O4 1075 cm－1 and 1032 cm－1.
A yellow-white to yellow, liquid or solid. Freely soluble in Purity Related substances—Dissolve 2 mg of ginsenoside
methanol, in ethanol (99.5) and in diethyl ether, and practi- Rg1 to be examined in 1 mL of methanol, and use this as the
cally insoluble in water. sample solution. Pipet 0.5 mL of the sample solution, add
Identification—Determine the absorption spectrum of a methanol to make exactly 25 mL, and use as the standard so-
solution of [6]-gingerol for thin-layer chromatography in lution. Perform the test with 2 mL each of the sample solu-
ethanol (99.5) (7 in 200,000) as directed under Ultraviolet- tion and standard solution as directed in the Identification
visible Spectrophotometry <2.24>: it exhibits a maximum be- (2) under Ginseng by developing the plate without entirely
tween 279 nm and 283 nm. drying after applying the solutions: any spot other than the
Purity Related substances—Dissolve 1.0 mg of [6]-gin- principal spot with an Rf value of about 0.5 obtained from
gerol for thin-layer chromatography in exactly 2 mL of the sample solution is not more intense than the spot ob-
methanol. Perform the test with 10 mL of this solution as di- tained from the standard solution.
rected in the Identification under Ginger: any spot other
Glacial acetic acid See acetic acid (100).
than the principal spot at the R f value of about 0.3 does not
appear. Glacial acetic acid for nonaqueous titration See acetic
acid for nonaqueous titration.
Ginsenoside Rb1 for thin-layer chromatography
C54H92O23 A white powder. Freely soluble in water and in Glacial acetic acid-sulfuric acid TS See acetic acid sul-
methanol, and sparingly soluble in ethanol (99.5). It is furic acid TS.
hygroscopic.
g-Globulin A plasma protein obtained from human se-
Identification Determine the infrared absorption spec-
rum as Cohn's II and III fractions. White crystalline pow-
trum of ginsenoside Rb1 for thin-layer chromatography as
der. It contains not less than 98z of g-globulin in the total
directed in the potassium bromide disk method under In-
protein.
frared Spectrophotometry <2.25>: it exhibits absorption at
the wave numbers of about 3390 cm－1, 1650 cm－1, D-Glucosamine hydrochloride C6H13NO5.HCl White,
1077cm－1 and 1038 cm－1. crystals or crystalline powder.
Purity Related substances—Dissolve 2 mg of ginsenoside Content: not less than 98z. Assay—Dissolve about
Rb1 for thin-layer chromatography in 1 mL of methanol, 0.4 g of D-glucosamine hydrochloride, accurately weighed,
and use this solution as the sample solution. Pipet 0.5 mL of in 50 mL of water, add 5 mL of diluted nitric acid (1 in 3),
the sample solution, add methanol to make exactly 25 mL, and titrate <2.50> with 0.1 mol/L silver nitrate VS (potentio-
and use this solution as the standard solution. Perform the metric titration).
test with 2 mL each of the sample solution and standard solu-
Each mL of 0.1 mol/L silver nitrate VS
tion as directed in the Identification (2) under Ginseng by de-
＝ 21.56 mg of C6H13NO5.HCl
veloping the plate without entirely drying after applying the
solutions: any spot other than the principal spot with an R f Glucose C6H12O6 [Same as the namesake monograph]
value of about 0.3 obtained from the sample solution is not
Glucose detection TS Dissolve 1600 units of glucose oxi-
more intense than the spot obtained from the standard solu-
dase, 16 mg of 4-aminoantipyrine, 145 units of peroxidase
tion.
and 0.27 g of p-hydroxybenzoic acid in tris buffer solution
Ginsenoside Rc C53H90O22 A white crystalline powder. (pH 7.0) to make 200 mL.
It is odorless.
Glucose detection TS for penicillium origin b-galactosi-
Purity—Dissolve 1 mg of ginsenoside Rc in diluted metha-
dase Dissolve glucose oxidase (not less than 500 units),
nol (3 in 5) to make 10 mL. Perform the test with 10 mL of
peroxidase (not less than 50 units), 10 mg of 4-aminoantipy-
this solution as directed under Liquid Chromatography

rine and 0.1 g of phenol in phosphate buffer (pH 7.2) to Glutathione C10H17N3O6S [Same as the namesake
make 100 mL. monograph]
Glucose oxidase Obtained from Aspergillus nigar. Glycerin C3H8O3 [K 8295, Glycerol, Special class, or
White powder. It is freely soluble in water. It contains about same as the monograph Concentrated Glycerin]
200 Units per mg. One unit indicates an amount of the en-
85z Glycerin C 3 H8 O 3 [Same as the monograph Glyce-
zyme which produces 1 mmol of D-glucono-d-lactone in 1
rin]
minute at 259C and pH 7.0 from glucose used as the sub-
strate. Glycerin for gas chromatography C3H8O3 [K 8295,
Special class] When perform the test as directed in the
Glucose TS Dissolve 30 g of glucose in water to make
Purity (11) under Concentrated Glycerin, it does not show
100 mL. Prepare as directed under Injections.
any peak at the retention times corresponding to ethylene
4?-O-Glucosyl-5-O-methylvisamminol for thin-layer chro- glycol and diethylene glycol.
matography C22H28O10 White, crystals or crystalline pow-
Glycine H2NCH2COOH [K 8291, Special class]
der. Freely soluble in methanol and in ethanol (99.5), and
sparingly soluble in water. Glycolic acid C2H4O3 Purity: not less than 98.0z.
Identification—Determine the absorption spectrum of a
N-Glycolylneuraminic acid C11H19NO10 White needle
solution of 4?-O-glucosyl-5-O-methylvisamminol for thin-
crystalline powder.
layer chromatography in ethanol (99.5) (1 in 50,000) as di-
rected under Ultraviolet-visible Spectrophotometry <2.24>: it 0.1 mmol/L N-Glycolylneuraminic acid TS Weigh accu-
exhibits a maximum between 286 nm and 290 nm. rately about 16.5 mg of N-glycolylneuraminic acid, and dis-
Purity Related substances—Dissolve 1 mg of 4?-O- solve in water to make exactly 50 mL. To exactly V mL of
glucosyl-5-O-methylvisamminol for thin-layer chromatogra- this solution add water to make exactly 100 mL.
phy in 1 mL of methanol. Perform the test with 5 mL of this
V (mL) ＝ 325.3 × 0.5/amount (mg) of
solution directed in the Identification under Saposhnikovia
N-glycolylneuraminic acid taken
Root and Rhizome: no spots other than the principal spot at
around R f value of 0.3 appears. Glycyrrhizic acid monoammonium salt for resolution
check See monoammonium glycyrrhizinate for solution
L-Glutamic acid HOOC(CH2)2CH(NH2)COOH
check
[K 9047, Special class]
Glycyrrhizic acid for thin-layer chromatography
L-Glutamine H2NCO(CH2)2CH(NH2)COOH
C42H62O16 White, crystals or crystalline powder. Freely
[K 9103, L(＋)-glutamine, Special class]
soluble in ethanol (99.5), and practically insoluble in water.
Glutamine TS Dissolve 2.92 g of L-glutamine in water to Identification Determine the infrared absorption spec-
make 100 mL, and sterilize by filtration through a mem- trum as directed in the potassium bromide disk method
brane filter with a pore size not exceeding 0.22 mm. under Infrared Spectrophotometry <2.25>: it exhibits absorp-
tion at the wave numbers of about 3420 cm－1, 1722 cm－1,
7-(Glutarylglycyl-L-arginylamino)-4-methylcoumarin
1654 cm－1 and 1389 cm－1.
C23H30N6O7 White powder. It is freely soluble in acetic acid
Purity Related substances—Dissolve 4 mg of glycyrrhizic
(100), sparingly soluble in dimethylsulfoxide, and practically
acid for thin-layer chromatography in 2 mL of ethanol
insoluble in water.
(99.5), and use this solution as the sample solution. Pipet 1
Absorbance <2.24> E 11zcm (325 nm): 310 – 350 [2 mg,
mL of the sample solution, add ethanol (99.5) to make ex-
diluted acetic acid (100) (1 in 500), 200 mL].
actly 100 mL, and use this solution as the standard solution.
Optical rotation <2.49> [a]20
D : －50 – －609[0.1 g, diluted
Perform the test with 10 mL each of the sample solution and
acetic acid (100) (1 in 2), 10 mL, 100 mm].
standard solution as directed in the Identification under
Purity Related substances—Prepare the sample solution
Glycyrrhiza: the spots other than the principal spot with an
by dissolving 5 mg of 7-(glutarylglycyl-L-arginylamino)-4-
Rf value of about 0.3 obtained from the sample solution are
methylcoumarin in 0.5 mL of acetic acid (100), and perform
not more intense than the spot obtained from the standard
the test as directed under Thin-layer Chromatography
solution.
<2.03>. Spot 5 mL of the sample solution on a plate of silica
gel for thin-layer chromatography. Develop the plate with a Goat anti-ECP antibody Combine 1 volume of ECP
mixture of 1-butanol, water, pyridine and acetic acid (100) standard substance (equivalent to about 1 mg of protein) and
(15:12:10:3) to a distance of about 10 cm, air-dry the plate, 1 volume of Freund's complete adjuvant, and immunize
and dry more at 809 C for 30 minutes. After cooling, allow goats subcutaneously in the back region with this solution 5
the plate to stand for 30 minutes in a box filled with iodine times at 2 week intervals. Harvest blood on the 10th day after
vapors: any observable spot other than the principal spot at completing the immunization to obtain goat antiserum. Goat
the R f value of about 0.6 does not appear. anti-ECP antibody is obtained by preparing an immobilized
ECP column in which ECP standard substance is bound to
7-(Glutarylglycyl-L-arginylamino)-4-methylcoumarin TS
sepharose 4B and then purifying by affinity column chroma-
Dissolve 5 mg of 7-(glutarylglycyl-L-arginylamino)-4-methyl-
tography.
coumarin in 0.5 to 1 mL of acetic acid (100), lyophilize, dis-
Description: Clear and colorless solution.
solve this in 1 mL of dimethylsulfoxide, and use this solution
Identification: When sodium lauryl sulfate-supplemented
as solution A. Dissolve 30.0 g of 2-amino-2-hydroxymethyl-
polyacrylamide gel electrophoresis is conducted under non-
1,3-propanediol and 14.6 g of sodium chloride in 400 mL of
reducing conditions, the molecular weight of the major band
water, adjust the pH to 8.5 with dilute hydrochloric acid,
is within the range of 1.30 × 105 to 1.70 × 105.
add water to make 500 mL, and use this solution as solution
Protein content: When determining the protein content
B. Mix 1 mL of the solution A and 500 mL of the solution B
using Assay (1) under Celmoleukin (Genetical Recombina-
before use.
tion), the protein content per mL is 0.2 to 1.0 mg.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
262 General Tests / 9.41 Reagents, Test Solutions JP XVII
Goat anti-ECP antibody TS Dilute goat anti-ECP anti- Carrier gas: Helium.
body with 0.1 mol/L carbonate buffer solution (pH 9.6) to Flow rate: Adjust so that the retention time of guaiacol is
prepare a solution containing 50 mg protein per mL. about 8 minutes.
Split ratio: 1:50.
Griess-Romijin's nitric acid reagent Triturate thoroughly
System suitability
1 g of 1-naphthylamine, 10 g of sulfanilic acid and 1.5 g of
Test for required detectability: Weigh accurately about 70
zinc dust in a mortar.
mg of guaiacol for assay, add methanol to make exactly 100
Storage—Preserve in tight, light-resistant containers.
mL, and use this solution as the solution for system suita-
Griess-Romijin's nitrous acid reagent Triturate thor- bility test. Confirm that the peak area of guaiacol obtained
oughly 1 g of 1-naphthylamine, 10 g of sulfanilic acid and from 1 mL of the solution for system suitability test is
89 g of tartaric acid in a mortar. equivalent to 0.08 to 0.16z of that of guaiacol obtained
Storage—Preserve in tight, light-resistant containers. when 0.5 mL of guaiacol for assay is injected.
System performance: When the procedure is run with 1 mL
Guaiacol CH3OC6H4OH Clear, colorless to yellow, liq-
of the solution for system suitability test under the above
uid or colorless crystals, having a characteristic aroma. Spar-
operating conditions, the number of theoretical plates and
ingly soluble in water, and miscible with ethanol (95), with
the symmetry factor of the peak of guaiacol are not less than
chloroform and with diethyl ether. Melting point: about
200,000 and not more than 1.5, respectively.
289 C.
System repeatability: When the test is repeated 6 times
Purity—Perform the test with 0.5 mL of guaiacol as di-
with 1 mL of the solution for system suitability test under the
rected under Gas Chromatography <2.02> according to the
above operating conditions, the relative standard deviation
following conditions. Measure each peak area by the auto-
of the peak area of guaiacol is not more than 2.0z.
matic integration method, and calculate the amount of
guaiacol by the area percentage method:It showed the purity Guaifenesin C10H14O4 [Same as the namesake mono-
of not less than 99.0z. graph]
Operating conditions
Guanine C5H5N5O White to pale yellowish white pow-
Detector: A hydrogen flame-ionization detector
der.
Column: A glass column about 3 mm in inside diameter
Absorbance <2.24> Weigh accurately about 10 mg of
and about 2 m in length, packed with siliceous earth for gas
guanine, dissolve in 20 mL of dilute sodium hydroxide TS,
chromatography, 150- to 180-mm in particle diameter, coated
and add 2 mL of 1 mol/L hydrochloric acid TS and 0.1
with polyethylene glycol 20 M at the ratio of 20z.
mol/L hydrochloric acid TS to make exactly 1000 mL. De-
Column temperature: A constant temperature of about
termine the absorbances, E 11z
z, of this solution at 248 nm and
2009C.
273 nm: they are between 710 and 770, and between 460 and
Carrier gas: Nitrogen.
500, respectively.
Flow rate: Adjust so that the retention time of guaiacol is
Loss on drying <2.41>: Not more than 1.5z (0.5 g, 1059C,
4 to 6 minutes.
4 hours).
Detection sensitivity: Adjust the detection sensitivity so
that the peak height of guaiacol obtained from 0.5 mL of Haloperidol for assay C21H23ClFNO2 [Same as the
guaiacol is about 90z of the full scale. monograph Haloperidol]
Time span of measurement: About 3 times as long as the
Hanus' TS Dissolve 20 g of iodine monobromide in 1000
retention time of guaiacol, beginning after the solvent peak.
mL of acetic acid (100).
Guaiacol for assay C7H8O2 Colorless to yellow clear Preserve in light-resistant, glass-stoppered bottles, in a
liquid or colorless crystals with a characteristic, aromatic cold place.
odor. Miscible with methanol and with ethanol (99.5), and
Heart infusion agar medium Prepared for biochemical
sparingly soluble in water. Congealing point: 25 – 309C.
tests.
Identification—Determine the infrared absorption spec-
trum of guaiacol for assay as directed in the ATR method Heavy hydrogenated solvent for nuclear magnetic reso-
under Infrared Spectrophotometry <2.25>: it exhibits absorp- nance spectroscopy Prepared for nuclear magnetic reso-
tion at the wave numbers of about 1595 cm－1, 1497 cm－1, nance spectroscopy. Heavy hydrogenated chloroform
1443 cm－1, 1358 cm－1, 1255 cm－1, 1205 cm－1, 1108 cm－1, (CDCl3), heavy hydrogenated dimethyl sulfoxide
1037 cm－1, 1020 cm－1, 916 cm－1, 833 cm－1, and 738 cm－1. [(CD3)2SO], heavy water (D2O), and heavy hydrogenated
Purity Related substances—Perform the test with 0.5 mL pyridine (C5D5N) are available.
of guaiacol for assay as directed under Gas Chromatography
Heavy water for nuclear magnetic resonance spectroscopy
<2.02> according to the following conditions. Determine each
D2O Prepared for nuclear magnetic resonance spectros-
peak area by the automatic integration method: the total
copy.
area of the peaks other than the peak of guaiacol is not more
than 2.0z. Helium He Not less than 99.995 volz.
Operating conditions
Hematoxylin C16H14O6.xH2O White or light yellow to
Detector: A hydrogen flame-ionization detector.
brownish, crystals or crystalline powder. It is soluble in hot
Column: A fused silica column 0.25 mm in inside diameter
water and in ethanol (95), and sparingly soluble in cold
and 60 m in length, coated inside with polymethylsiloxane
water.
for gas chromatography in 0.25 to 0.5 mm in thickness.
Residue on ignition <2.44>: not more than 0.1z (1 g).
Column temperature: Raise the temperature from 1009C
to 1309C at a rate of 59C per minute, raise to 1409C at a rate Hematoxylin TS Dissolve 1 g of hematoxylin in 12 mL
of 29C per minute, raise to 2009 C at a rate of 159C per of ethanol (99.5). Dissolve 20 g of aluminum potassium sul-
minute, and maintain at 2009C for 2 minutes. fate 12-water in 200 mL of warm water, cool, and filter.
Injection port temperature: 2009 C. After 24 hours, mix these two prepared solutions. Allow to
Detector temperature: 2509C. stand for 8 hours in a wide-mouthed bottle without using a

stopper, and filter. Test for required detectability: To exactly 1 mL of the

standard solution add the mobile phase to make exactly 20
Heparin sodium [Same as the namesake monograph]
mL. Confirm that the peak area of hesperidin obtained with
HEPES buffer solution (pH 7.5) Dissolve 2.38 g of N-2- 10 mL of this solution is equivalent to 3.5 to 6.5z of that ob-
hydroxyethylpiperazine-N?-2-ethanesulfonic acid in 90 mL tained with 10 mL of the standard solution.
of water, adjust to pH 7.5 with diluted 6 mol/L sodium hy-
Hesperidin for component determination See hesperidin
droxide TS (5 in 6), and add water to make 100 mL.
for assay.
Heptafluorobutylic acid C4HF7O2 A clear and colorless
Hesperidin for thin-layer chromatography C28H34O15
liquid.
A white to light brown-yellow, crystalline powder or pow-
Content: Not less than 98.0z. Assay—Take 30 mL of
der. Very slightly soluble in methanol and in ethanol (99.5),
water in a glass-stoppered flask, weigh accurately the mass
and practically insoluble in water. Melting point: about
of the flask, add about 4.3 g of heptafluorobutylic acid, and
2459C (with decomposition).
weigh accurately the mass of this flask. Then, add 40 mL of
Absorbance <2.24> E 11zcm (284 nm): 310 – 340 (8 mg dried in
water, and titrate <2.50> with 1 mol/L sodium hydroxide VS
a desiccator (silica gel) for 24 hours, methanol, 500 mL).
(indicator: 2 drops of phenolphthalein TS).
Purity Related substances—Dissolve 1 mg in 2 mL of
Each mL of 1 mol/L sodium hydroxide VS methanol. Proceed the test with 20 mL of this solution as di-
＝ 214.0 mg of C4HF7O2 rected in the Identification (6) under Hochuekkito Extract:
no spot other than the principle spot of around R f value of
Heptane CH3(CH2)5CH3 [K 9701, Special class]
0.3 appears.
Heptane for liquid chromatography C7H16 Clear and
Hexaammonium heptamolybdate-cerium (IV) sulfate TS
colorless solution.
Dissolve 2.5 g of hexaammonium heptamolybdate tetrahy-
Purity Ultraviolet-absorbing substances—Perform the test
drate and 1.0 g of cerium (IV) sulfate tetrahydrate in diluted
as directed under Ultraviolet-visible Spectrophotometry
sulfuric acid (3 in 50) to make 100 mL. Prepare before use.
<2.24>, and determine the absorbances of heptane for liquid
chromatography at 210 nm, 220 nm, 230 nm and 240 nm, Hexaammonium heptamolybdate-sulfuric acid TS Dis-
using water as the control solution: the absorbance is not solve 1.0 g of hexaammonium heptamolybdate tetrahydrate
more than 0.35, not more than 0.15, not more than 0.05 and in diluted sulfuric acid (3 in 20) to make 40 mL. Prepare
not more than 0.03, respectively. before use.
Heptyl parahydroxybenzoate C14H20O3 White, crystals Hexaammonium heptamolybdate tetrahydrate
or crystalline powder. (NH4)6Mo7O24.4H2O [K 8905, Special class]
Melting point <2.60>: 45 – 509C
Hexaammonium heptamolybdate TS dissolve 21.2 g of
Content: Not less than 98.0z Assay—Weigh accurately
hexaammonium heptamolybdate tetrahydrate in water to
about 3.5 g of heptyl parahydroxybenzoate, dissolve in 50
make 200 mL (10z). Prepare before use.
mL of diluted N, N-dimethylformamide (4 in 5), and titrate
<2.50> with 1 mol/L sodium hydroxide VS (potentiometric 1,1,1,3,3,3-Hexamethyldisilazane (CH3)3SiNHSi(CH3)3
titration). Perform a blank determination in the same man- A colorless or practically colorless, liquid. Very soluble in
ner, and make any necessary correction. diethyl ether, and reactable with water or with ethanol. Boil-
ing point: about 1259 C.
Each mL of 1 mol/L sodium hydroxide VS
＝ 236.3 mg of C14H20O3 Hexamethylenetetramine (CH2)6N4 [K 8847, Special
class]
Hesperidin for assay C28H34O15 Hesperidin for thin-
layer chromatography. It meets the following requirement. Hexamethylenetetramine TS Dissolve 2.5 g of hex-
Optical rotation <2.49> [a]20
D : －100 – －1209(5 mg dried amethylenetetramine in exactly 25 mL of water.
with silica gel for 24 hours, methanol, 50 mL, 100 mm).
Hexamine See hexamethylenetetramine.
Purity Related substances—Dissolve 2 mg of hesperidin
for assay in 10 mL of methanol, and use this solution as the Hexane C6H14 [K 8848, Special class]
sample solution. Pipet 1 mL of the sample solution, add the
Hexane for liquid chromatography C6H14 Colorless,
mobile phase to make exactly 100 mL, and use this solution
clear liquid. Miscible with ethanol (95), with diethyl ether,
as the standard solution. Perform the test with exactly 10 mL
with chloroform and with benzene.
each of the sample solution and standard solution as directed
Boiling point: about 699C.
under Liquid Chromatography <2.01> according to the
Purity (1) Ultraviolet absorptive substances—Read the
following conditions, and determine each peak area by the
absorbances of hexane for liquid chromatography as di-
automatic integration method: the total area of the peaks
rected under Ultraviolet-visible Spectrophotometry <2.24>,
other than hesperidin and the solvent is not larger than the
using water as the blank: not more than 0.3 at the wave-
peak area of hesperidin obtained with the standard solution.
length of 210 nm, and not more than 0.01 between 250 nm
Operating conditions
and 400 nm.
Detector, column, column temperature, mobile phase, and
(2) Peroxide—To a mixture of 100 mL of water and 25
flow rate: Proceed as directed in the operating conditions in
mL of dilute sulfuric acid add 25 mL of a solution of potas-
the Assay (1) under Hochuekkito Extract.
sium iodide (1 in 10) and 20 g of hexane for liquid chroma-
Time span of measurement: About 6 times as long as the
tography. Stopper tightly, shake, and allow to stand in a
retention time of hesperidin.
dark place for 15 minutes. Titrate <2.50> this solution, while
System suitability
shaking well, with 0.01 mol/L sodium thiosulfate VS (indi-
System performance, and system repeatability: Proceed as
cator: 1 mL of starch TS). Perform a blank determination in
directed in the system suitability in the Assay (1) under
the same manner (not more than 0.0005z).
Hochuekkito Extract.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
264 General Tests / 9.41 Reagents, Test Solutions JP XVII
n-Hexane for liquid chromatography See hexane for liq- Absorbance <2.24>: E 11zcm (245 nm): 354 – 389 (5 mg calcu-
uid chromatography. lated on the anhydrous basis, a mixture of methanol and
dilute acetic acid (7:3), 500 mL).
Hexane for purity of crude drug C6H14 [K 8848, Spe-
Purity Related substances—Dissolve 5 mg of hirsutine
cial class] Use hexane meeting the following additional spe-
for assay in 100 mL of a mixture of methanol and dilute
cification. Evaporate 300.0 mL of hexane for purity of crude
acetic acid (7:3), use this solution as the sample solution.
drug in vacuum at a temperature not higher than 409C, add
Pipet 1 mL of the sample solution, add a mixture of metha-
the hexane to make exactly 1 mL, and use this solution as the
nol and dilute acetic acid (7:3) to make exactly 50 mL, and
sample solution. Separately, dissolve 2.0 mg of g-BHC in
use this solution as the standard solution. Perform the test
hexane to make exactly 100 mL. Pipet 1 mL of this solution,
with exactly 20 mL each of the sample solution and standard
and add hexane to make exactly 100 mL. Further pipet 2 mL
solution as directed under Liquid-chromatography <2.01> ac-
of this solution, add hexane to make exactly 100 mL, and
cording to the following conditions. Determine the peak area
use this solution as the standard solution I. Perform the test
of each solution by the automatic integration method: the
with exactly 1 mL each of the sample solution and standard
total area of the peaks other than hyrsutine obtained from
solution I as directed under Gas Chromatography <2.02> ac-
the sample solution is not larger than the peak area of hirsu-
cording to the following operating conditions, and determine
tine obtained from the standard solution.
each peak area by the automatic integration method: the
Operating conditions
total area of peak other than the solvent peak from the sam-
Detector, column, column temperature, mobile phase, and
ple solution is not larger than the peak area of g-BHC from
flow rate: Proceed as directed in the operating conditions in
the standard solution I.
the Assay under Uncaria Hook.
Operating conditions
Time span of measurement: About 1.5 times as long as the
Proceed the operating conditions in 4.3. under Crude
retention time of hirsutine, beginning after the solvent peak.
Drugs Test <5.01> except detection sensitivity and time span
System suitability
of measurement.
System performance: Proceed as directed in the system
Detection sensitivity: Pipet 1 mL of the standard solution
suitability in the Assay under Uncaria Hook.
I, add hexane to make exactly 20 mL, and use this solution
Test for required detectability: Pipet 1 mL of the standard
as the standard solution II. Adjust the detection sensitivity
solution, add a mixture of methanol and dilute acetic acid
so that the peak area of g-BHC obtained from 1 mL of the
(7:3) to make exactly 20 mL. Confirm that the peak area of
standard solution II can be measured by the automatic in-
hirsutine obtained with 20 mL of this solution is equivalent to
tegration method, and the peak height of g-BHC from 1 mL
3.5 to 6.5z of that obtained with 20 mL of the standard so-
of the standard solution I is about 20z of the full scale.
lution.
Time span of measurement: About three times as long as
System repeatability: When the test is repeated 6 times
the retention time of g-BHC, beginning after the solvent
with 20 mL of the standard solution under the above operat-
peak.
ing conditions, the relative standard deviation of the peak
Hexane for ultraviolet-visible spectrophotometry C6H14 area of hirsutine is not more than 1.5z.
[K 8848, Special class]. When determining the absorbance
Hirsutine for thin-layer chromatography C22H28N2O3
of hexane for ultraviolet-visible spectrophotometry as di-
A white or light orange, crystalline powder or powder. Very
rected under Ultraviolet-visible Spectrophotometry <2.24>,
soluble in methanol, freely soluble in ethanol (99.5), and
using water as the blank solution, its value is not more than
practically insoluble in water. Melting point: about 1059C.
0.10 at 220 nm and not more than 0.02 at 260 nm, and it has
Identification—Determine the absorption spectrum of a
no characteristic absorption between 260 nm and 350 nm.
solution of hirsutine for thin-layer chromatography in a mix-
n-Hexane for ultraviolet-visible spectrophotometry See ture of methanol and dilute acetic acid (7:3) (1 in 100,000) as
hexane for ultraviolet-visible spectrophotometry. directed under Ultraviolet-visible Spectrophotometry <2.24>:
it exhibits a maximum between 287 nm and 291 nm.
1-Hexanol C6H14O A clear and colorless liquid.
Purity Related substances—Dissolve 1.0 mg of hirsutine
Specific gravity d 20
20: 0.816 – 0.821
for thin-layer chromatography in 1 mL of methanol, and use
Boiling point 156 – 1589C.
this solution as the sample solution. Perform the test with
Hexyl parahydroxybenzoate C13H18O3 White, crystals this solution as directed under Thin-layer Chromatography
or crystalline powder. <2.03>. Spot 10 mL of the sample solution on a plate of silica
Melting point <2.60>: 49 – 539C gel with fluorescent indicator for thin-layer chromatogra-
Content: not less than 98.0z. Assay—Weigh accurately phy. Develop the plate with a mixture of 1-butanol, water
about 1 g of hexyl parahydroxybenzoate, add exactly 20 mL and acetic acid (100) (7:2:1) to a distance of about 10 cm,
of 1 mol/L sodium hydroxide VS, heat at about 709 C for 1 and air-dry the plate. Examine under ultraviolet light (main
hour, and immediately cool in ice. Titrate <2.50> the excess wavelength: 254 nm): no spot other than the principal spot at
sodium hydroxide with 0.5 mol/L sulfuric acid VS up to the around R f value of 0.55 appears.
second equivalent point (potentiometric titration). Perform a
L-Histidine C 6 H 9 N3 O2 [Same as the namesake mono-
blank determination.
graph]
Each mL of 1 mol/L sodium hydroxide VS
L-Histidine hydrochloride See L-histidine hydrochloride
＝ 222.3 mg of C13H18O3
monohydrate.
High-density polyethylene film Prepared for cytotoxicity
L-Histidine hydrochloride monohydrate
test. It does not show cytotoxicity.
C6H9N3O2.HCl.H2O [K 9050, Special class]
Hirsutine See hirsutine for thin-layer chromatography.
Homatropine hydrobromide C16H21NO3.HBr [Same as
Hirsutine for assay C22H28N2O3 Hirsutine for thin- the namesake monograph]
layer chromatography. It meets the following requirements.
Honokiol C18H18O2 Odorless white, crystals or crystal-

line powder. ampoule by adding exactly 2 mL of cold water. Dilute this

Purity—Dissolve 1 mg of honokiol in the mobile phase to solution with cold water so that each mL contains exactly 1.3
make 10 mL, and use this solution as the sample solution. to 3.8 units of hyaluronidase. Prepare before use, and store
Perform the Liquid Chromatography <2.01> with 10 mL of in a cold place.
the sample solution as directed in the Assay under Magnolia (ii) Substrate solution: To exactly 50 mg of hyaluronic
Bark: when measure the peak areas for 2 times as long as the acid add 40 mL of 0.02 mol/L acetate buffer solution (pH
retention time of magnorole, the total area of peaks other 6.0), stir for 5 hours to dissolve, and add 0.02 mol/L acetate
than honokiol is not larger than 1/10 times the total area of buffer solution (pH 6.0) to make exactly 50 mL.
the peaks other than the solvent peak. (iii) 4-Dimethylaminobenzaldehyde solution: To a mix-
ture of 0.6 mL of water and 11.9 mL of hydrochloric acid
Horseradish peroxidase An oxidase (Molecular weight:
add acetic acid to make exactly 100 mL, and dissolve 10.0 g
about 40,000) derived from horseradish.
of 4-dimethylaminobenzaldehyde in this solution. To exactly
Horse serum Collect the blood from horse in a flask, 1 mL of this solution add exactly 9 mL of acetic acid. Pre-
coagulate, and allow to stand at room temperature until the pare before use.
serum is separated. Transfer the separated serum in glass (iv) Borate solution: Dissolve 4.95 g of boric acid in 40
containers, and preserve at －209 C. mL of water, adjust to pH 9.1 with potassium hydroxide TS,
and add water to make 100 mL.
Human anti-thrombin A serine protease inhibitor ob-
(v) Procedure: Pipet 0.5 mL of substrate solution, warm
tained from healthy human plasma. A protein that inhibits
at 60 ± 0.59C for 10 minutes, add exactly 0.5 mL of sample
activities of activated blood coagulation factor II (thrombin)
solution, and shake immediately. After allowing this solu-
and activated blood coagulation factor X. It contains not
tion to stand at 60 ± 0.59 C for exactly 30 minutes, add ex-
less than 6 IU per mg of protein.
actly 0.2 mL of borate solution, shake, heat the vessel co-
Human antithrombin III Serine protease inhibition fac- vered with a marble in a water bath for exactly 3 minutes,
tor obtained from normal plasma of health human. It is a and cool the vessel with running water. Then, add exactly 3
protein, which inhibits the activities of thrombin and activat- mL of 4-dimethylaminobenzaldehyde solution, shake, and
ed blood coagulation factor X. It contains not less than 300 allow to stand at 37 ± 0.59C for exactly 20 minutes. Deter-
Units per mg protein. One unit indicates an amount of the mine the absorbance, A1, at 585 nm of this solution as di-
antithrombin III which inhibits 1 unit of thrombin at 259C rected under Ultraviolet-visible Spectrophotometry <2.24>,
under the existence of heparin. using water as the blank. Separately, pipet 0.5 mL of sub-
strate solution, allow to stand at 60 ± 0.59 C for 40 minutes,
Human chorionic gonadotrophin TS Weigh accurately a
add exactly 0.2 mL of borate solution, and shake. Add ex-
suitable amount of Human Chorionic Gonadotrophin ac-
actly 0.5 mL of sample solution, and shake immediately.
cording to the labeled amount, and dissolve in bovine serum
Heat the vessel covered with a marble in a water bath for ex-
albumin-sodium chloride-phosphate buffer solution (pH 7.2)
actly 3 minutes, and cool the vessel with running water.
so that each 1.0 mL contains 80 human chorionic gonadotro-
Then, proceed in the same manner as above, and determine
phin Units.
the absorbance, A0, of this solution.
Human insulin desamido substance-containing TS Dis- (vi) Calculation: Calculate the enzyme activity in 1 am-
solve 1.5 mg of Insulin Human in 1 mL of 0.01 mol/L hy- poule by the following equation, where 1 unit means the
drochloric acid TS, allow to stand at 259C for 3 days, and amount of enzyme which decreases 50z in absorbance at
when the procedure is run with this solution according to the 660 nm of hyaluronic acid in 30 minutes at 609C and pH 6.0.
conditions as directed in the Purity (1) under Insurin Human
Hyaluronidase unit in 1 ampoule
(Genetical Recombination), the solution contains about 5z
＝ (A1 － A0) × Dm × 3.2 × 4
of the desamido substance.
Dm: Dilution factor for sample solution
Human insulin dimer-containing TS Allow to stand In-
3.2: Conversion factor to turbidity reduction unit
sulin Human (Genetical Recombination) at 259 C for 10 days
or more, and dissolve 4 mg of this in 1 mL of 0.01 mol/L hy- Hydralazine hydrochloride C8H8N4.HCl [Same as the
drochloric acid TS. namesake monograph]
Human normal plasma Dissolve an amount of dried hu- Hydralazine hydrochloride for assay C8H8N4.HCl
man normal plasma powder, equivalent to 1 mL of the nor- [Same as the monograph Hydralazine Hydrochloride. When
mal plasma of human, in 1 mL of water. Store between 29C dried, it contains not less than 99.0z of hydralazine hydro-
and 109 C, and use within one week. chloride (C8H8N4.HCl).]
Human serum albumin for assay White to pale-yellow Hydrazine monohydrate NH2NH2.H2O Colorless liq-
powder. Albumin content is at least 99z. Convert to the de- uid, having a characteristic odor.
hydrate using the following water determination method.
Hydrazine sulfate See hydrazinium sulfate.
Water <2.48>: (0.2 g, volumetric titration, direct titration).
However, in a dehydration solvent, use a mixture of pyridine Hydrazinium sulfate N2H6SO4 [K 8992, Special class]
for water determination and ethylene glycol for water deter-
Hydrazinium sulfate TS Dissolve exactly 1.0 g of
mination (5:1).
hydrazinium sulfate in exactly 100 mL of water. Use after
Hyaluronic acid (C14H21NO11)n A white powder. standing for 4 – 6 hours.
Hyaluronidase Obtained from Streptomyces albogriseo- Hydriodic acid HI [K 8917, Special class]
lus. A lyophilized white powder.
Hydrobromic acid HBr [K 8509, Special class]
Content: Not less than 100 units of hyaluronidase per
ampule. Hydrochloric acid HCl [K 8180, Special class]
Assay (i) Sample solution: Dissolve the content of 1
Hydrochloric acid-ammonium acetate buffer solution (pH

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
266 General Tests / 9.41 Reagents, Test Solutions JP XVII
3.5) Dissolve 25 g of ammonium acetate in 45 mL of 6 36.46) for amino acid automatic analysis (constant boiling
mol/L hydrochloric acid TS, and add water to make 100 hydrochloric acid).
mL.
Hydrochlorothiazide C7H8ClN3O4S2 [Same as the
Hydrochloric acid, dilute Dilute 23.6 mL of hydrochlo- namesake monograph]
ric acid with water to make 100 mL (10z).
Hydrocortisone C21H30O5 [Same as the namesake
Hydrochloric acid-ethanol TS Dilute 23.6 mL of hydro- monograph]
chloric acid with ethanol (95) to make 100 mL.
Hydrocortisone acetate C23H32O6 [Same as the name-
0.01 mol/L Hydrochloric acid-methanol TS To 20 mL sake monograph]
of 0.5 mol/L hydrochloric acid TS add methanol to make
Hydrocotarnine hydrochloride for assay See hydrocorta-
1000 mL.
nine hydrochloride hydrate for assay.
0.05 mol/L Hydrochloric acid-methanol TS To 100 mL
Hydrocotarnine hydrochloride hydrate for assay
of 0.5 mol/L hydrochloric acid add methanol to make 1000
C12H15NO3.HCl.H2O [Same as the monograph Hydro-
mL.
cotarnine Hydrochloride Hydrate. When dried, it contains
Hydrochloric acid-2-propanol TS Add 0.33 mL of hy- not less than 99.0z of hydrocotarnine hydrochloride
drochloric acid to 100 mL of 2-propanol, mix, and store in a (C12H15NO3.HCl).]
dark and cool place.
Hydrofluoric acid HF [K 8819, Special class] It con-
Hydrochloric acid-potassium chloride buffer solution (pH tains not less than 46.0z of HF.
2.0) To 10.0 mL of 0.2 mol/L hydrochloric acid VS add
Hydrogen H2 [K 0512, Standard substance, Third
88.0 mL of 0.2 mol/L potassium chloride TS, adjust the pH
class] It contains not less than 99.99z of H2.
to 2.0 ± 0.1 with 0.2 mol/L hydrochloric acid VS or 0.2
mol/L potassium chloride TS, then add water to make 200 Hydrogen chloride-ethanol TS Pass dry hydrogen chlo-
mL. ride, which is generated by slowly adding 100 mL of sulfuric
acid dropwise to 100 mL of hydrochloric acid and dried by
Hydrochloric acid, purified Add 0.3 g of potassium per-
washing with sulfuric acid, through 75 g of ethanol (99.5)
manganate to 1000 mL of diluted hydrochloric acid (1 in 2),
cooled in an ice bath until the increase in mass has reached
distil, discard the first 250 mL of the distillate, and collect
25 g. Prepare before use.
the following 500 mL of the distillate.
Hydrogen hexachloroplatinate (IV) hexahydrate
0.001 mol/L Hydrochloric acid TS Dilute 10 mL of 0.1
H2PtCl6.6H2O [K 8153, Special class]
mol/L hydrochloric acid TS with water to make 1000 mL.
Hydrogen hexachloroplatinate (IV)-potassium iodide TS
0.01 mol/L Hydrochloric acid TS Dilute 100 mL of 0.1
To 3 mL of hydrogen hexachloroplatinate (IV) TS add 97
mol/L hydrochloric acid TS with water to make 1000 mL.
mL of water and 100 mL of a solution of potassium iodide
0.02 mol/L Hydrochloric acid TS Dilute 100 mL of 0.2 (3 in 50). Prepare before use.
mol/L hydrochloric acid TS with water to make 1000 mL.
Hydrogen hexachloroplatinate (IV) TS Dissolve 2.6 g of
0.05 mol/L Hydrochloric acid TS Dilute 100 mL of 0.5 hydrogen hexachloroplatinate (IV) hexahydrate in water to
mol/L hydrochloric acid TS with water to make 1000 mL. make 20 mL (0.25 mol/L).
0.1 mol/L Hydrochloric acid TS Dilute 100 mL of 1 Hydrogen peroxide (30) H2O2 [K 8230, Hydrogen
mol/L hydrochloric acid TS with water to make 1000 mL. peroxide, Special class, Concentration: 30.0 – 35.5z.]
0.2 mol/L Hydrochloric acid TS Dilute 18 mL of hydro- Hydrogen peroxide-sodium hydroxide TS To a mixture
chloric acid with water to make 1000 mL. of water and hydrogen peroxide (30) (9:1) add 3 drops of
bromophenol blue TS, and then add 0.01 mol/L sodium hy-
0.5 mol/L Hydrochloric acid TS Dilute 45 mL of hydro-
droxide TS until a purple-blue color develops. Prepare be-
chloric acid with water to make 1000 mL.
fore use.
1 mol/L Hydrochloric acid TS Dilute 90 mL of hydro-
Hydrogen peroxide TS Dilute 1 volume of hydrogen
chloric acid with water to make 1000 mL.
peroxide (30) with 9 volumes of water. Prepare before use
2 mol/L Hydrochloric acid TS Dilute 180 mL of hydro- (3z).
chloric acid with water to make 1000 mL.
Hydrogen peroxide TS, dilute Dilute 1 mL of hydrogen
3 mol/L Hydrochloric acid TS Dilute 270 mL of hydro- peroxide (30) with 500 mL of water, and dilute 5 mL of this
chloric acid with water to make 1000 mL. solution with water to make 100 mL. Prepare before use.
5 mol/L Hydrochloric acid TS Dilute 450 mL of hydro- Hydrogen peroxide water, strong See hydrogen per-
chloric acid with water to make 1000 mL. oxide (30).
6 mol/L Hydrochloric acid TS Dilute 540 mL of hydro- Hydrogen sulfide H2S Colorless, poisonous gas, heavi-
chloric acid with water to make 1000 mL. er than air. It dissolves in water. Prepare by treating iron (II)
sulfide with dilute sulfuric acid or dilute hydrochloric acid.
7.5 mol/L Hydrochloric acid TS Dilute 675 mL of hy-
Other sulfides yielding hydrogen sulfide with dilute acids
drochloric acid with water to make 1000 mL.
may be used.
10 mol/L Hydrochloric acid TS Dilute 900 mL of hydro-
Hydrogen sulfide TS A saturated solution of hydrogen
chloric acid with water to make 1000 mL.
sulfide. Prepare by passing hydrogen sulfide into cold water.
6 mol/L Hydrochloric acid TS for amino acid automatic Preserve in well-filled, light-resistant bottles, in a dark, cold
analysis Contains 19 – 21z hydrogen chloride (HCl: place.

Hydrogen tetrachloroaurate (III) tetrahydrate tion as directed under Liquid Chromatography <2.01> ac-
HAuCl4.4H2O [K 8127, Special class] cording to the following conditions. Determine each peak
area from both solutions by the automatic integration
Hydrogen tetrachloroaurate (III) TS Dissolve 1 g of
method: the total area of the peaks other than 10-hydroxy-2-
hydrogen tetrachloroaurate (III) tetrahydrate in 35 mL of
(E )-decenoic acid from sample solution is not larger than the
water.
peak area of 10-hydroxy-2-(E )-decenoic acid from the stand-
Hydroquinone C6H4(OH)2 [K 8738, Special class] ard solution.
Operating conditions
Hydroxocobalamin acetate C62H89CoN13O15P.C2H4O2
Detector, column, column temperature, mobile phase and
Dark red, crystals or powder.
flow rate: Proceed as directed in the operating conditions in
Loss on drying <2.41>: not more than 12z (50 mg, in
the Assay under Royal Jelly.
vacuum not exceeding 0.67 kPa, phosphorus (V) oxide,
Time span of measurement: About 4 times as long as the
1009C, 6 hours).
retention time of 10-hydroxy-2-(E )-decenoic acid, beginning
Content: not less than 98.0z. Assay—Proceed as di-
after the solvent peak.
rected in the Assay under Hydroxocobalamin Acetate.
System suitability
m-Hydroxyacetophenone C8H8O2 White to light yel- System performance and system repeatability: Proceed as
lowish white crystalline powder. directed in the system suitability in the Assay under Royal
Melting point <2.60>: about 969C Jelly.
Purity Related substances—Perform the test with 10 mL Test for required detectability: Pipet 1 mL of the standard
of a solution of m-hydroxyacetophenone in 0.1 mol/L phos- solution, and add methanol to make exactly 20 mL. Confirm
phate buffer solution (pH 4.5) (1 in 15,000) as directed in the that the peak area of 10-hydroxy-2-(E )-decenoic acid ob-
Assay under Cefalexin: Any obstructive peaks for determi- tained from 10 mL of this solution is equivalent to 3.5 to
nation of cefalexin are not observed. 6.5z of that of 10-hydroxy-2-(E )-decenoic acid obtained
from 10 mL of the standard solution.
p-Hydroxyacetophenone C8H8O2 White to pale yellow,
crystals or crystalline powder. It is freely soluble in metha- 10-Hydroxy-2-(E )-decenoic acid for component determi-
nol. nation See 10-hydroxy-2-(E )-decenoic acid for assay.
Melting point <2.60>: 107 – 1119 C
10-Hydroxy-2-(E )-decenoic acid for thin-layer chromatog-
Purity—Weigh 1 mg of p-hydroxyacetophenone, add
raphy C10H18O3 White crystalline powder. Very soluble in
methanol and dissolve to make exactly 10 mL, and use this
methanol, freely soluble in ethanol (99.5), soluble in diethyl
solution as the sample solution. Perform the test with 20 mL
ether, and slightly soluble in water.
of the sample solution as directed under Liquid Chromatog-
Identification—Determine the absorption spectrum of a
raphy <2.01> according to the Assay under Peony Root: the
solution of 10-hydroxy-2-(E )-decenoic acid for thin-layer
total area of the peaks other than p-hydroxyacetophenone
chromatography in ethanol (99.5) (1 in 125,000) as directed
from the sample solution is not larger than the total area of
under Ultraviolet-visible Spectrophotometry <2.24>: it exhib-
the peaks other than the solvent peak.
its a maximum between 206 nm and 210 nm.
3-Hydroxybenzoic acid HOC6H4COOH White, crys- Melting point <2.60>: 63 – 669C
tals or crystalline powder. Purity Related substances—Dissolve 5.0 mg of 10-
Identification—Determine the infrared absorption spec- hydroxy-2-(E )-decenoic acid for thin-layer chromatography
trum according to the paste method under Infrared Spectro- in 1 mL of diethyl ether. Perform the test with 20 mL of this
photometry <2.25>: it exhibits absorption at the wave num- solution as directed in the Identification under Royal Jelly:
bers of about 3300 cm－1, 1690 cm－1, 1600 cm－1, 1307 cm－1, no spot other than the principal spot at around R f value of
1232 cm－1 and 760 cm－1. 0.5 appears.
Melting point <2.60>: 203 – 2069
C
d-3-Hydroxy-cis-2,3-dihydro-5-[2-(dimethylamino)ethyl]-
Purity Clarity of solution—Dissolve 1.0 g of 3-hydrox-
2-(4-methoxyphenyl)-1,5-benzothiazepine-4(5H )-one hydro-
ybenzoic acid in 20 mL of methanol: the solution is clear.
chloride C20H24N2O3S.HCl To 9 g of diltiazem hydro-
Content: not less than 99.0z. Assay—Weigh accurately
chloride add 50 mL of ethanol (99.5), and dissolve by heat-
about 0.2 g of 3-hydroxybenzoic acid, dissolve in 20 mL of
ing at 809C. To this solution add slowly 50 mL of a solution
diluted ethanol (95) (1 in 2), and titrate <2.50> with 0.1
of potassium hydroxide in ethanol (99.5) (33 in 500) drop-
mol/L sodium hydroxide VS (indicator: 3 drops of cresol red
wise, and heat for 4 hours with stirring. Cool in an ice bath,
TS) until the color of the solution changes from yellow to
filter, and evaporate the filtrate to dryness. Dissolve the
dark orange-red. Perform a blank determination and make
residue in ethanol (99.5), add slowly a solution of hydrochlo-
any necessary correction.
ric acid in ethanol (99.5) (59 in 250) to make acidic, and
Each mL of 0.1 mol/L sodium hydroxide VS filter. Add diethyl ether slowly to the filtrate, and filter the
＝ 13.81 mg of C7H6O3 crystals produced. To the crystals add ethanol (99.5), heat to
dissolve, add 0.5 g of activated charcoal, allow to stand, and
p-Hydroxybenzoic acid See parahydroxybenzoic acid.
filter. After cooling the filtrate in an ice-methanol bath,
10-Hydroxy-2-(E )-decenoic acid for assay C10H18O3 10- filter the crystals formed, and wash with diethyl ether. Fur-
hydroxy-2-(E )-decenoic acid for thin-layer chromatography ther, add ethanol (99.5) to the crystals, and heat to dissolve.
meeting the following additional specifications. After cooling, filter the crystals produced, and dry under
Purity Related substances—Dissolve 10 mg of 10-hy- reduced pressure. White, crystals or crystalline powder, hav-
droxy-2-(E )-decenoic acid for assay in 100 mL of methanol, ing a slight, characteristic odor.
and use this solution as the sample solution. Pipet 1 mL of Purity—Dissolve 50 mg of d-3-hydroxy-cis-2,3-dihydro-
this solution, add methanol to make exactly 100 mL, and use 5 - [2 - (dimethylamino)ethyl] - 2 - (4 - methoxyphenyl) - 1,5 - ben-
this solution as the standard solution. Perform the test with zothiazepine-4-(5H )-one hydrochloride in chloroform to
exactly 10 mL each of the sample solution and standard solu- make exactly 10 mL, and use this solution as the sample so-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
268 General Tests / 9.41 Reagents, Test Solutions JP XVII
lution. Perform the test with the sample solution as directed 4-Hydroxyisophthalic acid HOC6H3(COOH)2 White,
under Thin-layer Chromatography <2.03>. Spot 20 mL of the crystals or powder.
sample solution on a plate of silica gel for thin-layer chroma- Content: not less than 98.0z. Assay—Weigh accurately
tography. Develop the plate with a mixture of ethanol (99.5), about 0.14 g of 4-hydroxyisophthalic acid, dissolve in 50 mL
chloroform, water and acetic acid (100) (12:10:3:1) to a dis- of ethanol (95), and titrate <2.50> with 0.1 mol/L sodium
tance of about 13 cm, and air-dry the plate. Spray evenly hydroxide VS (potentiometric titration). Perform a blank de-
iodine TS on the plate: any spot other than the principal spot termination in the same manner, and make any necessary
does not appear. correction.
Water <2.48>: not more than 1.0z (0.5 g).
Each mL of 0.1 mol/L sodium hydroxide VS
Content: not less than 99.0z, calculated on the anhydrous
＝ 9.107 mg of C8H6O5
basis. Assay—Weigh accurately about 0.5 g of d-3-
hydroxy- cis -2,3-dihydro-5-[2-(dimethylamino)ethyl]-2-(4- Hydroxylamine hydrochloride See hydroxylammonium
methoxyphenyl)-1,5-benzothiazepine-4-(5H )-one hydrochlo- chloride.
ride, dissolve in 2.0 mL of formic acid, add 60 mL of acetic
Hydroxylamine hydrochloride-ferric chloride TS See hy-
anhydride, and titrate <2.50> with 0.1 mol/L perchloric acid
droxylammonium chloride-iron (III) chloride TS.
VS (potentiometric titration). Perform a blank determina-
tion in the same manner. Hydroxylamine hydrochloride TS See hydroxylammo-
nium chloride TS.
Each mL of 0.1 mol/L perchloric acid VS
＝ 40.89 mg of C20H24N2O3S.HCl Hydroxylamine perchlorate NH2OH.HClO4 Hygro-
scopic, white crystals. Dissolves in water and in ethanol (95).
d -3-Hydroxy- cis -2,3-dihydro-5-[2-(dimethylamino)ethyl]-
Melting point <2.60>: 87.5 – 909C
2-(p-methoxyphenyl)-1,5-benzothiazepine-4 (5H)-one hydro-
chloride See d-3-hydroxy-cis-2,3-dihydro-5-[2-(dimethyl- Hydroxylamine perchlorate-dehydrated ethanol TS See
amino)ethyl]-2-(4-methoxyphenyl)-1,5-benzothiazepine-4 hydroxylamine perchlorate-ethanol TS.
(5H )-one hydrochloride.
Hydroxylamine perchlorate-ethanol TS Dilute 2.99 mL
N-(2-Hydroxyethyl)isonicotinamide nitric ester of hydroxylamine perchlorate TS with ethanol (99.5) to
C8H9N3O4 A white crystalline powder. make 100 mL.
Identification—Determine the infrared absorption spec- Storage—Preserve in tight containers, in a cold place.
trum of N-(2-hydroxyethyl)isonicotinamide nitric ester as di-
Hydroxylamine perchlorate TS An ethanol (99.5) solu-
rected in the potassium bromide disk method under Infrared
tion which contains 13.4z of hydroxylamine perchlorate.
Spectrophotometry <2.25>: it exhibits absorption at the wave
Storage—Preserve in tight containers, in a cold place.
numbers of about 3270 cm－1, 1653 cm－1, 1546 cm－1 and
1283 cm－1. Hydroxylamine TS Dissolve 10 g of hydroxylammonium
chloride in 20 mL of water, and add ethanol (95) to make
N-2-Hydroxyethylpiperazine-N?-2-ethanesulfonic acid
200 mL. To this solution add, with stirring, 150 mL of 0.5
C8H18N2O4S White crystalline powder.
mol/L potassium hydroxide-ethanol VS, and filter. Prepare
Purity Clarity and color of solution—Dissolve 11.9 g of
before use.
N-2-hydroxyethylpiperazine-N?-2-ethanesulfonic acid in 50
mL of water: the solution is clear and colorless. Hydroxylamine TS, alkaline Mix equal volumes of a so-
Content: not less than 99.0z. Assay—Weigh accurately lution of hydroxylammonium chloride in methanol (7 in 100)
about 1 g of N-2-hydroxyethylpiperazine-N?-2-ethanesulfon- and a solution of sodium hydroxide in methanol (3 in 25),
ic acid, dissolve in 60 mL of water, and titrate <2.50> with and filter. Prepare before use.
0.5 mol/L sodium hydroxide VS (Potentiometric titration).
Hydroxylammine hydrochloride TS (pH 3.1) See hy-
Each mL of 0.5 mol/L sodium hydroxide VS droxylammonium chloride TS (pH 3.1).
＝ 119.2 mg of C8H18N2O4S
Hydroxylammonium chloride NH2OH.HCl [K 8201,
1-(2-Hydroxyethyl)-1H-tetrazol-5-thiol C3H6N4OS Special class]
White, crystals or powder.
Hydroxylammonium chloride-ethanol TS Dissolve 34.8 g
Melting point <2.60>: 136 – 1419 C
of hydroxylammonium chloride in water to make 100 mL,
Purity Related substances—Dissolve 0.10 g of 1-(2-
and use this solution as Solution A. Dissolve 10.3 g of so-
hydroxyethyl)-1H-tetrazol-5-thiol in 1 mL of water, and use
dium acetate trihydrate and 86.5 g of sodium hydroxide in
this solution as the sample solution. Pipet 0.5 mL of the
water to make 1000 mL, and use this solution as Solution B.
sample solution, add water to make exactly 25 mL, and use
Mix 1 volume of Solution A, 1 volume of Solution B and 4
this solution as the standard solution. Perform the test with
volumes of ethanol (95).
these solutions as directed under Thin-layer Chromatogra-
phy <2.03>. Spot 1 mL each of the sample solution and stand- Hydroxylammonium chloride-iron (III) chloride TS
ard solution on a plate of silica gel with fluorescent indicator Acidify 100 mL of a solution of iron (III) chloride hexahy-
for thin-layer chromatography, develop with a mixture of drate in ethanol (95) (1 in 200) with hydrochloric acid, and
ethyl acetate, water, methanol and formic acid (60:10:7:6) to dissolve 1 g of hydroxylammonium chloride in the solution.
a distance of about 10 cm, and air-dry the plate. Examine
Hydroxylammonium chloride TS Dissolve 20 g of hy-
under ultraviolet light (main wavelength: 254 nm): the spot
droxylammonium chloride in water to make 65 mL, transfer
other than the principal spot obtained from the sample solu-
it to a separator, add 2 to 3 drops of thymol blue TS, then
tion is not more intense than the spot obtained from the
add ammonia solution (28) until the solution exhibits a yel-
standard solution.
low color. Shake well after adding 10 mL of a solution of so-
2-Hydroxy-1-(2-hydroxy-4-sulfo-1-naphthylazo)-3-naph- dium N, N-diethyldithiocarbamate trihydrate (1 in 25), allow
thoic acid C21H14N2O7S [K 8776, Special class] to stand for 5 minutes, and extract this solution with 10 to 15

mL portions of chloroform. Repeat the extraction until 5 Hyodeoxycholic acid for thin-layer chromatography
mL of the extract does not exhibit a yellow color, upon add- C24H40O4 White to pale brown, crystalline powder or pow-
ing 5 drops of a solution of copper (II) sulfate pentahydrate der. Freely soluble in methanol an in ethanol (99.5), and
(1 in 100) and shaking it. Add 1 to 2 drops of thymol blue practically insoluble in water.
TS, add dropwise dilute hydrochloric acid to this aqueous Identification—Determine the infrared absorption spec-
solution until it exhibits a red color, then add water to make trum of hyodeoxycholic acid for thin-layer chromatography
100 mL. as directed in the potassium bromide disk method under
Infrared Spectrophotometry <2.25>: it exhibits absorption
Hydroxylammonium chloride TS (pH 3.1) Dissolve 6.9 g
at the wave numbers of about 2940 cm－1, 2840 cm－1, 1740
of hydroxylammonium chloride in 80 mL of water, adjust
cm－1, 1460 cm－1, 1340 cm－1, 1200 cm－1, 1160 cm－1, 1040
the pH to 3.1 by adding dilute sodium hydroxide TS, and
cm－1 and 600 cm－1.
add water to make 100 mL.
Optical rotation <2.49> [a]20D : ＋7 – ＋109(0.4 g, ethanol
4-Hydroxy-3-methoxybenzyl nonylic acid amide (99.5), 20 mL, 100 mm).
C17H27NO3 A white crystalline powder, having a faint, Melting point <2.60>: 198 – 2059C
characteristic odor. Purity Related substances—Dissolve 20 mg of hyodeoxy-
Purity Related substances—Dissolve 10 mg of 4-Hydro- cholic acid for thin-layer chromatography in 1 mL of metha-
xy-3-methoxybenzyl nonylic acid amide in 50 mL of methanol, and use this solution as the sample solution. Pipet 0.2
nol, and use this solution as the sample solution. Pipet 1 mL mL of this solution, add methanol to make exactly 10 mL,
of the sample solution, add methanol to make exactly 20 and use this solution as the standard solution. Perform the
mL, and use this solution as the standard solution. Perform test as directed under Thin-layer Chromatography <2.03>.
the test with exactly 20 mL each of the sample solution and Spot 5 mL each of the sample solution and standard solution
standard solution as directed in the Assay under Capsicum: on a plate of silica gel for thin-layer chromatography. De-
when measure the peak areas 2 times as long as the retention velop the plate with a mixture of chloroform, acetone and
time of capsaicin, the total area of the peaks other than 4- acetic acid (100) (7:2:1) to a distance of about 10 cm, and
hydroxy-3-methoxybenzyl nonylic acid amide from the sam- air-dry the plate. Splay evenly dilute sulfuric acid on the
ple solution is not larger than the peak area of 4-hydroxy-3- plate, and heat at 1059 C for 10 minutes: the spots other than
methoxybenzyl nonylic acid amide from the standard solu- the principal spot at the R f value of about 0.3 obtained from
tion. the sample solution are not more intense than the spot ob-
tained from the standard solution.
3-(3-Hydroxy-4-methoxyphenyl)-2-(E )-propenic acid See
(E )-isoferulic acid. Hypaconitine for purity C33H45NO10 White, crystals or
crystalline powder. Soluble in acetonitrile, sparingly soluble
3-(3-Hydroxy-4-methoxyphenyl)-2-(E )-propenic acid-(E )-
in ethanol (99.5) and in diethyl ether, and practically insolu-
ferulic acid TS for thin-layer chromatography See (E )-
ble in water. Melting point: about 1759 C (with decomposi-
isoferulic acid-(E )-ferulic acid TS for thin-layer chromatog-
tion).
raphy.
Identification—Determine the infrared absorption spec-
2-[4-(2-Hydroxymethyl)-1-piperazinyl] propanesulfonic trum of hypaconitine for purity as directed in the potassium
acid C8H18N2O4S A white crystalline powder. bromide disk method under Infrared Spectrophotometry
Residue on ignition <2.44>: not more than 0.1z. <2.25>: it exhibits absorption at the wave numbers of about
Content: not less than 99z. 3500 cm－1, 1728 cm－1, 1712 cm－1, 1278 cm－1, 1118 cm－1,
1099 cm－1 and 714 cm－1.
N-(3-Hydroxyphenyl)acetamide C8H9NO2 White to
Absorbance <2.24> E 11zcm (230 nm): 217 – 252 [5 mg,
pale yellowish white crystals. It is freely soluble in ethanol
ethanol (99.5), 200 mL].
(95), and sparingly soluble in water.
Purity Related substances—(1) Dissolve 5.0 mg of
Melting point <2.60>: 146 – 1499 C
hypaconitine for purity in 2 mL of acetonitrile, and use as
Purity (1) Clarity and color of solution—Dissolve 0.5 g
the sample solution. Pipet 1 mL of the sample solution, add
of N-(3-hydroxyphenyl)acetamide in 50 mL of water: the so-
acetonitrile to make exactly 50 mL, and use as the standard
lution is clear and colorless.
solution. Perform the test with these solutions as directed
(2) Related substances—Dissolve 0.1 g of N-(3-hydrox-
under Thin-layer Chromatography <2.03>. Spot 20 mL each
yphenyl)acetamide in 1000 mL of water. Pipet 10 mL of this
of the sample solution and standard solution on a plate of
solution, add 6.5 mL of acetonitrile and water to make ex-
silica gel for thin-layer chromatography, and proceed the test
actly 50 mL, and use this solution as the sample solution.
as directed in the Identification in Processed Aconite Root:
Perform the test with 10 mL of the sample solution as di-
the spot other than the principal spot obtained with the sam-
rected in the Assay under Aspoxicillin Hydrate: any peak
ple solution is not more intense than the spot obtained with
other than those of N-(3-hydroxyphenyl)acetamide and the
the standard solution.
solvent does not appear.
(2) Dissolve 5.0 mg of hypaconitine for purity in 5 mL of
3-( p-Hydroxyphenyl)propionic acid C9H10O3 White to acetonitrile, and use as the sample solution. Pipet 1 mL of
light yellow-brown, crystals or crystalline powder, having a the sample solution, add acetonitrile to make exactly 50 mL,
faint, characteristic odor. and use as the standard solution. Perform the test with ex-
Content: not less than 99.0z. Assay—Weigh accurately actly 10 mL each of the sample solution and standard solu-
about 0.2 g of 3-( p-hydroxyphenyl)propionic acid, previ- tion as directed under Liquid Chromatography <2.01> ac-
ously dried (in vacuum, 609C, 4 hours), dissolve in 5 mL of cording to the following conditions, and determine each
methanol, add 45 mL of water, and titrate <2.50> with 0.1 peak area by the automatic integration method: the total
mol/L sodium hydroxide VS (indicator: 5 drops of bromo- area of the peaks other than hypaconitine obtained with the
thymol blue TS). sample solution is not larger than the peak area of hypaconi-
tine obtained with the standard solution.
Each mL of 0.1 mol/L sodium hydroxide VS
＝ 16.62 mg of C9H10O3

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
270 General Tests / 9.41 Reagents, Test Solutions JP XVII
Operating conditions using phosphate buffer solution (pH 7.0) as the blank solu-
Detector, column, and column temperature: Proceed as tion.
directed in the operating conditions in the Purity under
Amount (mg) of hypoxanthine (C5H4N4O)
Processed Aconite Root.
A
Mobile phase: A mixture of phosphate buffer solution for ＝ × 250,000
779
processed aconite root and tetrahydrofuran (9:1).
Flow rate: Adjust so that the retention time of hypaconi- Ibuprofen C13H18O2 [Same as the namesake mono-
tine is about 23 minutes. graph]
Time span of measurement: About 3 times as long as the
Ibuprofen piconol C19H23NO2 [Same as the namesake
retention time of hypaconitine, beginning after the solvent
monograph]
peak.
System suitability Ibuprofen piconol for assay C19H23NO2 [Same as the
Test for required detectability: Pipet 1 mL of the standard monograph Ibuprofen Piconol. It contains not less than
solution, and add acetonitrile to make exactly 20 mL. Con- 99.0z of ibuprofen piconol (C19H23NO2), calculated on the
firm that the peak area of hypaconitine obtained from 10 mL anhydrous basis, and meets the following additional require-
of this solution is equivalent to 3.5 to 6.5z of that obtained ment.]
from 10 mL of the standard solution. Purity Related substances—Dissolve 0.15 g of ibuprofen
System performance: Dissolve 1 mg each of aconitine for piconol for assay in the mobile phase to make 100 mL. To 10
purity, hypaconitine for purity and mesaconitine for purity, mL of this solution add the mobile phase to make 30 mL,
and 8 mg of jesaconitine for purity in 200 mL of acetonitrile. and use this as the sample solution. Pipet 1 mL of the sample
When the procedure is run with 10 mL of this solution under solution, add the mobile phase to make exactly 100 mL, and
the above operating conditions, mesaconitine, hypaconitine, use this solution as the standard solution. Perform the test
aconitine and jesaconitine are eluted in this order, and each with exactly 5 mL each of the sample solution and standard
resolution between these peaks is not less than 1.5, respec- solution as directed under Liquid Chromatography <2.01>
tively. according to the following conditions, and determine the
System repeatability: When the test is repeated 6 times peak areas by the automatic integration method: the total
with 10 mL of the standard solution under the above operat- area of the peaks other than ibuprofen piconol obtained
ing conditions, the relative standard deviation of the peak from the sample solution is not larger than the peak area of
area of hypaconitine is not more than 1.5z. ibuprofen piconol obtained from the standard solution.
Water <2.48>: not more than 1.0z (5 mg, coulometric Operating conditions
titration). Detector, column, column temperature, mobile phase, and
flow rate: Perform as directed in the operating conditions in
Hyperoside for thin-layer chromatography C21H20O12
the Assay under Ibuprofen Piconol Ointment.
Yellow, crystals or crystalline powder. Slightly soluble in
Time span of measurement: About 2 times as long as the
methanol, very slightly soluble in ethanol (99.5), and practi-
retention time of ibuprofen piconol.
cally insoluble in water. Melting point: about 2209C (with
System suitability
decomposition).
Test for required detectability: To exactly 1 mL of the
Identification—Determine the absorption spectrum of a
standard solution add the mobile phase to make exactly 20
solution of hyperoside for thin-layer chromatography in
mL. Confirm that the peak area of ibuprofen piconol ob-
methanol (1 in 100,000) as directed under Ultraviolet-visible
tained with 5 mL of this solution is equivalent to 3.5 to 6.5z
Spectrophotometry <2.24>: it exhibits a maximum between
of that obtained with 5 mL of the standard solution.
255 nm and 259 nm.
System performance: When the procedure is run with 5 mL
Purity Related substances—Dissolve 1 mg of hyperoside
of the standard solution under the above operating condi-
for thin-layer chromatography in 20 mL of methanol. Per-
tions, the number of theoretical plates and the symmetry fac-
form the test with 10 mL of this solution as directed in the
tor of the peak of ibuprofen piconol are not less than 5000
Identification 2) under Crataegus Fruit: any spot other than
and not more than 1.3, respectively.
the principal spot of around R f value of 0.5 does not appear.
System repeatability: When the test is repeated 6 times
Hypophosphorus acid See phosphinic acid. with 5 mL of the standard solution under the above operating
conditions, the relative standard deviation of the peak area
Hypoxanthine C5H4N4O White, crystals or crystalline
of ibuprofen piconol is not more than 2.0z.
powder. Freely soluble in ammonia TS, sparingly soluble in
dilute hydrochloric acid and in hot water, very slightly solu- Icariin for thin-layer chromatography C33H40O15 Light
ble in water, and practically insoluble in methanol. yellow crystals. Very slightly soluble in methanol and in
Purity Related substances—Dissolve 5.0 mg of hypoxan- ethanol (99.5), and practically insoluble in water. Melting
thine in 100 mL of a solution of ammonia solution (28) in point: about 2349C (with decomposition).
methanol (1 in 10) to make exactly 100 mL. Proceed with Purity Related substances—Dissolve 1.0 mg of icariin
this solution as directed in the Purity (4) under Mercaptopu- for thin-layer chromatography in 1 mL of methanol. Per-
rine Hydrate: any spot other than the principal spot at the form the test with 10 mL of this solution as directed in the
R f value of about 0.2 does not appear. Identification under Epimedium Herb: no spot other than
Content: not less than 97.0z and not more than 103.0z. the principal spot having R f value about 0.4 appears.
Assay—Weigh accurately about 0.15 g of hypoxanthine,
Ifenprodil tartrate for assay (C21H27NO2)2.C4H6O6
previously dried at 1059C for 3 hours, and dissolve in phos-
[Same as the monograph Ifenprodil Tartrate. It contains not
phate buffer solution (pH 7.0) to make exactly 1000 mL.
less than 99.5z of ifenprodil tartrate [(C21H27NO2)2.C4H6O6],
Pipet 10 mL of this solution, and dilute with phosphate
calculated on the anhydrous basis, and meets the following
buffer solution (pH 7.0) to make exactly 250 mL. Read the
additional requirement.]
absorbance A of this solution at the wavelength of 250 nm as
Purity Related substances—Dissolve 20 mg of ifenprodil
directed under Ultraviolet-visible Spectrophotometry <2.24>,
tartrate for assay in 200 mL of the mobile phase A, and use

this solution as the sample solution. Pipet 1 mL of the sam- ane.

ple solution, add the mobile phase A to make exactly 100 Melting point <2.60>: 89 – 929C
mL, and use this solution as the standard solution. Perform Purity Related substances—Dissolve 10 mg of imidazole
the test with exactly 20 mL each of the sample solution and for thin-layer chromatography in exactly 20 mL of dichloro-
standard solution as directed under Liquid Chromatography methane, and proceed with this solution as directed in the
<2.01> according to the following conditions. Determine each Purity (6) under Clotrimazole: any spot other than the prin-
peak area by the automatic integration method: the total cipal spot does not appear.
area of the peaks other than ifenprodil obtained from the
Imidazole for water determination See Water Determi-
sample solution is not larger than 1/2 times the peak area of
nation <2.48>.
ifenprodil obtained from the standard solution. For the area
of the peak, having the relative retention time of about 0.55 Imidazole TS Dissolve 8.25 g of imidazole in 65 mL of
to ifenprodil, multiply the relative response factor, 7.1. water, adjust the pH to 6.8 with 5 mol/L hydrochloric acid
Operating conditions TS, and add water to make 100 mL.
Detector, column, column temperature, and flow rate:
Iminodibenzyl C14H13N White to light brown, crystals
Proceed as directed in the operating conditions in the Assay
or crystalline powder, having a slight, characteristic odor.
under Ifenprodil Tartrate Fine Granules.
Melting point <2.60>: 104 – 1109C
Mobile phase A: Dissolve 6.8 g of potassium dihydrogen
Purity (1) Clarity of solution—Dissolve 1.0 g of im-
phosphate in 900 mL of water, adjust to pH 6.5 with potas-
inodibenzyl in 20 mL of methanol by heating on a water
sium hydroxide TS, and add water to make 1000 mL. To 420
bath: the solution is clear.
mL of this solution, add 320 mL of methanol for liquid
(2) Related substances—Proceed as directed in the Purity
chromatography and 260 mL of acetonitrile for liquid chro-
(6) under Carbamazepine: any spot other than the principal
matography.
spot at the R f value of about 0.9 does not appear.
Mobile phase B: Methanol for liquid chromatography.
Nitrogen <1.08>: 6.8 – 7.3z.
Flowing of mobile phase: Control the gradient by mixing
the mobile phases A and B as directed in the following table. 2,2?-Iminodiethanol hydrochloride C4H11NO2.HCl
A pale yellow liquid.
Time after injection Mobile phase Mobile phase Refractive index <2.45> n 20
D : 1.515 – 1.519
of sample (min) A (volz) B (volz) Specific gravity <2.56> d 20
20: 1.259 – 1.263
Water <2.48>: less than 0.1z.
0.0 – 15.0 100 0
15.0 – 15.1 100 → 0 0 → 100 Imipramine hydrochloride C19H24N2.HCl [Same as the
15.1 – 35.0 0 100 namesake monograph]
Immature orange [Same as the namesake monograph]
Time span of measurement: For 35 minutes after injection
Indigo carmine C16H8N2Na2O8S2 [K 8092, Special class]
of the sample solution.
System suitability Indigo carmine TS Dissolve 0.20 g of indigo carmine in
Test for required detectability: Pipet 1 mL of the standard water to make 100 mL. Use within 60 days.
solution, add the mobile phase A to make exactly 10 mL.
2,3-Indolinedione C8H5NO2 [K 8089, Special class]
Confirm that the peak area of ifenprodil obtained from 20
mL of this solution is equivalent to 7 to 13z of that of ifen- Indometacin C19H16ClNO4 [Same as the namesake
prodil obtained from the standard solution. monograph]
System performance: When the procedure is run with 20
Insulin human [Same as the monograph Insulin Human
mL of the standard solution under the above operating con-
(Genetical Recombination)]
ditions, the number of theoretical plates and the symmetry
factor of the peak of ifenprodil are not less than 3500 and Interleukin-2 dependent mouse natural killer cell NKC3
not more than 2.0, respectively. Fractionate using discontinuous concentration gradient
System repeatability: When the test is repeated 6 times method cells obtained by removing adhesive cells and
with 20 mL of the standard solution under the above operat- phagocyteic cells from C3H/He mouse spleen cells. Then,
ing conditions, the relative standard deviation of the peak cultivate in soft agar containing interleukin-2 the cell frac-
area of ifenprodil is not more than 2.0z. tion with potent NK activity and obtain the colonies. From
among the cell lines obtained, one of the cell lines dependent
Imidapril hydrochloride C20H27N3O6.HCl [Same as the
on interleukin-2 that grows in liquid medium and serially
namesake monograph] subcultured in liquid medium containing interleukin-2 is
Imidapril hydrochloride for assay C20H27N3O6.HCl identified as NKC3.
[Same as the monograph Imidapril Hydrochloride. When
Iodine I [K 8920, Special class]
dried, it contains not less than 99.0z of imidapril hydro-
chloride (C20H27N3O6.HCl).] Iodine bromide (II) TS Dissolve 20 g of iodine monobro-
mide in acetic acid (100) to make 1000 mL. Store protected
Imidazole C3H4N2 White crystalline powder. Very
from light.
soluble in water and in methanol.
Absorbance <2.24> E 11zcm (313 nm): not more than 0.031 Iodine for assay I [Same as the monograph Iodine]
(8 g, water, 100 mL).
Iodine monobromide IBr Blackish brown, crystals or
Melting point <2.60>: 89 – 929C masses. It dissolves in water, in ethanol (95), in acetic acid
Imidazole for thin-layer chromatography C3H4N2 (100), in diethyl ether and in carbon disulfide.
White crystalline powder. Very soluble in water and in meth- Melting point <2.60>: 37 – 439C
anol, and freely soluble in ethyl acetate and in dichlorometh- Storage—Preserve in light-resistant glass containers, in a
cold place.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
272 General Tests / 9.41 Reagents, Test Solutions JP XVII
Iodine-starch TS To 100 mL of starch TS add 3 mL of trophotometry <2.24>, and determine the absorbance A at
dilute iodine TS. the wavelength of maximum absorption at about 282 nm.
Iodine trichloride ICl3 [K 8403, Special class] Amount (mg) of 5-iodouracil (C4H3IN2O2)
A
Iodine TS Dissolve 14 g of iodine in 100 mL of a solu- ＝ × 2500
265
tion of potassium iodide (2 in 5), add 1 mL of dilute hydro-
chloric acid, and dilute with water to make 1000 mL (0.05 Iopamidol for assay C17H22I3N3O8 [Same as the mono-
mol/L). graph Iopamidol]
Storage—Preserve in light-resistant containers.
Iotalamic acid for assay C11H9I3N2O4 [Same as the
Iodine TS, dilute To 1 volume of iodine TS add 4 monograph Iotalamic Acid]
volumes of water.
Iron Fe Iron in the forms of strips, sheets, granules or
0.0002 mol/L Iodine TS Measure exactly 1 mL of 0.5 wires. Fe: not less than 97.7z. It is attracted by a magnet.
mol/L iodine TS, add water to make exactly 250 mL, pipet
Iron (II) sulfate heptahydrate FeSO4.7H2O [K 8978,
10 mL of the solution, and add water to make exactly 100
Special class]
mL. Prepare before use.
Iron (II) sulfate TS Dissolve 8 g of iron (II) sulfate hep-
0.5 mol/L Iodine TS To 12.7 g of iodine and 25 g of
tahydrate in 100 mL of freshly boiled and cooled water. Pre-
potassium iodide add 10 mL of water, triturate, and add
pare before use.
water to make 100 mL.
Iron (II) sulfide FeS [K 8948, for hydrogen sulfide de-
Iodoacetic acid ICH2COOH White or practically white
velopment]
crystals.
Iron (II) tartrate TS Dissolve 1 g of iron (II) sulfate hep-
Iodoethane C2H5I A colorless or a dark-brown, clear
tahydrate, 2 g of potassium sodium tartrate tetrahydrate and
liquid, having diethyl ether-like odor.
0.1 g of sodium hydrogen sulfite in water to make 100 mL.
Distilling range <2.57>: 71.0 – 72.59C, not less than 94
volz. Iron (II) thiocyanate TS Add 3 mL of dilute sulfuric acid
to 35 mL of water, and remove the dissolved oxygen by boil-
Iodomethane CH3I [K 8919, Special class]
ing the solution. Dissolve 1 g of iron (II) sulfate heptahy-
Iodomethane for assay CH3I Clear, colorless liquid. drate in this hot solution, cool, and then dissolve 0.5 g of po-
On exposure to light, it liberates iodine and becomes brown. tassium thiocyanate. When the solution is pale red in color,
Miscible with ethanol (95) and with diethyl ether, and spar- decolorize by adding reduced iron, separate the excess of
ingly soluble in water. Use the distillate obtained between reduced iron by decanting, and preserve the solution with
42.29C and 42.69C. protection from oxgen. Do not use a solution showing a pale
Specific gravity <2.56> d 25
25: 2.27 – 2.28. red color.
Purity—Perform the test with 1 mL of iodomethane for
Iron (II) trisodium pentacyanoamine TS To 1.0 g of so-
assay as directed under Gas Chromatography <2.02> accord-
dium pentacyanonitrosylferrate (III) dihydrate add 3.2 mL
ing to the operating conditions in the Assay under
of ammonia TS, shake, stopper closely, and allow to stand
Hypromellose. Measure each peak area by the automatic in-
in a refrigerator overnight. Add this solution to 10 mL of
tegration method, and calculate the amount of iodomethane
ethanol (99.5), filter a yellow colored precipitate by suction,
by the area percentage method: it shows the purity of not
wash with dehydrated diethyl ether, dry, and preserve in a
less than 99.8z. Adjust the detection sensitivity so that the
desiccator. Before using, dissolve in water to make a solu-
peak height of iodomethane from 1 mL of methyl iodide for
tion of 1.0 mg/mL, and store in a refrigerator. Use within 7
assay is about 80z of the full scale.
days after preparation.
Content: not less than 98.0z. Assay—Proceed as di-
rected in the Assay under isopropyl iodide for assay. Iron (III) chloride-acetic acid TS Dissolve 0.1 g of iron
(III) chloride hexahydrate in diluted acetic acid (31) (3 in
Each mL of 0.1 mol/L silver nitrate VS
100) to make 100 mL.
＝ 14.19 mg of CH3I
Iron (III) chloride hexahydrate FeCl3.6H2O [K 8142,
5-Iodouracil for liquid chromatography C4H3IN2O2
Special class]
White crystalline powder. Melting point: about 2759 C (with
decomposition). Iron (III) chloride-iodine TS Dissolve 5 g of iron (III)
Purity—Dissolve 3 mg of 5-iodouracil for liquid chroma- chloride hexahydrate and 2 g of iodine in a mixture of 50 mL
tography in diluted methanol (1 in 25) to make 10 mL. Per- of acetone and 50 mL of a solution of tartaric acid (1 in 5).
form the test with 10 mL of this solution as directed under
Iron (III) chloride-methanol TS Dissolve 1 g of iron (III)
Liquid Chromatography <2.01>, according to the operating
chloride hexahydrate in methanol to make 100 mL.
conditions in the Purity under Idoxuridine Ophthalmic Solu-
tion. Determine each peak area by the automatic integration Iron (III) chloride-potassium hexacyanoferrate (III) TS
method over a time span of twice as long as the retention Dissolve 0.1 g of potassium hexacyanoferrate (III) in 20 mL
time of the principal peak, and calculate the amount of 5- of iron (III) chloride TS. Prepare before use.
iodouracil by the area percentage method: It shows the
Iron (III) chloride-pyridine TS, anhydrous Heat gradu-
purity of not less than 98.5z.
ally 1.7 g of iron (III) chloride hexahydrate by direct applica-
Content: not less than 98.5z. Assay—Weigh accurately
tion of flame, melt, and solidify. After cooling, dissolve the
about 5 mg of 5-iodouracil for liquid chromatography,
residue in 100 mL of chloroform, add 8 mL of pyridine, and
previously dried at 609C for 3 hours under reduced pressure,
filter.
dissolve in water to make exactly 250 mL. Perform the test
with this solution as directed under Ultraviolet-visible Spec- Iron (III) chloride TS Dissolve 9 g of iron (III) chloride

hexahydrate in water to make 100 mL (0.33 mol/L). namesake monograph]

Iron (III) chloride TS, acidic To 60 mL of acetic acid Irsogladine maleate for assay C9H7Cl2N5.C4H4O4
(100) add 5 mL of sulfuric acid and 1 mL of iron (III) chlo- [Same as the monograph Irsogladine Maleate. When dried,
ride hexahydrate TS. it contains not less than 99.5z of irsogladine maleate
(C9H7Cl2N5.C4H4O4).]
Iron (III) chloride TS, dilute Dilute 2 mL of iron (III)
chloride TS with water to make 100 mL. Prepare before use. Isatin See 2,3-indolinedione.
Iron (III) nitrate enneahydrate Fe(NO3)3.9H2O Iscove's modified Dulbecco's fluid medium for filgrastim
[K 8559, Special class] A fluid medium for cell culture, containing 0.165 g of anhy-
drous calcium chloride, 97.67 mg of anhydrous magnesium
Iron (III) nitrate TS Dissolve 1 g of iron (III) nitrate en-
sulfate, 0.330 g of potassium chloride, 76 mg of potassium
neahydrate in hydrochloric acid-potassium chloride buffer
nitrate, 4.5 g of sodium chloride, 0.125 g of sodium dihydro-
solution (pH 2.0) to make 300 mL.
gen phosphate monohydrate, 17.3 mg of sodium selenite pen-
Iron (III) perchlorate-ethanol TS Dissolve 0.8 g of iron tahydrate, 30 mg of glycine, 25 mg of L-alanine, 84 mg of L-
(III) perchlorate hexahydrate in perchloric acid-ethanol TS arginine hydrochloride, 25 mg of L-asparagine, 30 mg of L-
to make 100 mL. aspartic acid, 91.4 mg of L-cystine dihydrochloride, 75 mg of
Storage—Preserve in tight containers, in a cold place. L-glutamic acid, 0.584 g of L-glutamine, 42 mg of L-histidine
hydrochloride monohydrate, 0.105 g of L-isoleucine, 0.105 g
Iron (III) perchlorate hexahydrate Fe(ClO4)3.6H2O
of L-leucine, 0.146 g of L-lysine hydrochloride, 30 mg of L-
Hygroscopic, light purple crystals, and a solution in ethanol
methionine, 66 mg of L-phenylalanine, 40 mg of L-proline,
(99.5) (1 in 125) is clear and orange in color.
42 mg of L-serine, 95 mg of L-threonine, 16 mg of L-trypto-
Iron (III) sulfate n-hydrate Fe2(SO4)3.x H2O [K 8981, phan, 0.104 g of disodium L-tyrosine, 94 mg of L-valine, 13
Special class] mg of biotin, 4 mg of choline chloride, 4 mg of calcium D-
pantothenate, 4 mg of folic acid, 4 mg of nicotinic acid
Iron (III) sulfate TS Dissolve 50 g of iron (III) sulfate n-
amide, 4 mg of pyridoxal hydrochloride, 0.4 mg of
hydrate in an excess of water, and add 200 mL of sulfuric
riboflavin, 4 mg of thiamine hydrochloride, 13 mg of
acid and water to make 1000 mL.
cyanocobalamin, 7.2 mg of myoinositol, 4.5 g of glucose,
Iron-phenol TS Dissolve 1.054 g of ammonium iron (II) 5.958 g of N-2-hydroxyethylpiperazine-N?-2-ethanesulfonic
sulfate hexahydrate in 20 mL of water, add 1 mL of sulfuric acid, 15 mg of phenol red, 0.110 g of sodium pyruvate and
acid and 1 mL of hydrogen peroxide (30), heat until efferves- 3.024 g of sodium hydrogen carbonate in 1 L.
cence ceases, and dilute with water to make 50 mL. To 3
Iscove's modified Dulbecco's powder medium A powder
volumes of this solution contained in a volumetric flask add
to make fluid medium for cell culture, containing 0.165 g of
sulfuric acid, with cooling, to make 100 volumes, yielding
anhydrous calcium chloride, 97.67 mg of anhydrous magne-
the iron-sulfuric acid solution. Purify phenol by distillation,
sium sulfate, 0.330 g of potassium chloride, 76 mg of potas-
discarding the first 10z and the last 5z, and collect the dis-
sium nitrate, 4.5 g of sodium chloride, 0.125 g of sodium
tillate, with exclusion of moisture, in a dry, tared, glass-
dihydrogen phosphate monohydrate, 17.3 mg of sodium
stoppered flask of about twice the volume of the phenol.
selenite pentahydrate, 30 mg of glycin, 25 mg of L-alanine,
Stopper the flask, solidify the phenol in an ice bath, break-
84 mg of L-arginine hydrochloride, 25 mg of L-asparagine,
ing the top crust with a glass rod to ensure complete crystalli-
30 mg of L-aspartic acid, 91.4 mg of L-cystine dihydrochlo-
zation, and after drying, weigh the flask. To the glass-
ride, 75 mg of L-glutamic acid, 0.584 g of L-glutamine, 42
stoppered flask add 1.13 times the mass of phenol of the
mg of L-histidine hydrochloride monohydrate, 0.105 g of L-
iron-sulfuric acid solution, insert the stopper in the flask,
isoleucine, 0.105 g of L-leucine, 0.146 g of L-lysine hydro-
and allow to stand, without cooling but with occasional
chloride, 30 mg of L-methionine, 66 mg of phenylalanine, 40
shaking, until the phenol is liquefied, then shake the mixture
mg of L-proline, 42 mg of L-serine, 95 mg of L-threonine, 16
vigorously. Allow to stand in a dark place for 16 to 24 hours.
mg of L-tryptophan, 0.104 g of disodium L-tyrosine, 94 mg
To the mixture add diluted sulfuric acid (10 in 21) equivalent
of L-valine, 13 mg of biotin, 4 mg of choline chloride, 4 mg
to 23.5z of its mass, mix well, transfer to dry glass-
of calcium D-pantothenate, 4 mg of folic acid, 4 mg of nico-
stoppered bottles, and preserve in a dark place, with protec-
tinic acid amide, 4 mg of pyridoxal hydrochloride, 0.4 mg of
tion from atmospheric moisture. Use within 6 months.
riboflavin, 4 mg of thiamine hydrochloride, 13 mg of
Iron-phenol TS, dilute To 10 mL of iron-phenol TS add cyanocobalamin, 7.2 mg of myoinositol, 4.5 g of glucose,
4.5 mL of water. Prepare before use. 5.958 g of N-2-hydroxyethylpyperadine-N-2-ethanesul-
fonate, 15 mg of phenol red and 0.110 g of sodium pyruvate
Iron powder Fe A lusterless, gray to grayish black
in each L.
powder, being attracted by a magnet.
Identification—To 1 mL of a solution of iron powder in Isoamyl acetate See 3-methylbutyl acetate.
hydrochloric acid (1 in 50) add water to make 15 mL, and
Isoamyl alcohol See 3-methyl-1-butanol.
add 0.1 mL of potassium hexacyanoferrate (III) TS: a blue
color appears. Isoamyl benzoate C12H16O2
Specific gravity <2.56> d 15
4 : 0.993
Iron salicylate TS Dissolve 0.1 g of ammonium iron (III)
Boiling point <2.57>: 260 – 2629 C
sulfate dodecahydrate in 50 mL of diluted sulfuric acid (1 in
250), and add water to make 100 mL. Measure 20 mL of this Isoamyl parahydroxybenzoate C12H16O3 White crystal-
solution, and add 10 mL of a solution of sodium salicylate line powder, having a faint characteristic odor.
(23 in 2000), 4 mL of dilute acetic acid, 16 mL of sodium It is very soluble in acetonitrile, in ethanol (95), in acetone
acetate TS and water to make 100 mL. Prepare before use. and in diethyl ether, and practically insoluble in water.
Melting point <2.60>: 62 – 649C
Irsogladine maleate C9H7Cl2N5.C4H4O4 [Same as the

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
274 General Tests / 9.41 Reagents, Test Solutions JP XVII
Isobutanol See 2-methyl-1-propanol. length: 365 nm): no spot other than the principal spot (R f
value of about 0.6) appears.
Isobutyl parahydroxybenzoate C11H14O3 Colorless crys-
tals or white crystalline powder. Freely soluble in ethanol (E )-Isoferulic acid-(E )-ferulic acid TS for thin-layer chro-
(95), and practically insoluble in water. matography Dissolve 1 mg each of (E )-isoferulic acid and
Melting point <2.60>: 74 – 789C (E )-ferulic acid in 2 mL of methanol.
Residue on ignition <2.44>: not more than 0.1z.
Isoelectric point markers for teceleukin Dissolve 0.02 to
Content: not less than 99.0z. Assay—Weigh accurately
0.05 mg of cytochrome C, trypsinogen, lentil-lectin basic
about 1 g of isobuthyl parahydroxybenzoate, add exactly 20
band, lentil-lectin middle band, lentil-lectin acidic band,
mL of 1 mol/L sodium hydroxide VS, heat at about 709 C
horse myoglobin basic band, horse myoglobin acidic band,
for 1 hour, and immediately cool in ice. Titrate <2.50> the
human carbonic anhydrase B, bovine carbonic anhydrase B,
excess sodium hydroxide with 0.5 mol/L sulfuric acid VS up
and b-lactoglobulin A, in 0.1 mL of saccharose solution (3 in
to the second equivalent point (potentiometric titration).
10).
Perform a blank determination.
L-Isoleucine C6H13NO2 [Same as the namesake mono-
Each mL of 1 mol/L sodium hydroxide VS
graph]
＝ 194.2 mg of C11H14O3
L-Isoleucine for assay C6H13NO2 [Same as the mono-
Isobutyl salicylate C11H14O3 Colorless, clear liquid,
graph L-Isoleucine. When dried, it contains not less than
having a characteristic odor.
99.0z of L-isoleucine (C6H13NO2).]
Refractive index <2.45> n 20
D : 1.506 – 1.511
Specific gravity <2.56> d 20
4 : 1.068 – 1.073 Isomalt C12H24O11 White, powder or grain. Very solu-
Boiling point <2.57>: 260 – 2629C ble in water, and practically insoluble in ethanol (99.5).
Purity—Perform the test with 1 mL of isobutyl salicylate
Isoniazid C 6 H 7 N3 O [Same as the namesake mono-
as directed under Gas Chromatography <2.02> according to
graph]
the following conditions. Determine each peak area by the
automatic integration method, and calculate the amount of Isoniazid for assay C6H7N3O [Same as the monograph
isobutyl salicylate by the area percentage method: It shows Isoniazid. When dried, it contains not less than 99.0z of
the purity of not less than 97.0z. isoniazid (C6H7N3O).]
Operating conditions
Isoniazid TS Dissolve 0.1 g of isoniazid for assay in a
Detector: A thermal conductivity detector.
mixture of 50 mL of methanol and 0.12 mL of hydrochloric
Column: A column about 3 mm in inside diameter and
acid, and add methanol to make 200 mL.
about 2 m in length, packed with siliceous earth for gas chro-
matography, 180 to 250 mm in particle diameter, coated with Isonicotinic acid White, crystals or powder. Melting
polythylene glycol 20 M for gas chromatography at the ratio point: about 3159C (decomposition).
of 10z.
Isonicotinic acid amide C6H6N2O White, crystals or
Column temperature: A constant temperature of about
crystalline powder.
2209C.
Melting point <2.60>: 155 – 1589C
Carrier gas: Helium.
Purity Clarity of solution—Dissolve 1.0 g of the sub-
Flow rate: About 20 mL per minute.
stance to be tested in 20 mL of methanol: the solution is
Detection sensitivity: Adjust the detection sensitivity so
clear.
that the peak height of isobutyl salicylate obtained from 1
Content: not less than 99.0z. Assay—Weigh accurately
mL of the isobutyl salicylate is about 60 to 80z of the full
about 0.3 g of isonicotinic acid amide, previously dried, and
scale.
dissolve in 20 mL of acetic acid (100) by heating. After cool-
Time span of measurement: About 3 times as long as the
ing, add 100 mL of benzene, and titrate <2.50> with 0.1
retention time of isobutyl salicylate, beginning after the sol-
mol/L perchloric acid VS until the color of the solution
vent peak.
changes from purple to blue-green (indicator: 3 drops of
(E )-Isoferulic acid C10H10O4 White to light yellow, crystal violet TS). Perform a blank determination and make
crystals or crystalline powder. Sparingly soluble in methanol any necessary correction.
and in ethanol (99.5), and practically insoluble in water.
Each mL of 0.1 mol/L perchloric acid VS
Melting point: about 2309 C (with decomposition).
＝ 11.21 mg of C6H6N2O
Identification—Determine the absorption spectrum of a
solution of (E )-isoferulic acid in methanol (1 in 200,000) as Isooctane See octane, iso.
directed under Ultraviolet-visible Spectrophotometry <2.24>:
Isopromethazine hydrochloride for thin-layer chromatog-
it exhibits maxima between 215 nm and 219 nm, between 238
raphy C17H20N2S.HCl White, odorless crystalline pow-
nm and 242 nm, between 290 nm and 294 nm, and between
der. Freely soluble in water, in ethanol (95) and in chlo-
319 nm and 323 nm.
roform, and practically insoluble in diethyl ether.
Purity Related substances—Conduct this procedure
Melting point <2.60>: 193 – 1979C
without exposure to light, using light-resistant vessels. Dis-
Purity Related substances—Dissolve 5.0 mg of
solve 1 mg of (E )-isoferulic acid in 1 mL of methanol, and
isopromethazine hydrochloride for thin-layer chromatogra-
use this solution as the sample solution. Perform the test
phy in exactly 25 mL of ethanol (95), and perform the test
with the sample solution as directed under Thin-layer Chro-
with this solution as directed in the Purity (3) under
matography <2.03>. Spot 2 mL of the sample solution on a
Promethazine Hydrochloride: any spot other than the princi-
plate of silica gel for thin-layer chromatography. Develop
pal spot at the R f value of about 0.65 does not appear.
the plate with a mixture of ethyl acetate, acetone and water
(20:12:3) to a distance of about 7 cm, and air-dry the plate. Isopropanol See 2-propanol.
Spray evenly sulfuric acid on the plate, heat at 1059 C for 5
Isopropanol for liquid chromatography See 2-propanol
minutes, and examine under ultraviolet light (main wave-

for liquid chromatography. 100 mL of twice-distilled water, and shake vigorously for 10
minutes. Then centrifuge at a rate of 1800 revolutions per
Isopropylamine See propylamine, iso.
minute for 20 minutes, separate the supernatant liquid
Isopropylamine-ethanol TS To 20 mL of isopropylamine (isopropyl myristate layer), and determine the pH of the
add ethanol (99.5) to make 100 mL. Prepare before use. residual water layer: not less than 5.5.
Treat isopropyl myristate which meets the requirements of
Isopropyl p-aminobenzoate See isopropyl 4-aminoben-
pH determination as follows: 500 mL of isopropyl myristate,
zoate.
which has met the requirements of pH determination, is per-
Isopropyl 4-aminobenzoate NH2C6H4COOCH(CH3)2 colated through a 15-cm high layer of activated alumina
Pale brown crystals. filled in a glass column 20 mm in diameter and 20 cm in
Melting point <2.60>: 83 – 869C length with a slightly positive pressure in order to facilitate
adequate flow, and then sterilized by filtration.
Isopropyl benzoate C6H5COOCH(CH3)2 A clear, col-
orless liquid, having a characteristic odor. Isopropyl parahydroxybenzoate C10H12O3 Colorless fine
Referactive index <2.45> n 20D : 1.490 – 1.498 crystals, or white crystalline powder. Freely soluble in
Specific gravity <2.56> d 20
20: 1.008 – 1.016 ethanol (95), and very slightly soluble in water.
Melting point <2.60>: 84 – 869C
Isopropylether See propylether, iso.
Residue on ignition <2.44>: not more than 0.1z.
Isopropyl p-hydroxybenzoate See isopropyl parahy- Content: not less than 99.0z. Assay—Weigh accurately
droxybenzoate. about 1 g of isopropyl parahydroxybenzoate, add exactly 20
mL of 1 mol/L sodium hydroxide VS, heat at about 709 C
Isopropyl iodide for assay C3H7I Colorless, clear liq-
for 1 hour, and immediately cool in ice. Titrate <2.50> the
uid. On exposure to light it liberates iodine and becomes
excess sodium hydroxide with 0.5 mol/L sulfuric acid VS up
brown. Miscible with ethanol (95), with diethyl ether and
to the second equivalent point (potentiometric titration).
with petroleum benzin, and not miscible with water. Use the
Perform a blank determination.
distillate obtained between 89.09C and 89.59C.
Specific gravity <2.56> d 20
4 : 1.700 – 1.710 Each mL of 1 mol/L sodium hydroxide VS
Purity—Perform the test with 1 mL of isopropyl iodide for ＝ 180.2 mg of C10H12O3
assay as directed under Gas Chromatography <2.02> accord-
4-Isopropylphenol C9H12O White to reddish yellow,
ing to the operating conditions in the Assay under
crystals or crystalline powder.
Hypromellose. Measure each peak area by the automatic
Melting point <2.60>: 59 – 639C
integration method, and calculate the amount of isopropyl
iodide by the area percentage method: It shows the purity of Isosorbide dinitrate for assay C6H8N2O8 [Same as the
not less than 99.8z. Adjust the detection sensitivity so that monograph Isosorbide Dinitrate. It contains not less than
the peak height of isopropyl iodide from 1 mL of isopropyl 99.0z of isosorbide dinitrate (C6H8N2O8), calculated on the
iodide for assay is about 80z of the full scale. anhydrous basis, meeting the following additional specifica-
Content: not less than 98.0z. Assay—Transfer 10 mL tions.]
of ethanol (95) into a brown volumetric flask, weigh accu- Purity Related substances—Dissolve 50 mg of isosorbide
rately, add 1 mL of isopropyl iodide for assay, and weigh dinitrate for assay in 50 mL of a mixture of water and meth-
accurately again. Add ethanol (95) to make exactly 100 mL, anol (1:1), and use this solution as the sample solution. Pipet
pipet 20 mL of this solution into the second brown volumet- 1 mL of this solution, add a mixture of water and methanol
ric flask, add exactly 50 mL of 0.1 mol/L silver nitrate VS (1:1) to make exactly 200 mL, and use this solution as the
and then 2 mL of nitric acid, stopper, shake occasionally for standard solution. Perform the test with exactly 10 mL each
2 hours in a dark place, and allow to stand overnight in a of the sample solution and standard solution as directed
dark place. Shake occasionally for 2 hours, add water to under Liquid Chromatography <2.01> according to the fol-
make exactly 100 mL, and filter through dry filter paper. lowing conditions, and determine each peak area of both
Discard the first 20 mL of the filtrate, pipet the next 50 mL, solutions by the automatic integration method: the total area
and titrate <2.50> the excess silver nitrate with 0.1 mol/L am- of the peaks other than the peak of isosorbide dinitrate
monium thiocyanate VS (indicator: 2 mL of ammonium iron obtained from the sample solution is not larger than the peak
(III) sulfate TS). Perform a blank determination in the same area of isosorbide dinitrate obtained from the standard solu-
manner. tion.
Operating conditions
Each mL of 0.1 mol/L silver nitrate VS
Detector, column, column temperature, mobile phase and
＝ 17.00 mg of C3H7I
flow rate: Proceed as directed in the operating conditions in
Isopropyl myristate C17H34O2 Colorless, clear, oily liq- the Assay under Isosorbide Dinitrate Tablets.
uid, and odorless. Congeals at about 59C. Soluble in 90z Time span of measurement: About 2 times as long as the
alcohol, miscible with many organic solvents and with solid retention time of isosorbide dinitrate, beginning after the
oils, and insoluble in water, in glycerin and in propylene solvent peak.
glycol. System suitability
Refractive index <2.45> n 20
D : 1.432 – 1.436 Test for required detectability: Pipet 5 mL of the standard
Specific gravity <2.56> d 20
20: 0.846 – 0.854 solution, and add a mixture of water and methanol (1:1) to
Acid value <1.13>: not more than 1. make exactly 50 mL. Confirm that the peak area of isosor-
Saponification value <1.13>: 202 – 212 bide dinitrate obtained from 10 mL of this solution is equiva-
Iodine value <1.13>: not more than 1. lent to 7 to 13z of that of isosorbide dinitrate obtained
Residue on ignition <2.44>: not more than 0.1z (1 g). from 10 mL of the standard solution.
System performance: When the procedure is run with 10
Isopropyl myristate for sterility test C17H34O2 Transfer
mL of the standard solution under the above operating con-
100 mL of isopropyl myristate into a centrifuge tube, add
ditions, the number of theoretical plates and the symmetry

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
276 General Tests / 9.41 Reagents, Test Solutions JP XVII
factor of the peak of isosorbide dinitrate are not less than assay, put in a Kjeldahl flask, dissolve in 10 mL of water,
3000 and not more than 1.5, respectively. add 3 g of Devarda's alloy and 40 mL of water, and set the
System repeatability: When the test is repeated 6 times flask on the apparatus as shown in the figure under Nitrogen
with 10 mL of the standard solution under the above operat- Determination <1.08>. Put exactly 25 mL of 0.05 mol/L sul-
ing conditions, the relative standard deviation of the peak furic acid VS and 5 drops of bromocresol green-methyl red
area of isosorbide dinitrate is not more than 2.0z. TS in a absorption flask, and set to the apparatus to immerse
the lower end of the condenser. Add 15 mL of sodium hy-
Isosorbide mononitrate for assay C6H9NO6 Odorless
droxide solution (1 in 2) through the funnel, rinse cautiously
white crystals.
the funnel with 20 ml of water, immediately close the clamp
Method of purification: To Isosorbide Mononitrate 70z/
attached to the rubber tubing, then begin the distillation with
Lactose 30z add not less than 3-fold volume of ethyl ace-
steam, and continue until the distillate measures about 100
tate, shake vigorously, filter through a membrane filter with
mL. Remove the absorption flask from the lower end of the
a pore size not exceeding 0.5 mm, and evaporate the filtrate
condenser, rinse the end part of the condenser with a small
to dryness on a water bath under reduced pressure.
quantity of water, and titrate <2.50> with 0.1 mol/L sodium
Recrystallize the residue from a mixture of hexane and ethyl
hydroxide VS until the color of the solution changes from
acetate (3:2), and dry under reduced pressure on silica gel for
red to light blue-green through a light red-purple. Perform a
4 hours.
blank determination in the same manner.
Identification—Determine the infrared absorption spec-
trum of isosorbide mononitrate for assay, previously dried, Each mL of 0.05 mol/L sulfuric acid VS
as directed in the potassium bromide disk method under In- ＝ 19.11 mg of C6H9NO6
frared Spectrophotometry <2.25>: it exhibits absorption at
Isotonic sodium chloride solution [Same as the name-
the wave numbers of between 3210 cm－1 and 3230 cm－1, and
sake monograph]
about 1651 cm－1, 1635 cm－1, 1282 cm－1, 1093 cm－1 and 852
cm－1. Isoxsuprine hydrochloride for assay C18H23NO3.HCl
Optical rotation <2.49> [a]20 D : ＋171 – ＋1769(after dry- [Same as the monograph Isoxsuprine Hydrochloride]
ing, 1 g, ethanol (95), 100 mL, 100 mm).
Japanese acid clay Natural hydrous aluminum silicate,
Melting point <2.60>: 89 – 929C
grayish white powder, having a particle size of about 75 mm.
Purity Related substances—Dissolve 50 mg of isosorbide
Loss on drying <2.41>: not more than 10z (1 g, 1059C,
mononitrate for assay in 5 mL of water, and use this solu-
4 hours).
tion as the sample solution. Pipet 1 mL of the sample solu-
Water adsorbing capacity: not less than 2.5z. Weigh ac-
tion, add water to make exactly 100 mL. Pipet 5 mL of this
curately about 10 g of Japanese acid clay in weighing bottle,
solution, add water to make exactly 50 mL, and use this so-
allow to stand for 24 hours with cover in a chamber, in
lution as the standard solution. Perform the test with exactly
which humidity is maintained to 80z by means of sulfuric
10 mL each of the sample solution and standard solution as
acid (specific gravity 1.19), reweigh, and determine the in-
directed under Liquid Chromatography <2.01> according to
crease of mass of the sample.
the following conditions. Determine each peak area by the
automatic integration method: the area of the peak other Japanese zanthoxylum peel [Same as the namesake
than isosorbide mononitrate obtained from the sample solu- monograph]
tion is not larger than the peak area of isosorbide mononi-
Jesaconitine for purity C35H49NO12 A white powder.
trate obtained from the standard solution, and the total area
Freely soluble in acetonitrile, in ethanol (99.5) and in diethyl
of the peaks other than isosorbide mononitrate from the
ether, and practically insoluble in water.
sample solution is not larger than 2 times the peak area of
Identification—Determine the infrared absorption spec-
isosorbide mononitrate from the standard solution. For the
trum of jesaconitine for purity as directed in the potassium
area of the peak, having a relative retention time of about
bromide disk method under Infrared Spectrophotometry
4.5 to isosorbide mononitrate, multiply its relative response
<2.25>: it exhibits absorption at the wave numbers of about
factor, 0.62.
3500 cm－1, 1715 cm－1, 1607 cm－1, 1281 cm－1, 1259 cm－1,
Operating conditions
1099 cm－1 and 772 cm－1.
Detector, column, column temperature, mobile phase, and
Absorbance <2.24> E 11zcm (258 nm): 270 – 291 [5 mg,
flow rate: Proceed as directed in the operating conditions in
ethanol (99.5), 200 mL].
the Assay under Isosorbide Mononitrate 70z/Lactose 30z.
Purity Related substances—(1) Dissolve 5.0 mg of
Time span of measurement: About 5 times as long as the
jesaconitine for purity in 2 mL of acetonitrile, and use this
retention time of isosorbide mononitrate, beginning after the
solution as the sample solution. Pipet 1 mL of the sample so-
solvent peak.
lution, add acetonitrile to make exactly 50 mL, and use this
System suitability
solution as the standard solution. Perform the test with these
System performance: When the procedure is run with 10
solutions as directed under Thin-layer Chromatography
mL of the standard solution under the above operating con-
<2.03>. Spot 20 mL each of the sample solution and standard
ditions, the number of theoretical plates and the symmetry
solution on a plate of silica gel for thin-layer chromatogra-
factor of the peak of isosorbide mononitrate are not less
phy, and proceed the test as directed in the Identification in
than 2000 and not more than 1.5, respectively.
Processed Aconite Root: the spot other than the principal
System repeatability: When the test is repeated 6 times
spot with the sample solution is not more intense than the
with 10 mL of the standard solution under the above operat-
spot with the standard solution.
ing conditions, the relative standard deviation of the peak
(2) Dissolve 5.0 mg of jesaconitine for purity in 5 mL of
area of isosorbide mononitrate is not more than 2.0z.
acetonitrile, and use this solution as the sample solution.
Loss on drying <2.41>: not more than 0.5z (1 g, in vacu-
Pipet 1 mL of the sample solution, add acetonitrile to make
um, silica gel, 4 hours).
exactly 50 mL, and use this solution as the standard solution.
Content: not less than 99.0z. Assay—Weigh accurately
Perform the test with exactly 10 mL each of the sample solu-
about 0.2 g of previously dried isosorbide mononitrate for
tion and standard solution as directed under Liquid Chroma-

tography <2.01> according to the following conditions, and phosphate buffer solution (pH 8.0) so that 10 mL of the so-
determine each peak area by the automatic integration lution contains 1 mg of kininogen, and use this solution as
method: the total area of the peaks other than jesaconitine the sample solution. Perform the following tests with the
with the sample solution is not larger than the peak area of sample solution: it meets the requirement of each test.
jesaconitine with the standard solution. (i) Immediately after the sample solution is prepared,
Operating conditions add 0.1 mL of a solution of trichloroacetic acid (1 in 5) to
Detector, column, and column temperature: Proceed as 0.5 mL of the sample solution, shake, and centrifuge. To 0.5
directed in the operating conditions in the Purity under mL of the supernatant liquid add 0.5 mL of gelatin-tris
Processed Aconite Root. buffer solution (pH 8.0), and shake. To 0.1 mL of this solu-
Mobile phase: A mixture of phosphate buffer solution for tion add 1.9 mL of trichloroacetic acid-gelatin-tris buffer so-
processed aconite root and tetrahydrofuran (9:1). lution. Proceed with 0.1 mL of this solution as directed in
Flow rate: Adjust so that the retention time of jesaconitine the Purity (2) under Kallidinogenase, and determine the
is about 36 minutes. amount of kinin: kinin is not detected.
Time span of measurement: About 3 times as long as the (ii) Warm 0.5 mL of the sample solution at 30 ± 0.59C
retention time of jesaconitine, beginning after the solvent for 20 minutes, and proceed as directed in (i): kinin is not de-
peak. tected.
System suitability (iii) Perform the test with 0.5 mL of the sample solution
Test for required detectability: Pipet 1 mL of the standard as directed in the Purity (2) under Kallidinogenase: the de-
solution, and add acetonitrile to make exactly 20 mL. Con- composition of bradykinin is not observed.
firm that the peak area of jesaconitine obtained from 10 mL (iv) To 0.5 mL of the sample solution add 0.5 mL of
of this solution is equivalent to 3.5 to 6.5z of that obtained 0.02 mol/L phosphate buffer solution (pH 8.0) containing
from 10 mL of the standard solution. 500 mg of crystalline trypsin, previously warmed at 30 ±
System performance: Dissolve 5 mg each of aconitine for 0.59C for 5 minutes, warm this solution at 30 ± 0.59C for 5
purity, hypaconitine for purity and mesaconitine for purity, minutes, add 0.2 mL of a solution of trichloroacetic acid
and 1 mg of jesaconitine for purity in 200 mL of acetonitrile. (1 in 5), and shake. Then boil for 3 minutes, cool in ice im-
When the procedure is run with 10 mL of this solution under mediately, and centrifuge. To 0.5 mL of the supernatant liq-
the above operating conditions, mesaconitine, hypaconitine, uid add 0.5 mL of gelatin-tris buffer solution (pH 8.0), and
aconitine and jesaconitine are eluted in this order, and each shake. To 0.1 mL of this solution add 0.9 mL of trichloroa-
resolution between these peaks is not less than 1.5, respec- cetic acid-gelatin-tris buffer solution. To 0.1 mL of this solu-
tively. tion add trichloroacetic acid-gelatin-tris buffer solution to
System repeatability: When the test is repeated 6 times make 20 mL, then proceed as directed in (i), and determine
with 10 mL of the standard solution under the above operat- the amount, BK, of kinin per well. Calculate the kinin-releas-
ing conditions, the relative standard deviation of the peak ing activity per mg by the following equation: not less than
area of jesaconitine is not more than 1.5z. 10 mg bradykinin equivalent per mg.
Water <2.48>: not more than 1.0z (5 mg, coulometric
Kinin-releasing activity per mg (mg bradykinin
titration).
equivalent/mg) ＝ BK × 0.96
Josamycin C42H69NO15 [Same as the namesake mono-
Kininogen TS Dissolve a sufficient quantity of kininogen
graph]
in 0.02 mol/L phosphate buffer solution (pH 8.0) to prepare
Josamycin propionate C45H73NO16 [Same as the name- a solution having an ability in each mL to release kinin cor-
sake monograph] responding to not less than 1 mg of bradykinin.
Kainic acid See kainic acid hydrate. Labetalol hydrochloride C19H24N2O3.HCl [Same as the
namesake monograph]
Kainic acid for assay See kainic acid hydrate.
Labetalol hydrochloride for assay C19H24N2O3.HCl
Kainic acid hydrate C10H15NO4.H2O [Same as the
[Same as the monograph Labetalol Hydrochloride.
namesake monograph]
However, when dried, it contains not less than 99.0z of
Kainic acid hydrate for assay See kainic acid hydrate. labetalol hydrochloride (C19H24N2O3.HCl).]
Kanamycin sulfate C18H36N4O11.xH2SO4 [Same as the Lactic acid CH3CH(OH)COOH [K 8726, Special class]
namesake monograph]
Lactic acid TS Dissolve 12.0 g of lactic acid in water to
Karl Fischer TS for water determination See Water De- make 100 mL.
termination <2.48>.
a-Lactoalbumin White powder. Derived from milk.
Kerosene It is mainly a mixture of hydrocarbons in the Molecular weight of about 14,200.
methane series, and a colorless, clear liquid, having not a
Lactobionic acid C12H22O12 Colorless crystals or white
disagreeable, characteristic odor.
crystalline powder, having no odor.
Specific gravity <2.56>: about 0.80
Melting point <2.60>: 113 – 1189C
Distilling range <2.57>: 180 – 3009C
Purity—Dissolve 0.10 g of lactobionic acid in 10 mL of a
Ketoconazole C26H28Cl2N4O4 [Same as the namesake mixture of methanol and water (3:2), and perform the test
monograph] with 10 mL of this solution as directed in the Identification
(2) under Erythromycin Lactobionate: the spot other than
Ketoconazole for assay C26H28Cl2N4O4 [Same as the
the principal spot is not found.
monograph Ketoconazole. When dried, it contains not less
than 99.5z of ketoconazole (C26H28Cl2N4O4).] b-Lactoglobulin Prepare from milk. White to light yel-
low powder.
Kininogen Produced by purifying from bovine plasma.
Nitrogen content <1.08>: not less than 14z (calculated on
Dissolve an appropriate amount of kininogen in 0.02 mol/L

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
278 General Tests / 9.41 Reagents, Test Solutions JP XVII
the dried basis). Lead monoxide See lead (II) oxide.
Lactose See lactose monohydrate. Lead nitrate See lead (II) nitrate.
a-Lactose and b-lactose mixture (1:1) Use a mixture of Lead dioxide See lead (IV) oxide.
lactose monohydrate and anhydrous lactose (3:5).
Lead subacetate TS Place the yellowish mixture, ob-
Lactose broth After adding lactose monohydrate to ordi- tained by triturating 3 g of lead (II) acetate trihydrate and 1 g
nary broth in the ratio of 0.5z, add about 12 mL of bromo- of lead (II) oxide with 0.5 mL of water, in a beaker, and heat
thymol blue-sodium hydroxide TS to 1000 mL of the me- on a water bath, covering with a watch glass, until it shows a
dium. Then dispense portions of about 10 mL into tubes for homogeneous, white to reddish white color. Then add 9.5
fermentation, and sterilize fractionally on each of three mL of hot water in small portions, cover it again with a
successive days for 15 to 30 minutes at 1009C by using an watch glass, and set it aside. Decant the supernatant liquid,
autoclave, or sterilize by autoclaving for not more than 20 and adjust the specific gravity to 1.23 to 1.24 (159C) by add-
minutes at 1219C, and cool quickly by immersing in cold ing water.
water. Storage—Preserve in tightly stoppered bottles.
Lactose broth, three times concentrated Add lactose Lead subacetate TS, dilute To 2 mL of lead subacetate
monohydrate to ordinary broth prepared by using 330 mL in TS add freshly boiled and cooled water to make 100 mL.
place of 1000 mL of water in the ratio of 1.5z, and prepare Prepare before use.
according to the method of preparation under lactose broth,
Lead (II) acetate trihydrate Pb(CH3COO)2.3H2O
with 25 mL portions in tubes for fermentation.
[K 8374, Special class]
Lactose broth, twice concentrated Add lactose monohy-
Lead (II) acetate TS To 9.5 g of lead (II) acetate trihy-
drate to ordinary broth prepared by using 500 mL in place of
drate add freshly boiled and cooled water to make 100 mL.
1000 mL of water in the ratio of 1.0z and prepare according
Storage—Preserve in tightly stoppered bottles (0.25 mol/
to the method of preparation under lactose broth.
L).
Lactose monohydrate C12H22O11.H2O [Same as the
Lead (II) nitrate Pb(NO3)2 [K 8563, Special class]
monograph Lactose].
Lead (II) oxide PbO [K 8090, Special class]
Lactose substrate TS Dissolve 6.0 g of lactose monohy-
drate in disodium hydrogen phosphate-citric acid buffer so- Lead (IV) oxide PbO2 A dark brown to black-brown,
lution (pH 4.5) to make 100 mL. powder or granules.
Identification—A supernatant liquid of a solution of lead
Lactose substrate TS for b-galactosidase (penicillium)
(IV) oxide in dilute acetic acid (1 in 100) responds to Qualita-
Dissolve 6.0 g of lactose monohydrate in diluted disodium
tive Tests <1.09> (3) for lead salt.
hydrogen phosphate-citric acid buffer solution (pH 4.5) (1 in
10) to make 100 mL. Lecithin A pale yellow to yellow-brawn, powder or
grains, having a characteristic odor.
Lafutidine for assay C22H29N3O4S [Same as the mono-
It is emulsified with water. Hygroscopic.
graph Lafutidine. When dried, it contains not less than
99.5z of lafutidine (C22H29N3O4S).] L-Leucine C6H13NO2 [Same as the namesake mono-
graph]
Lanthanum-alizarin complexone TS To 1 mL of ammo-
nia water (28) add 10 mL of water. To 4 mL of this solution L-Leucine for assay C6H13NO2 [Same as the mono-
add 4 mL of a solution of ammonium acetate (1 in 5) and graph L-Leucine. When dried, it contains not less than
192 mg of alizarin complexone, and label this solution as 99.0z of L-leucine (C6H13NO2).]
alizarin complexone stock solution. Dissolve 41 g of sodium
Levallorphan tartrate for assay C19H25NO.C4H6O6
acetate trihydrate in 400 mL of water, and add 24 mL of
[Same as the monograph Levallorphan Tartrate. When
acetic acid (100). To this solution add the total volume of the
dried, it contains not less than 99.0z of levallorphen tar-
alizarin complexone stock solution, add 400 mL of acetone,
trate (C19H25NO.C4H6O6).]
and label this solution as alizarin complexone solution. To
10 mL of diluted hydrochloric acid (1 in 6) add 163 mg of Levofloxacin hydrate for assay C18H20FN3O4.1/2H2O
lanthanum (III) oxide, heat to dissolve, and label this solu- [Same as the monograph Levofloxacin Hydrate]
tion as lanthanum (III) oxide solution. To the alizarin com-
Levothyroxine sodium See levothyroxine sodium hy-
plexone solution add the lanthanum (III) oxide solution, and
drate.
mix. After cooling, adjust to pH 4.7 with acetic acid (100) or
ammonia water (28), and add water to make 1000 mL. Pre- Levothyroxine sodium for thin-layer chromatography
pare before use. See levothyroxine sodium hydrate for thin-layer chromato-
graphy.
Lanthanum chloride TS To 58.65 g of lanthanum (III)
oxide add 100 mL of hydrochloric acid, and boil. After cool- Levothyroxine sodium hydrate C15H10I4NNaO4.xH2O
ing, add water to make 1000 mL. [Same as the namesake monograph]
Lanthanum (III) oxide La2O3 White crystals. Levothyroxine sodium hydrate for thin-layer chromatog-
Loss on ignition <2.43>: not more than 0.5z (1 g, 10009C, raphy C15H10I4NNaO4.xH2O [Same as the monograph
1 hour). Levothyroxine Sodium Hydrate. Proceed the test as directed
in the Purity (3) under Levothyroxine Sodium Hydrate: any
Lauromacrogol [Same as the namesake monograph]
spot other than the principal spot at the R f value of about
Lead acetate See lead (II) acetate trihydrate. 0.26 does not appear.]
Lead acetate TS See lead (II) acetate TS. Lidocaine for assay (C14H22N2O) [same as the mono-

graph Lidocaine] insoluble in water. Melting point: about 2109C (with decom-
position).
Limonene C10H16 Clear and colorless liquid, having a
Identification—Determine the absorption spectrum of a
specific perfume and a bitter taste.
solution of liquiritin for thin-layer chromatography in
Refractive index <2.45> n 20
D : 1.472 – 1.474
diluted methanol (1 in 2) (1 in 100,000) as directed under Ul-
Specific gravity <2.56> d 20
20: 0.841 – 0.846
traviolet-visible Spectrophotometry <2.24>: it exhibits maxi-
Melting point <2.60>: 176 – 1779 C
ma between 215 nm and 219 nm, and between 275 nm and
Purity Related substances—Dissolve 0.1 g of limonene in
279 nm.
25 mL of hexane and use this solution as the sample solu-
Purity Related substances—Dissolve 1.0 mg of liquiritin
tion. Perform the test with 2 mL of the sample solution as di-
for thin-layer chromatography in 1 mL of methanol, and
rected under Gas Chromatography <2.02> according to the
perform the test with 1 mL of this solution as directed in the
following conditions. Determine each peak area by the auto-
Identification (5) under Kakkonto Extract: no spot other
matic integration method and calculate the amount of limo-
than the principal spot with an R f value of about 0.4 ap-
nene: it is not less than 97.0z.
pears.
Operating conditions
Proceed the operating conditions in the Assay under Eu- Lisinopril See lisinopril hydrate.
calyptus Oil except detection sensitivity and time span of
Lisinopril for assay See lisinopril hydrate for assay.
measurement.
Detection sensitivity: Measure 1 mL of the sample solu- Lisinopril hydrate C21H31N3O5.2H2O [Same as the
tion, add hexane to make 100 mL, and adjust the detection namesake monograph]
sensitivity so that the peak height of limonene obtained from
Lisinopril hydrate for assay C21H31N3O5.2H2O [Same
2 mL of this solution is 40 to 60z of the full scale.
as the monograph Lisinopril Hydrate. It contains not less
Time span of measurement: About 3 times as long as the
than 99.5z of lisinopril (C21H31N3O5: 405.49), calculated on
retention time of limonene, beginning after the solvent peak.
the anhydrous basis.]
Limonin for thin-layer chromatography C26H30O8
Lithium acetate dihydrate CH3COOLi.2H2O Colorless
White, crystals or crystalline powder. Slightly soluble in
crystals.
methanol and in ethyl acetate, and practically insoluble in
Dilute acetic acid insoluble substances—To 40.0 g of lithi-
water and in ethanol (99.5). Melting point: about 2909C.
um acetate dihydrate add 45 mL of water, heat in a water
Identification Determine the infrared absorption spec-
bath to dissolve, cool, then dissolve in dilute acetic acid, and
trum as directed in the potassium bromide disk method
filter by suction. Wash the filter with water, dry the filter at
under Infrared-visible Spectrophotometry <2.25>: it exhibits
105 ± 29C for 1 hour, and weigh the mass of the residue
absorption at the wave numbers of about 1759 cm－1, 1709
after cooling: not more than 0.0025z.
cm－1, 1166 cm－1, 798 cm－1 and 601 cm－1.
Content: not less than 97.0z. Assay—Weigh accurately
Purity Related substances—Dissolve 1 mg of limonin for
0.3 g of lithium acetate dihydrate, add exactly 50 mL of
thin-layer chromatography in 1 mL of ethyl acetate, and per-
acetic acid (100) and exactly 5 mL of acetic anhydride, dis-
form the test with 1 mL of this solution as directed in the
solve by heating in a water bath, and titrate <2.50> with 0.1
Identification (2) under Orengedokuto Extract: no spot
mol/L perchloric acid VS after cooling (potentiometric titra-
other than the principal spot (R f value is about 0.4) appears.
tion). Perform a blank determination in the same manner,
(Z )-Ligustilide for thin-layer chromatography C12H14O2 and make any necessary correction.
A clear, yellow-grown liquid, having a characteristic odor.
Each mL of 0.1 mol/L perchloric acid VS
Miscible with methanol and with ethanol (99.5), and practi-
＝ 10.20 mg of CH3COOLi.2H2O
cally insoluble in water.
Identification—Determine the absorption spectrum of a Lithium bromide LiBr White, crystals or crystalline
solution in methanol (1 in 100,000) as directed under Ultravi- powder. It is hygroscopic.
olet-visible Spectrophotometry <2.24>: it exhibits a maxi- Purity (1) Chloride <1.03>: not more than 0.1z.
mum between 320 nm and 324 nm. (2) Sulfate <1.14>: not more than 0.01z.
Purity Related substances—Dissolve 1 mg of (Z )-
Lithium chloride LiCl White, crystals or masses.
ligustilide for thin-layer chromatography in 10 mL of metha-
Identification—Perform the test as directed under Flame
nol. Proceed the test with 1 mL of this solution as directed in
Coloration Test <1.04> (1): a persistent red color appears.
the Identification (5) under Hochuekkito Extract: no spot
other than the principle spot of around R f value of about 0.6 Lithium hydroxide monohydrate LiOH.H2O White,
appears. crystals or crystalline powder, having a hygroscopicity.
Liothyronine sodium C15H11I3NNaO4 [Same as the Lithium perchlorate LiClO4 White, crystals or crystal-
namesake monograph] line powder.
Content: not less than 98z. Assay—Accurately weigh
Liothyronine sodium for thin-layer chromatography
about 0.2 g of lithium perchlorate, dissolve in 30 mL of
C15H11I3NNaO4 [Same as the monograph Liothyronine So-
water. Transfer the solution to a chromatographic column,
dium. Proceed as directed for the Identification (1) under
prepared by pouring about 25 mL of strongly acidic ion-
Liothyronine Sodium Tablets: any spot other than the prin-
exchange resin (H type) for column chromatography into a
cipal spot at the R f value of about 0.3 to 0.4 does not ap-
chromatographic tube about 11 mm in inside diameter and
pear.]
about 300 mm in height (after adding 200 mL of 1 mol/L hy-
Liquid paraffin See paraffin, liquid. drochloride TS and flowing at a flow rate of 3 – 4 mL per
minute, wash the chromatographic column with water until
Liquiritin for thin-layer chromatography C21H22O9
the color of the rinse water changes to yellowish red when
White, crystals or crystalline powder. Sparingly soluble in
adding methyl orange TS to the eluate), and flow at a flow
methanol, slightly soluble in ethanol (99.5), and practically
rate of 3 – 4 mL per minute. Then, wash the column with

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
280 General Tests / 9.41 Reagents, Test Solutions JP XVII
about 30 mL of water at a flow rate of 3 – 4 mL per minute 5 larger than the peak area of loganin from the standard solu-
times. Combine the rinse water and the eluate, and titrate tion.
<2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 3 Operating conditions
drops of bromothymol blue TS). Perform a blank determi- Detector, column, column temperature, mobile phase and
nation, and make any necessary correction. flow rate: Proceed as directed in the operating conditions in
the Assay (1) under Goshajinkigan Extract.
Each mL of 0.1 mol/L sodium hydroxide VS
Time span of measurement: About 3 times as long as the
＝ 10.64 mg of LiClO4
retention time of loganin.
Lithium sulfate See lithium sulfate monohydrate. System suitability
System performance and system repeatability: Proceed as
Lithium sulfate monohydrate Li2SO4.H2O [K 8994,
directed in the system suitability in the Assay (1) under
Special class]
Goshajinkigan Extract.
Lithocholic acid for thin-layer chromatography Test for required detectability: Pipet 1 mL of the standard
C24H40O3 White, crystals or crystalline powder. Soluble in solution, and add the mobile phase to make exactly 20 mL.
ethanol (95), in acetic acid (100) and in acetone, slightly solu- Confirm that the peak area of loganin obtained from 10 mL
ble in chloroform, and practically insoluble in water. Melt- of this solution is equivalent to 3.5 to 6.5z of that of loga-
ing point: about 1869C. nin obtained from 10 mL of the standard solution.
Purity Related substances—Dissolve 25 mg of litho-
Loganin for component determination See loganin for
cholic acid for thin-layer chromatography in a mixture of
assay.
chloroform and ethanol (95) (9:1) to make exactly 25 mL.
Dilute 1.0 mL of this solution with a mixture of chloroform Loganin for thin-layer chromatography C17H26O10
and ethanol (95) (9:1) to make exactly 100 mL. Perform the White, crystals or crystalline powder. Soluble in water, spar-
test with 10 mL of this solution as directed in the Purity ingly soluble in methanol, and very slightly soluble in
(4) under Ursodeoxycholic Acid: any spot other than the ethanol (99.5). Melting point: 221 – 2279C.
principal spot with the R f value of about 0.7 does not Purity Related substances—Dissolve 1.0 mg of loganin
appear. for thin-layer chromatography in 2 mL of methanol.
Content: 98.0z. Assay—Weigh accurately about 0.5 g Perform the test with 10 mL of this solution as directed in
of lithocholic acid for thin-layer chromatography, previously the Identification under Cornus Fruit: any spot other than
dried at 809C for 4 hours under reduced pressure (phospho- the principal spot at the R f value of about 0.4 does not
rus (V) oxide), dissolve in 40 mL of neutralized ethanol and appear.
20 mL of water. Add 2 drops of phenolphthalein TS, titrate
Losartan potassium C22H22ClKN6O [Same as the
<2.50> with 0.1 mol/L sodium hydroxide VS, add 100 mL of
namesake monograph]
freshly boiled and cooled water near the end point, and con-
tinue the titration. Lovastatin C24H36O5 White, crystals or crystalline
powder. Soluble in acetonitrile and in methanol, sparingly
Each mL of 0.1 mol/L sodium hydroxide VS
soluble in ethanol (99.5), and practically insoluble in water.
＝ 37.66 mg of C24H40H3
Optical rotation <2.49> [a]20
D : ＋325 – ＋3409(50 mg calcu-
Locke-Ringer's TS lated on the dried basis, acetonitrile, 10 mL, 100 mm).
Loss on drying <2.41>: Not more than 1.0z (1 g, under
Sodium chloride 9.0 g
reduced pressure not exceeding 0.67 kPa, 609 C, 3 hours).
Potassium chloride 0.42 g
Calcium chloride dihydrate 0.24 g Low-molecular mass heparin for calculation of molecular
Magnesium chloride hexahydrate 0.2 g mass.
Sodium hydrogen carbonate 0.5 g It is a low-molecular mass heparin with a disaccharide unit
Dextrose 0.5 g prepared, and display the molecular mass distribution be-
Water, freshly distilled with tween 600 and more than 10,000. When the average of mo-
a hard-glass apparatus a sufficient quantity lecular mass of Low-molecular mass heparin international
To make 1000 mL standard is determined as a reference with this, the differ-
ence compared as a reference with the Low-molecular mass
Prepare before use. The constituents except dextrose and heparin international standard is not less than 5z.
sodium hydrogen carbonate can be made up in concentrated
Luteolin for thin-layer chromatography C15H10O6
stock solutions, stored in a dark place, and diluted before
use. Light yellow to yellow-brown crystalline powder. Slightly
soluble in methanol and in ethanol (99.5), and practically in-
Loganin for assay Loganin for thin-layer chromatogra- soluble in water. Melting point: about 3109C (with decom-
phy meeting the following additional specifications. position).
Absorbance <2.24> E 11zcm (235 nm): 275 – 303 (dried in a Purity Related substances—Dissolve 1.0 mg of luteolin
desiccator (silica gel) for 24 hours, 5 mg, methanol, 500 mL). for thin-layer chromatography in 1 mL of methanol. Pro-
Purity Related substances—Dissolve 2 mg of loganin for ceed the test with 10 mL of this solution as directed in the
assay in 5 mL of the mobile phase, and use this solution as Identification under Chrysanthemum Flower: any spot other
the sample solution. Pipet 1 mL of this solution, add the mo- than the principal spot of R f value of about 0.7 does not
bile phase to make exactly 100 mL, and use this solution as appear.
the standard solution. Perform the test with exactly 10 mL
Lysate reagent A lyophilized product obtained from
each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol- amebocyte lysate of horseshoe crab (Limulus polyphemus or
Tachypleus tridentatus). Amebocyte lysate preparations
lowing conditions. Determine each peak area of both solu-
which do not react to b-glucans are available: they are pre-
tions by the automatic integration method: the total area of
pared by removing the G factor reacting to b-glucans from
the peaks other than loganin from the sample solution is not

amebocyte lysate or by inhibiting the G factor reacting sys- mide disk method under Infrared Spectrophotometry <2.25>:
tem of amebocyte lysate. it exhibits absorption at the wave numbers of about 3170
cm－1, 3000 cm－1, 2840 cm－1, 1459 cm－1, 1231 cm－1, 1122
Lysate TS Dissolve a lysate reagent in water for bacterial
cm－1 and 833 cm－1.
endotoxins test, or in a suitable buffer, by gentle stirring.
Absorbance <2.24> E 11zcm (223 nm): 1066 – 1132 (5 mg,
Lysil endopeptidase White, powder or masses, An ex- methanol, 1000 mL).
otoxin produced by Achromobacter. Molecular weight: Purity Related substances—Dissolve 5 mg of magnoflo-
27,500. rine iodide for assay in 2 mL of a mixture of water and
methanol (1:1), and use this solution as the sample solution.
L-Lysine hydrochloride C6H14N2O2.HCl [Same as the
Pipet 1 mL of the sample solution, add a mixture of water
namesake monograph]
and methanol (1:1) to make exactly 100 mL, and use this so-
Lysyl endopeptidase A protease obtained from Lysobac- lution as the standard solution. Perform the test with these
ter enzymogenes. It contains about 150 units per mg, where 1 solutions as directed under Thin-layer Chromatography
unit is an enzyme amount which hydrolyzes 1 mmol of tosyl- <2.03>. Spot 10 mL each of the sample solution and standard
glycyl-prolyl-lysine-4-nitroanilide acetate per minute at pH solution on a plate of silica gel for thin-layer chromatogra-
7.7 and 259C. phy. Develop the plate with a mixture of ethyl acetate, ace-
tone, water and formic acid (5:3:1:1) to a distance of about 7
Macrogol 600 HOCH2(CH2OCH2)nCH2OH, n ＝ 11 －
cm, and air-dry the plate. Spray evenly Dragendorff's TS for
13 Clear, colorless, viscous liquid or a white, petrolatum-
spraying on the plate, air-dry the plate, and spray evenly so-
like solid, having a faint, characteristic odor. Very soluble in
dium nitrite TS: the spot other than the principal spot at the
water, in ethanol (95), in acetone and in macrogol 400, solu-
R f value of about 0.3 obtained from the sample solution is
ble in diethyl ether, and practically insoluble in petroleum
not more intense than the spot obtained from the standard
benzine. Congealing point: 18 – 239 C
solution.
Average molecular mass: When perform the test as di-
Unity of peak: Dissolve 5 mg of magnoflorine iodide for
rected in the Average molecular mass test under Macrogol
assay in 10 mL of a mixture of water and methanol (1:1),
400, it is between 570 and 630.
and use this solution as the sample solution. Perform the test
Magnesia TS Dissolve 5.5 g of magnesium chloride hexa- with 10 mL of the sample solution as directed under Liquid
hydrate and 7 g of ammonium chloride in 65 mL of water, Chromatography <2.01> according to the following condi-
add 35 mL of ammonia TS, allow the mixture to stand for a tions, and compare the absorption spectra of at least 3 points
few days in tightly stoppered bottles, and filter. If the solu- including the top of magnoflorine peak and around the two
tion is not clear, filter before use. middle peak heights of before and after the top: no differ-
ence is observed in the shape between their spectra.
Magnesium Mg [K 8875, Special class]
Operating conditions
Magnesium chloride See magnesium chloride hexahy- Column, column temperature, and mobile phase: Proceed
drate. as directed in the operating conditions in the Assay (4) under
Kakkontokasenkyushin'i Extract.
Magnesium chloride hexahydrate MgCl2.6H2O
Detector: A photodiode array detector (wavelength: 303
[K 8159, Special class]
nm; measuring range of spectrum: 220 – 400 nm).
Magnesium nitrate See magnesium nitrate hexahydrate. Flow rate: Adjust so that the retention time of magnoflo-
rine is about 20 minutes.
Magnesium nitrate hexahydrate Mg(NO3)2.6H2O
System suitability
[K 8567, Special class]
System performance: To 1 mL of the sample solution add
Magnesium oxide MgO [K 8432, Special class] a mixture of water and methanol (1:1) to make 100 mL.
When the procedure is run with 10 mL of this solution under
Magnesium powder Mg [K 8876, Special class]
the above operating conditions, the number of theoretical
Magnesium sulfate See magnesium sulfate heptahy- plates and the symmetry factor of the peak of magnoflorine
drate. are not less than 5000 and not more than 1.5, respectively.
System repeatability: To 1 mL of the sample solution add
Magnesium sulfate heptahydrate MgSO4.7H2O
a mixture of water and methanol (1:1) to make 100 mL.
[K 8995, Special class]
When the test is repeated 6 times with 10 mL of this solution
Magnesium sulfate TS Dissolve 12 g of magnesium sul- under the above operating conditions, the relative standard
fate heptahydrate in water to make 100 mL (0.5 mol/L). deviation of the peak area of magnoflorine is not more than
1.5z.
Magnoflorine iodide for assay C20H24INO4 White to
Assay—Weigh accurately 5 mg of magnoflorine iodide for
light yellowish white, crystals or crystalline powder. Slightly
assay and 1 mg of DSS-d6 for nuclear magnetic resonance
soluble in water and in methanol, and very slightly soluble in
spectroscopy using an ultramicrobalance, dissolve in 1 mL of
ethanol (99.5). Melting point: about 2509C (with decomposi-
deuterated dimethylsulfoxide for nuclear magnetic resonance
tion).
spectroscopy, and use this solution as the sample solution.
It is used after correcting with the amount of magnoflo-
Transfer the sample solution into an NMR tube 5 mm in
rine iodide obtained in the Assay.
outer diameter, and measure 1H-NMR spectrum as directed
Identification (1) Determine the absorption spectrum
under Nuclear Magnetic Resonance Spectroscopy <2.21> and
of a solution of magnoflorine iodide for assay in methanol (1
Crude Drugs Test <5.01> according to the following condi-
in 200,000) as directed under Ultraviolet-visible Spectropho-
tions, using DSS-d6 for nuclear magnetic resonance spectros-
tometry <2.24>: it exhibits a maximum between 221 nm and
copy as the internal reference compound. Calculate the sig-
225 nm.
nal integrated intensity A (equivalent to 3 hydrogen) around
(2) Determine the infrared absorption spectrum of mag-
d 6.94 – d 7.05 ppm [the integrated intensities A1 (equivalent
noflorine iodide for assay as directed in the potassium bro-
to 2 hydrogen) and A2 (equivalent to 1 hydrogen) of the sig-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
282 General Tests / 9.41 Reagents, Test Solutions JP XVII
nals around d 6.96 ppm and d 7.04 ppm], assuming the sig- tion as directed under Liquid Chromatography <2.01> ac-
nal of the internal reference compound as d 0 ppm. cording to the following conditions, and determine the area
of each peak from these solutions by the automatic integra-
Amount (z) of magnoflorine iodide (C20H24INO4)
tion method: the total area of the peaks other than magnolol
＝ MS × I × P/(M × N) × 2.0918
obtained from the sample solution is not larger than the peak
M: Amount (mg) of magnoflorine iodide for assay taken area of magnolol obtained from the standard solution.
MS: Amount (mg) of DSS-d6 for nuclear magnetic reso- Operating conditions
nance spectroscopy taken Detector, column, column temperature, mobile phase, and
I: The signal integrated intensity, A, based on the signal flow rate: Proceed as directed in the operating conditions in
integrated intensity of DSS-d6 for nuclear magnetic the Assay under Magnolia Bark.
resonance spectroscopy as 9.000 Time span of measurement: About 3 times as long as the
N: Number of hydrogen derived from A. retention time of magnolol.
P: Purity (z) of DSS-d6 for nuclear magnetic resonance System suitability
spectroscopy System performance, and system repeatability: Proceed as
directed in the system suitability in the Assay under Magno-
Operating conditions
lia Bark.
Apparatus: A nuclear magnetic resonance spectroscopy
Test for required detectability: To exactly 1 mL of the
apparatus having 1H resonance frequency of not less than
standard solution add the mobile phase to make exactly 20
400 MHz.
mL. Confirm that the peak area of magnolol obtained with
Target nuclei: 1H.
10 mL of this solution is equivalent to 3.5 to 6.5z of that ob-
Digital resolution: 0.25 or lower.
tained with 10 mL of the standard solution.
Measuring spectrum width: 20 ppm or upper, including
2) Magnolol for assay 2 (Purity value by quantitative
between －5 ppm and 15 ppm.
NMR)
Spinning: off.
Unity of peak—Dissolve 5 mg of magnolol for assay 2 in
Pulse angle: 909.
13C decoupling: on. 10 mL of the mobile phase. To 1 mL of this solution add the
mobile phase to make 100 mL, and use this solution as the
Delay time: Repeating pulse waiting time 60 seconds or
sample solution. Perform the test with 10 mL of the sample
longer.
solution as directed under Liquid Chromatography <2.01>
Integrating times: 8 or more times.
according to the following conditions, and compare the ab-
Dummy scanning: 2 or more times.
sorption spectra of at least 3 points including the top of mag-
Measuring temperature: A constant temperature of 20 –
nolol peak and around the two middle peak heights of before
309 C.
and after the top: no difference in form is observed between
System suitability
their spectra.
Test for required detectability: When the procedure is run
Operating conditions
with the sample solution under the above operating condi-
Column, column temperature, mobile phase, and flow
tions, the S/N of the signal around d 6.94 – d 7.05 ppm is
rate: Proceed as directed in the operating conditions in the
not less than 100.
Assay under Magnolia Bark.
System performance: When the procedure is run with the
Detector: A photodiode array detector (wavelength: 289
sample solution under the above operating conditions, the
nm, measuring range of spectrum: 220 – 400 nm).
two signals of around d 6.96 – d 7.04 ppm are not over-
System suitability
lapped with any signal of obvious foreign substance, and the
System performance: Proceed as directed in the system
ratio of the integrated intensity of each signal (A1/2)/A2 is
suitability in the Assay under Magnolia Bark.
between 0.99 and 1.01.
Assay—Weigh accurately 5 mg of magnolol for assay 2
System repeatability: When the test is repeated 6 times
and 1 mg of 1,4-BTMSB-d4 for nuclear magnetic resonance
with the sample solution under the above operating condi-
spectroscopy using an ultramicrobalance, dissolve in 1 mL of
tions, the relative standard deviation of the ratio of the inte-
deuterated chloroform for nuclear magnetic resonance spec-
grated intensity A to that of the internal reference is not
troscopy, and use this solution as the sample solution.
more than 1.0z.
Transfer the sample solution into an NMR tube 5 mm in
Magnolia flower [Same as the namesake monograph] outer diameter, measure 1H-NMR as directed under Nuclear
Magnetic Resonance Spectroscopy <2.21> and Crude Drugs
Magnolol for assay C18H18O2 Use magnolol for thin-
Test <5.01> according to the following conditions, using 1,4-
layer chromatography meeting the following additional
BTMSB-d4 for nuclear magnetic resonance spectroscopy as
specifications, 1) magnolol for assay 1 or 2) magnolol for
the internal reference compound. Calculate the resonance in-
assay 2 (Purity valve by quantitative NMR). The former is
tensities, A1 (equivalent to 2 hydrogen) and A2 (equivalent
used after drying in a desiccator (silica gel) for 1 hour, and
to 2 hydrogen), of the signals around d 6.70 ppm and d 6.81
the latter is corrected the content based on the amount (z)
ppm assuming the signal of the internal reference compound
obtained in the Assay.
as d 0 ppm.
1) Magnolol for assay 1
Absorbance <2.24> E 11zcm (290 nm): 270 – 293 (10 mg, Amount (z) of magnolol (C18H18O2)
methanol, 500 mL). Use the sample dried in a desiccator ＝ MS × I × P/(M × N) × 1.1758
(silica gel) for not less than 1 hour.
M: Amount (mg) of magnolol for assay 2 taken
Purity Related substances—Dissolve 5.0 mg of magnolol
MS: Amount (mg) of 1,4-BTMSB-d4 for nuclear magnetic
for assay in 10 mL of the mobile phase, and use this solution
resonance spectroscopy taken
as the sample solution. Pipet 1 mL of the sample solution,
I: Sum of the signal resonance intensities, A1 and A2,
add the mobile phase to make exactly 100 mL, and use this
based on the signal resonance intensity of
solution as the standard solution. Perform the test with ex-
1,4-BTMSB-d4 for nuclear magnetic resonance spectros-
actly 10 mL each of the sample solution and standard solu-
copy as 18.000

N: Sum of numbers of the hydrogen derived from A1 and Identification—Determine the infrared absorption spec-
A2 trum of maleic acid as directed in the potassium bromide
P: Purity (z) of 1,4-BTMSB-d4 for nuclear magnetic disk method as directed under Infrared Spectrophotometry
resonance spectroscopy <2.25>: it exhibits absorption at the wave numbers of about
1706 cm－1, 1637 cm－1, 1587 cm－1, 1567 cm－1, 1436 cm－1,
Operating conditions
1263 cm－1, 876 cm－1 and 786 cm－1.
Apparatus: An apparatus of nuclear magnetic resonance
spectrum measurement having 1H resonance frequency of 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid N-
not less than 400 MHz. succinimidyl ester C16H18N2O2 Colorless crystals, which is
Target nucleus: 1H. decomposed by acid or alkali.
Digital resolution: 0.25 or lower.
Maltitol C12H24O11 A white crystalline powder. Very
Measuring spectrum range: 20 ppm or upper, including
soluble in water, and practically insoluble in ethanol (99.5).
between －5 ppm and 15 ppm.
Spinning: off. Maltose See maltose monohydrate.
Pulse angle: 909.
13C decoupling: on. Maltose monohydrate C12H22O11.H2O [Same as the
namesake monograph].
Delay time: Repeating pulse waiting time not less than 60
seconds. Maltotriose C18H32O16 A white powder.
Integrating times: 8 or more times. Identification—Determine the infrared absorption spec-
Dummy scanning: 2 or more times. trum of maltotriose as directed in the potassium bromide
Measuring temperature: A constant temperature between disk method under Infrared Spectrophotometry <2.25>: it ex-
209 C and 309C. hibits absorption at the wave numbers of about 3420 cm－1,
System suitability 1420 cm－1, 1153 cm－1 and 1024 cm－1.
Test for required detectability: When the procedure is run
Manganese dioxide MnO2 Black to black-brown,
with the sample solution under the above operating condi-
masses or powder.
tions, the S/N of the two signals of around d 6.70 ppm and d
Identification—To 0.5 g add 20 mL of water and 3 mL of
6.81 ppm is not less than 100.
hydrochloric acid, and 3 mL of hydrogen peroxide (30).
System performance: When the procedure is run with the
Alkalinize the solution with ammonia solution (28) while
sample solution under the above operating conditions, the
cooling, and add 25 mL of hydrogen sulfide TS: pale red
two signals of around d 6.70 ppm and d 6.81 ppm are not
precipitates appear.
overlapped with any signal of obvious foreign substance,
and the ratios of the resonance intensities, A1/A2, of each Manganese (II) nitrate hexahydrate Mn(NO3)2.6H2O
signal around d 6.70 ppm and d 6.81 ppm are between 0.99 [K 8568, Special class]
and 1.01, respectively.
Manninotriose for thin-layer chromatography C18H32O16
System repeatability: When the test is repeated 6 times
A white powder. Very soluble in water, and practically in-
with the sample solution under the above operating condi-
soluble in ethanol (99.5). It is hygroscopic. It is deliques-
tions, the relative standard deviation of the ratio of the reso-
cence with the atmospheric moisture.
nance intensity, A1 or A2, to that of the internal reference is
Optical rotation <2.49> [a]20
D : ＋159 – ＋1709(50 mg calcu-
not more than 1.0z.
lated on the anhydrous basis, diluted ammonia solution (28)
Magnolol for component determination See magnolol (1 in 1000), 5 mL, 100 mm).
for assay. Purity Related substances—Dissolve 3 mg of mannino-
triose for thin-layer chromatography in 1 mL of a mixture of
Magnolol for thin-layer chromatography C18H18O2
water and methanol (1:1), and use this solution as the sample
Odorless, white, crystals or crystalline powder. Freely solu-
solution. Perform the test with the sample solution as di-
ble in methanol and in ethanol (99.5), and practically insolu-
rected under Thin-layer chromatography <2.03>. Spot 2 mL
ble in water. Melting point: about 1029C.
of the sample solution on a plate of silica gel for thin-layer
Identification—Determine the absorption spectrum of a
chromatography. Develop the plate with a mixture of 2-
solution of magnolol for thin-layer chromatography in
propanol, water and methanol (3:2:2) to a distance of about
methanol (1 in 50,000) as directed under Ultraviolet-visible
7 cm, and air-dry the plate. Spray evenly 1,3-naphtha-
Spectrophotometry <2.24>: it exhibits a maximum between
lenediol TS to the plate, and heat at 1059C for 10 minutes: a
287 nm and 291 nm.
spot other than the principle spot with an R f value of about
Purity Related substances—Dissolve 1.0 mg of magnolol
0.4 is not observed.
for thin-layer chromatography in 1 mL of methanol, and use
this solution as the sample solution. Perform the test with D-Mannitol C6H14O6 [Same as the monograph D-
the sample solution as directed under Thin-layer Chromatog- Mannitol]
raphy <2.03>. Spot 10 mL of the sample solution on a plate of
D-Mannosamine hydrochloride C6H13NO5.HCl White
silica gel with fluorescent indicator for thin-layer chromatog-
powder. Melting point: about 1689 C (with decomposition).
raphy, develop the plate with a mixture of hexane, acetone
Optical rotation <2.49> [a]20
D : －4.2 – －3.29(0.4 g, water,
and acetic acid (100) (20:15:1) to a distance of about 10 cm,
20 mL, 100 mm).
and air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): any spot other than the principal spot D-Mannose C6H12O6 White, crystal or crystalline pow-
of around R f value of 0.5 does not appear. der. It is very soluble in water. Melting point: about 1329 C
(with decomposition).
Malachite green See malachite green oxalate.
Optical rotation <2.49> [a]20
D : ＋13.7 – ＋14.79(4 g, diluted
Malachite green oxalate C52H54N4O12 [K 8878, Mala- ammonia TS (1 in 200), 20 mL, 100 mm).
chite green (oxalate), Special class]
Marker protein for celmoleukin molecular mass deter-
Maleic acid C4H 4O 4 A white crystalline powder. mination Add 10 mL of cytochrome C prepared to a con-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
284 General Tests / 9.41 Reagents, Test Solutions JP XVII
centration of 2 mg per mL to 10 mL of a commercially total peak area other than l-menthol from the sample solu-
available marker protein with a known molecular weight tion is not larger than the peak area of l-menthol from the
(6 ingredients: phosphorylase b, bovine serum albumin, standard solution (1).
ovalbumin, carbonic dehydratase, soy trypsin inhibitor, and Operatin conditions
lysozyme) and then dilute 10-fold with buffer solution for Proceed the operating conditions in the Assay under Men-
celmoleukin. tha Oil except detection sensitivity and time span of meas-
urement.
Meat extract Concentrated extract of fresh meat of bo-
Detection sensitivity: Pipet 1 mL of the standard solution
vine, equine or other animals. A yellow-brown to dark
(1), add dichloromethane to make exactly 20 mL, and use
brown, paste-like mass, having a meat-like odor.
this solution as the standard solution (2). Adjust the detec-
Mebendazole C16H13N3O3 White powder. Practically tion sensitivity so that the peak area of l-menthol obtained
insoluble in water and in ethanol (95). from 5 mL of the standard solution (2) can be measured, and
the peak height of l-menthol from 5 mL of the standard solu-
Medium for float culture Dissolve 6.000 g of sodium
tion (1) is about 20z of the full scale.
chloride, 0.400 g of potassium chloride, 0.677 g of anhy-
Time span of measurement: About twice as long as the
drous sodium dihydrogen phosphate, 0.100 g of calcium
retention time of l-menthol, beginning after the solvent
nitrite tetrahydrate, 0.100 g of magnesium sulfate hepta-
peak.
hydrate, 2.000 g of glucose, 0.164 g of sodium succinate
hexahydrate, 46 mg of succinic acid, 0.240 g of L-arginine Mepivacaine hydrochloride for assay C15H22N2O.HCl
hydrochloride, 56.8 mg of L-asparagine monohydrate, 20 mg [Same as the monograph Mepivacaine Hydrochloride. When
of L-aspartic acid, 72.9 mg of L-cysteine hydrochloride dried, it contains not less than 99.0z of mepivacaine hydro-
monohydrate, 20 mg of L-glutamic acid, 1 mg of chloride (C15H22N2O.HCl).]
glutathione, 10 mg of glycine, 20.3 mg of L-histidine hydro-
Mequitazine for assay C20H22N2S [Same as the mono-
chloride monohydrate, 20 mg of L-hydroxyproline, 50 mg of
graph Mequitazine. When dried, it contains not less than
L-isoleucine, 40 mg of L-lysine hydrochloride, 15 mg of
99.5z of mequitazine (C20H22N2S).]
methionine, 20 mg of L-threonine, 5 mg of L-tryptophan, 20
mg of L-valine, 50 mg of L-leucine, 15 mg of L-phenylala- Mercapto acetic acid HSCH2COOH [K 8630, Special
nine, 20 mg of L-proline, 30 mg of L-serine, 20 mg of L-tyro- class] Place in an ampule, and preserve in a dark, cold
sine, 0.2 mg of D-biotin (crystals), 0.25 mg of calcium panto- place. Do not use after storing for a long period.
thenate, 3 mg of choline chloride, 35 mg of i-inositol, 1 mg
Mercaptoethanesulfonic acid C2H6O3S2 Prepared for
of 4-aminobenzoic acid, 5 mg of cyanocobalamin, 1 mg of
amino acid analysis or biochemistry.
folic acid, 1 mg of nicotinamide, 0.2 mg of riboflavin, 1 mg
of thiamine hydrochloride, 1 mg of pyridoxine hydrochlo- 2-Mercaptoethanol HSCH2CH2OH Clear and colorless
ride, and 5 mg of phenol red in a suitable amount of water, liquid.
add 1 mL of kanamycin sulfate solution (3 in 50), add water Specific gravity <2.56> d 20
4 : 1.112 – 1.117
to make 1000 mL, and then sterilize by autoclaving for 15 Content: not less than 97.0z. Assay—Perform the test
minutes at 1219C. After cooling, add 10 mL of L-glutamine with 0.6 mL of the substance to be examined as directed
solution (3 in 100) and 20 mL of 7z sodium bicarbonate in- under Gas Chromatography <2.02> according to the follow-
jection, and then mix. Store at 49C. ing conditions, and determine the peak areas of each compo-
nent by the automatic integration method.
Mefloquin hydrochloride C17H16F6N2O.HCl [Same as
Content (z) ＝ (the peak area of 2-mercaptoethanol/the
the namesake monograph]
total of the peak areas of each component) × 100
Mefruside for assay C13H19ClN2O5S2 [Same as the Operating conditions
monograph Mefruside. When dried, it contains not less than Detector: A hydrogen flame-ionization detector.
99.0z of mefruside (C13H19ClN2O5S2).] Column: A glass column 3 mm in inside diameter and 2 m
in length, packed with siliceous earth for gas chromatogra-
Meglumine C7H17NO5 [same as the namesake mono-
phy (177–250 mm in particle diameter) coated in 20z with
graph]
50z phenyl-methyl silicone polymer for gas chromatogra-
Mentha herb [Same as the namesake monograph] phy.
Column temperature: A constant temperature of about
Mentha oil [Same as the namesake monograph]
1209C.
Menthol C10H20O [Same as the monograph dl-Menthol Carrier gas: Helium.
or l-Menthol] Flow rate: about 50 mL per minute (the retention time of
2-mercaptoethanol is 3 to 4 minutes.)
l-Menthol for assay C10H20O [Same as the monograph
Time span of measurement: About 7 times as long as the
l-Menthol. It contains not less than 99.0z of l-menthol
retention time of 2-mercaptoethanol.
(C10H20O) and meets the following additional specifications.]
Optical rotation <2.49> [a]20D : －48.0 – －51.09 (2.5 g, 2-Mercaptoethanol for epoetin beta HSCH2CH2OH
ethanol (95), 25 mL, 100 mm). Prepared for study of sulfoprotein.
Purity Related substances—Dissolve 0.10 g of l-menthol
Mercaptopurine See mercaptopurine hydrate.
for assay in 10 mL of dichloromethane, and use this solution
as the sample solution. Pipet 1 mL of this solution, add Mercaptopurine hydrate C5H4N4S.H2O [Same as the
dichloromethane to make exactly 100 mL, and use this solu- namesake monograph]
tion as the standard solution (1). Perform the test with ex-
Mercuric acetate See mercury (II) acetate.
actly 5 mL each of the sample solution and standard solution
(1) as directed under Gas Chromatography <2.02> according Mercuric acetate TS for nonaqueous titration See mer-
to the following conditions, determine each peak area of cury (II) acetate TS for nonaqueous titration.
these solutions by the automatic integration method: the

Mercuric chloride See mercury (II) chloride. peak.

System suitability
Mercury Hg [K 8572, Special class]
Test for required detectability: Pipet 1 mL of the standard
Mercury (II) acetate Hg(CH3COO)2 White, crystals or solution, and add acetonitrile to make exactly 20 mL. Con-
crystalline powder. firm that the peak area of mesaconitine obtained from 10 mL
Identification—(1) Dissolve 1 g of mercury (II) chloride of this solution is equivalent to 3.5 to 6.5z of that obtained
in 1 mL of diluted nitric acid (1 in 7), add 20 mL of water, from 10 mL of the standard solution.
and use this solution as the sample solution. To 10 mL of the System performance: Dissolve 1 mg each of aconitine for
sample solution add 0.8 mL of iron (III) chloride TS: a red- purity, hypaconitine for purity and mesaconitine for purity,
brown color is developed. and 8 mg of jesaconitine for purity in 200 mL of acetonitrile.
(2) To 10 mL of the sample solution obtained in (1) add When the procedure is run with 10 mL of this solution under
2 mL of potassium iodate TS: a red precipitate is produced. the above operating conditions, mesaconitine, hypaconitine,
Preserve in a light-resistant tight container. aconitine and jesaconitine are eluted in this order, and each
resolution between these peaks is not less than 1.5, respec-
Mercury (II) acetate TS for nonaqueous titration Dis-
tively.
solve 5 g of mercury (II) acetate in acetic acid (100) for nona-
System repeatability: When the test is repeated 6 times
queous titration to make 100 mL.
with 10 mL of the standard solution under the above operat-
Mercury (II) chloride HgCl2 [K 8139, Special class] ing conditions, the relative standard deviation of the peak
area of mesaconitine is not more than 1.5z.
Mercury (II) chloride TS Dissolve 5.4 g of mercury (II)
Water <2.48>: not more than 1.0z (5 mg, coulometric
chloride in water to make 100 mL.
titration).
Mesaconitine for purity C33H45NO11 White, crystals or
Mesityl oxide CH3COCH ＝ C(CH3)2 A colorless or
crystalline powder. Slightly soluble in acetonitrile and in
pale yellow, clear liquid, having a characteristic odor.
ethanol (99.5), very slightly soluble in diethyl ether, and
Specific gravity <2.56> d 20
20: 0.850 – 0.860
practically insoluble in water. Melting point: about 1909C
(with decomposition). Metacresol purple C21H18O5S [K 8889, Special class]
Identification—Determine the infrared absorption spec-
Metacresol purple TS Dissolve 0.10 g of metacresol pur-
trum of mesaconitine for purity as directed in the potassium
ple in 13 mL of 0.01 mol/L sodium hydroxide TS, and add
bromide disk method under Infrared Spectrophotometry
water to make 100 mL.
<2.25>: it exhibits absorption at the wave numbers of about
3510 cm－1, 1713 cm－1, 1277 cm－1, 1116 cm－1, 1098 cm－1 Metacycline hydrochloride C22H22N2O8.HCl Yellow to
and 717 cm－1. dark yellow, crystals or crystalline powder.
Absorbance <2.24> E 11zcm (230 nm): 211 – 247 (5 mg, Purity Related substances—Dissolve 20 mg of metacy-
ethanol (99.5), 200 mL). cline hydrochloride in 25 mL of 0.01 mol/L hydrochloric
Purity Related substances—(1) Dissolve 5.0 mg of acid TS, and use this solution as the sample solution. Pro-
mesaconitine for purity in 2 mL of acetonitrile, and use this ceed the test with 20 mL of the sample solution as directed in
solution as the sample solution. Pipet 1 mL of the sample so- the Purity (2) under Doxycycline Hydrochloride Hydrate,
lution, add acetonitrile to make exactly 50 mL, and use this determine each peak area by the automatic integration
solution as the standard solution. Perform the test with these method, and calculate the amounts of them by the area per-
solutions as directed under Thin-layer Chromatography centage method: the total area of peaks other than metacy-
<2.03>. Spot 20 mL each of the sample solution and standard cline is not more than 10z.
solution on a plate of silica gel for thin-layer chromatogra-
Metallic sodium See sodium.
phy, and proceed the test as directed in the Identification
under Processed Aconite Root: the spot other than the prin- Metanil yellow C18H14N3NaO3S Yellow-brown powder.
cipal spot with the sample solution is not more intense than Sparingly soluble in water, and very slightly soluble in
the spot with the standard solution. ethanol (95) and in N, N-dimethylformamide.
(2) Dissolve 5.0 mg of mesaconitine for purity in 5 mL of
Metanil yellow TS Dissolve 0.1 g of metanil yellow in
acetonitrile, and use this solution as the sample solution.
200 mL of N, N-dimethylformamide.
Pipet 1 mL of the sample solution, add acetonitrile to make
exactly 50 mL, and use this solution as the standard solution. Metaphosphoric acid HPO3 A colorless, deliquescent,
Perform the test with exactly 10 mL each of the sample solu- stick or masses.
tion and standard solution as directed under Liquid Chroma- Identification—(1) Dissolve 1 g of metaphosphoric acid
tography <2.01> according to the following conditions, and in 50 mL of water, and use this solution as the sample solu-
determine each peak area by the automatic integration tion. To 10 mL of the sample solution add 0.2 mL of ammo-
method: the total area of the peaks other than mesaconitine nia TS and 1 mL of silver nitrate TS: a yellowish white pre-
with the sample solution is not larger than the peak area of cipitate is produced.
mesaconitine with the standard solution. (2) To 10 mL of the sample solution obtained in (1) add
Operating conditions 10 mL of albumin TS: a white precipitate is produced.
Detector, column, and column temperature: Proceed as
Metaphosphoric acid-acetic acid TS Dissolve 15 g of
directed in the operating conditions in the Purity under
metaphosphoric acid and 40 mL of acetic acid (100) in water
Processed Aconite Root.
to make 500 mL. Preserve in a cold place, and use within 2
Mobile phase: A mixture of phosphate buffer solution for
days.
processed aconite root and tetrahydrofuran (9:1).
Flow rate: Adjust so that the retention time of mesaconi- Metenolone enanthate C27H42O3 [Same as the name-
tine is about 19 minutes. sake monograph]
Time span of measurement: About 3 times as long as the
Metenolone enanthate for assay C27H42O3 To 1 g of
retention time of mesaconitine, beginning after the solvent

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
286 General Tests / 9.41 Reagents, Test Solutions JP XVII
metenolone enanthate add 30 mL of water, and add slowly Methionin See L-methionine.
70 mL of methanol with warming to dissolve. Filter while
L-Methionine C5H11NO2S [Same as the namesake
hot, and allow the filtrate to stand on a water bath for 30
monograph]
minutes. Allow to stand overnight in a cold place, collect the
crystals thus formed, and wash with a small amount of Methotrexate C20H22N8O5 [Same as the namesake
diluted methanol (1 in 3). Recrystallize in the same manner, monograph]
and dry the crystals in a desiccator (in vacuum, phosphorus
4?-Methoxyacetophenone C9H10O2 White to light
(V) oxide) for 4 hours. It is white, odorless crystals.
brown, crystals or crystalline powder.
Absorbance <2.24> E 11zcm (242 nm): 321 – 328 (1 mg, meth-
Melting point <2.60>: 34 – 399C
anol, 100 mL).
Optical rotation <2.49> [a]20D : ＋40 – ＋429 (0.2 g, chlo- 4-Methoxybenzaldehyde C8H8O2 Clear, colorless to
roform, 10 mL, 100 mm). light yellow liquid. Miscible with ethanol (95) and with
Melting point <2.60>: 69 – 729C diethyl ether, and practically insoluble in water.
Purity Related substances—Dissolve 50 mg of meteno- Specific gravity <2.56> d 20
4 : 1.123 – 1.129
lone enanthate for assay in chloroform to make exactly 10 Content: not less than 97.0z. Assay—Weigh accurately
mL, and use this solution as the sample solution. Proceed about 0.8 g of 4-methoxybenzaldehyde, add exactly 75 mL
with 10 mL of this solution as directed in the Purity (3) under of hydroxylamine TS, shake well, allow to stand for 30
Metenolone Enanthate: any spot other than the principal minutes, and titrate <2.50> with 0.5 mol/L hydrochloric acid
spot does not appear. VS (indicator: 3 drops of bromophenol blue TS) until the
color of the solution changes from blue through green to yel-
Metformin hydrochloride for assay C4H11N5.HCl
low-green. Perform a blank determination.
[Same as the monograph Metformin Hydrochloride. When
dried, it contains not less than 99.0z of metformin hydro- Each mL of 0.5 mol/L hydrochloric acid VS
chloride (C4H11N5.HCl).] ＝ 68.08 mg of C8H8O2
Methanesulfonic acid CH3SO3H Clear, colorless liquid 4-Methoxybenzaldehyde-acetic acid TS To 0.5 mL of 4-
or colorless or white, crystalline mass, having a characteristic methoxybenzaldehyde add acetic acid (100) to make 100 mL.
odor. Miscible with water, with ethanol (95) and with diethyl
4-Methoxybenzaldehyde-sulfuric acid TS To 9 mL of
ether.
ethanol (95) add 0.5 mL of 4-methoxybenzaldehyde and 0.5
Congealing point <2.42>: 15 – 209 C
mL of sulfuric acid, and mix thoroughly.
Specific gravity <2.56> d 20
20: 1.483 – 1.488
Content: not less than 99.0z. Assay—Weigh accurately 4-Methoxybenzaldehyde-sulfuric acid-acetic acid-ethanol
about 2 g of methanesulfonic acid, dissolve in 40 mL of TS for spray To 9 mL of ethanol (95) add 0.5 mL of 4-
water, and titrate <2.50> with 1 mol/L sodium hydroxide VS methoxybenzaldehyde, mix gently, add gently 0.5 mL of sul-
(indicator: 2 drops of bromothymol blue TS). furic acid and 0.1 mL of acetic acid (100) in this order, and
mix well.
Each mL of 1 mol/L sodium hydroxide VS
＝ 96.11 mg of CH3SO3H 4-Methoxybenzaldehyde-sulfuric acid-acetic acid TS To
50 mL of acetic acid (100) add 1 mL of sulfuric acid and 0.5
Methanesulfonic acid TS To 35 mL of methanesulfonic
mL of 4-methoxybenzaldehyde, and stir well. Prepare before
acid add 20 mL of acetic acid (100) and water to make 500
use.
mL.
(E )-2-Methoxycinnamaldehyde for thin-layer chromatog-
0.1 mol/L Methanesulfonic acid TS To 4.8 g of
raphy C10H10O2 White to yellow, crystalline powder or
methanesulfonic acid add water to make 500 mL.
powder. Freely soluble in methanol and in ethanol (99.5),
Methanol CH3OH [K 8891, Special class] and practically insoluble in water. Melting point: 44 – 509C.
Identification—(1) Determine the absorption spectrum
Methanol, anhydrous CH4O To 1000 mL of methanol
of a solution of (E )-2-methoxycinnamaldehyde for thin-layer
add 5 g of magnesium powder. If necessary, add 0.1 mL of
chromatography in methanol (1 in 200,000) as directed under
mercury (II) chloride TS to start the reaction. After the
Ultraviolet-visible Spectrophotometry <2.24>: it exhibits
evolving of a gas is stopped, distillate the solution, and
maxima between 282 nm and 286 nm, and between 331 nm
preserve the distillate protecting from moisture. Water con-
and 335 nm.
tent per mL is not more than 0.3 mg.
(2) Determine the infrared absorption spectrum of (E )-2-
Methanol for liquid chromatography CH3OH A clear, methoxycinnamaldehyde for thin-layer chromatography as
colorless liquid. Miscible with water. directed in the potassium bromide disk method under In-
Purity Ultraviolet-absorbing substances—Perform the frared Spectrophotometry <2.25>: it exhibits absorption at
test as directed in Ultraviolet-visible Spectrophotometry the wave numbers of about 1675 cm－1, 1620 cm－1, 1490
<2.24> using water as the blank: the absorbances at 210 nm, cm－1, 1470 cm－1, 1295 cm－1, 1165 cm－1, 1130 cm－1, 1025
at 220 nm, at 230 nm, at 240 nm and at 254 nm are not more cm－1 and 600 cm－1.
than 0.70, 0.30, 0.15, 0.07 and 0.02, respectively. Purity Related substances—Dissolve 10 mg of (E )-2-
methoxycinnamaldehyde for thin-layer chromatography in 5
Methanol for water determination See Water Determina-
mL of methanol, and use this solution as the sample solu-
tion <2.48>.
tion. Pipet 1 mL of the sample solution, add methanol to
Methanol-free ethanol See ethanol (95), methanol-free. make exactly 50 mL, and use this as the standard solution.
Perform the test with 5 mL each of the sample solution and
Methanol-free ethanol (95) See ethanol (95), methanol-
standard solution as directed in the Identification (5) (ii)
free.
under Goshajinkigan Extract: the spots other than the prin-
Methanol, purified Distil methanol before use. cipal spot appeared at R f value of about 0.4 obtained from
the sample solution are not more intense than the spot ob-

tained from the standard solution. 909C.

Carrier gas: Helium.
2-Methoxyethanol CH3OCH2CH2OH [K 8895, Special
Flow rate: A constant flow rate of 20 mL per minute.
class]
Methyl acetate CH3COOCH3 [K 8382, Special class]
2-Methoxy-4-methylphenol C8H10O2 Colorless to pale
yellow liquid. Miscible with methanol and with ethanol p-Methyl aminophenol sulfate See 4-methyl animo-
(99.5), and slightly soluble in water. Congealing point: 3 – phenol sulfate.
89C.
4-Methyl aminophenol sulfate (HOC6H4NHCH3)2.H2SO4
Identification—Determine the infrared absorption spec-
White to pale yellow or very pale grayish white, crystals or
trum of 2-methoxy-4-methylphenol as directed in the ATR
crystalline powder. Melting point: about 2609 C (with de-
method under Infrared Spectrophotometry <2.25>: it exhibits
composition).
absorption at the wave numbers of about 1511 cm－1, 1423
cm－1, 1361 cm－1, 1268 cm－1, 1231 cm－1, 1202 cm－1, 1148 p-Methyl aminophenol sulfate TS See 4-methyl amino-
cm－1, 1120 cm－1, 1031 cm－1, 919 cm－1, 807 cm－1 and 788 phenol sulfate TS.
cm－1.
4-Methyl aminophenol sulfate TS Dissolve 0.35 g of 4-
Purity Related substances—Perform the test with 0.2 mL
methyl aminophenol sulfate and 20 g of sodium hydrogen
of 2-methoxy-4-methylphenol as directed under Gas Chro-
sulfite in water to make 100 mL. Prepare before use.
matography <2.02> according to the following conditions.
Determine each peak area by the automatic integration 2-Methylaminopyridine C6H8N2 A pale yellow liquid.
method: the total area of the peaks other than 2-methoxy-4- Specific gravity <2.56> d 20
20: 1.050 – 1.065
methylphenol is not more than 3.0z. Boiling point <2.57>: 200 – 2029 C
Operating conditions Water <2.48>: less than 0.1z.
Detector: A hydrogen flame-ionization detector.
2-Methylamino pyridine for water determination See
Column: A fused silica column 0.25 mm in inside diameter
Water Determination <2.48>.
and 60 m in length, coated inside with polymethylsiloxane
for gas chromatography in 0.25 to 0.5 mm in thickness. Methyl arachidate for gas chromatography C21H42O2
Column temperature: Inject at a constant temperature of White to light yellow, crystals or crystalline masses.
about 1009 C, raise the temperature to 1309 C at a rate of 59C Melting point <2.60>: 45 – 509C
per minute, raise to 1409 C at a rate of 29C per minute, raise
Methyl behenate C23H46O2 White, odorless and taste-
to 2009C at a rate of 159C per minute, and maintain at
less, scaly crystals or powder. Dissolves in acetone, in diethyl
2009C for 2 minutes.
ether and in chloroform.
Injection port temperature: 2009 C.
Melting point <2.60>: 549C
Detector temperature: 2509C.
Saponification value <1.13>: 155.5 – 158.5
Carrier gas: Helium.
Flow rate: Adjust so that the retention time of 2-methoxy- Methyl benzoate C6H5COOCH3 Clear, colorless liquid.
4-methylphenol is about 10 minutes. Refractive index <2.45> n 20
D : 1.515 – 1.520
Split ratio: 1:50. Specific gravity <2.56> d 20
20: 1.087 – 1.095
System suitability Purity—Dissolve 0.1 mL of methyl benzoate in the mobile
System performance: Dissolve 60 mg of 2-methoxy-4- phase in Assay under Thiamine Chloride Hydrochloride to
methylphenol in methanol to make 100 mL, and use this so- make 50 mL. Perform the test as directed under Liquid
lution as the solution for system suitability test. Proceed Chromatography <2.01> with 10 mL of this solution accord-
with 1 mL of the solution for system suitability test under the ing to the Assay under Thiamine Chloride Hydrochloride.
above operating conditions, the symmetry factor of the peak Determine each peak area by the automatic integration
of 2-methoxy-4-methylphenol is not more than 1.5. method in a range about twice the retention time of methyl
System repeatability: When the test is repeated 6 times benzoate, and calculate the amount of methyl benzoate by
with 1 mL of the solution for system suitability test under the the area percentage method: it shows the purity of not less
above operating conditions, the relative standard deviation than 99.0z.
of the peak area of 2-methoxy-4-methylphenol is not more
Methyl benzoate for estriol test C6H5COOCH3 Clear,
than 2.0z.
colorless liquid, having a characterisic odor.
1-Methoxy-2-propanol C4H10O2 A colorless, clear liq- Refractive index <2.45> n 20
D : 1.515 – 1.520
uid. Specific gravity <2.56> d 20
20: 1.087 – 1.095
Refractive index <2.45> n 20
D : 1.402 – 1.405 Acid value <1.13>: not more than 0.5.
Specific gravity <2.56> d 20
4 : 0.920 – 0.925
4-Methylbenzophenone C14H12O White crystals.
Purity Clarity of solution—To 5 mL of 1-methoxy-2-
propanol add 20 mL of water, and mix: the solution is clear. D-(＋)-a-Methylbenzylamine C6H5CH(CH3)NH2 Color-
Water <2.48>: not more than 0.5z (5 g). less or pale yellow, clear liquid, having an amine like odor.
Content: not less than 98.0z. Assay—Proceed as di- Very soluble in ethanol (95) and in acetone, and slightly solu-
rected under Gas Chromatography <2.02> using the area per- ble in water.
centage method according to the following conditions: Refractive index <2.45> n 20
D : 1.524–1.529
Operating conditions Optical rotation <2.49> [a]20
D : ＋37 – ＋419(50 mm).
Detector: Thermal conductivity detector. Purity—Perform the test with 0.6 mL of D-(＋)-a-methyl-
Column: A glass column about 3 mm in inside diameter benzylamine as directed under Gas Chromatography <2.02>
and about 2 m in length, packed with siliceous earth for gas according to the following conditions. Determine each peak
chromatography (150 to 180 mm) coated with polyethylene area by the automatic integration method, and calculate the
glycol 20 M for gas chromatography in 20z. amount of D-(＋)-a-methylbenzylamine by the area percen-
Column temperature: A constant temperature of about tage method: not less than 98.0z.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
288 General Tests / 9.41 Reagents, Test Solutions JP XVII
Operating conditions Methylene blue trihydrate C16H18ClN3S.3H2O [K 8897,
Detector: A hydrogen flame-ionization detector. Special class]
Column: A glass column about 3 mm in inside diameter
Methylene blue TS Dissolve 0.1 g of methylene blue tri-
and about 2 m in length, packed with siliceous earth for gas
hydrate in water to make 100 mL. Filter if necessary.
chromatography (180 to 250 mm in particle diameter) coated
with polyethylene glycol 20 M for gas chromatography and dl-Methylephedrine hydrochloride C11H17NO.HCl
potassium hydroxide at the ratio of 10z and 5z, respec- [Same as the namesake monograph]
tively.
dl-Methylephedrine hydrochloride for assay [Same as
Column temperature: A constant temperature of about
the monograph dl-Methylephedrine Hydrochloride]
1409C.
Carrier gas: Helium. Methylergometrine maleate for assay C20H25N3O2.C4H4O4
Flow rate: Adjust so that the retention time of D-(＋)- [Same as the monograph Methylergometrine Maleate. When
a-methylbenzylamine is about 5 minutes. dried, it contains not less than 99.0z of methylergometrine
Selection of column: To 5 mL of D-(＋)-a-methylbenzyl- maleate (C20H25N3O2.C4H4O4).]
amine add 1 mL of pyridine. Proceed with 0.6 mL of this so-
Methyl ethyl ketone See 2-butanone.
lution under the above operating conditions, and calculate
the resolution. Use a column giving elution of pyridine and Methyl iodide See iodomethane.
D-(＋)-a-methylbenzylamine in this order with the resolution
Methyl iodide for assay See iodomethane for assay.
between these peaks being not less than 3.
Detection sensitivity: Adjust the detection sensitivity so Methyl isobutyl ketone See 4-methyl-2-pentanone.
that the peak height of D-(＋)-a-methylbenzylamine obtained
Methyl laurate for gas chromatography C13H26O2 A
from 0.6 mL of the D-( ＋ )-a-methylbenzylamine is not less
colorless to yellow, liquid.
than about 90z of the full scale.
Refractive index <2.45> n20 D : 1.431 – 1.433
Time span of measurement: About 3 times as long as the
Specific gravity <2.56> d 20
20: 0.870 – 0.872
retention time of D-(＋)-a-methylbenzylamine, beginning
after the solvent peak. Methyl lignocerate for gas chromatography C25H50O2
A white, crystalline powder.
3-Methyl-1-butanol C5H12O [K 8051, Special class]
Melting point <2.60>: 58 – 619C
3-Methylbutyl acetate CH3COOCH2CH2CH(CH3)2 A
Methyl linoleate for gas chromatography C19H34O2 A
clear and colorless liquid. Boiling point: about 1409C.
colorless to light yellow, liquid.
Specific gravity <2.56> d 20
20: 0.868 – 0.879
Specific gravity <2.56> d 20
20: 0.880 – 0.889
Preserve in a light-resistant tight container.
Methyl linolenate for gas chromatography C19H32O2 A
Methyl cellosolve See 2-methoxyethanol.
colorless to light yellow, liquid.
Methylcyclohexane C7H14 A clear and colorless liquid. Specific gravity <2.56> d 20
20: 0.890 – 0.901
Refractive index <2.45>: 1.420 – 1.425
3-O-Methylmethyldopa for thin-layer chromatography
Density <2.56> (209C): 0.766 – 0.772 g/mL
C11H15NO4
Methyl docosanate C23H46O2 White, tabular crystals or Purity Related substances—Dissolve 5 mg of 3-O-
crystalline powder. methylmethyldopa for thin-layer chromatography in metha-
Melting point <2.60>: 51.0 – 56.09C nol to make exactly 100 mL. Perform the test with 20 mL of
this solution as directed in the Purity (5) under Methyldopa
Methyldopa See methyldopa hydrate.
Hydrate: any spot other than the principal spot at the R f
Methyldopa for assay See methyldopa hydrate for assay. value of about 0.7 does not appear.
Methyldopa hydrate C10H13NO4.1 1/2 H2O [Same as the Methyl myristate for gas chromatography C15H30O2 A
namesake monograph] colorless to light yellow, liquid.
Specific gravity <2.56> d 20
20: about 0.866 – 0.874
Methyldopa hydrate for assay C10H13NO4.1 1/2 H2O
[Same as the monograph Methyldopa Hydrate. It contains 2-Methyl-5-nitroimidazole for thin-layer chromatography
not less than 99.0z of methyldopa (C10H13NO4), calculated C4H5N3O2 White crystalline powder. Slightly soluble in
on the anhydrous basis.] water and in acetone. Melting point: about 2539C (with de-
composition).
N, N?-Methylenebisacrylamide CH2(NHCOCHCH2)2
Purity Related substances—Dissolve 40 mg of 2-methyl-
White crystalline powder.
5-nitroimidazole for thin-layer chromatography in 8 mL of
Content: not less than 97.0z.
acetone, and use this solution as the sample solution. Pipet
Methyl eicosenoate for gas chromatography C21H40O2 2.5 mL of the sample solution, add acetone to make exactly
A clear and colorless, liquid. 100 mL, and use this solution as the standard solution. Per-
form the test as directed in the Purity (2) under Metronida-
Methylene blue See methylene blue trihydrate.
zole: the spots other than the principal spot from the sample
Methylene blue-sulfuric acid-monobasic sodium phos- solution are not more intense than the spot from the stand-
phate TS See methylene blue-sulfuric acid-sodium dihydro- ard solution.
genphosphate TS.
Methyl oleate for gas chromatography C19H36O2 A
Methylene blue-sulfuric acid-sodium dihydrogenphos- clear, colorless to light yellow, liquid.
phate TS To 30 mL of a solution of methylene blue (1 in Specific gravity <2.56> d 20
20: 0.866 – 0.882
1000) add 500 mL of water, 6.8 mL of sulfuric acid and 50 g
Methyl orange C14H14N3NaO3S [K 8893, Special class]
of sodium dihydrogenphosphate dihydrate, dissolve, and
add water to make 1000 mL. Methyl orange-boric acid TS Add 0.5 g of methyl orange

and 5.2 g of boric acid in 500 mL of water, and dissolve by 4-Methylpentan-2-ol C6H14O A clear and colorless,
warming on a water bath. After cooling, wash this solution volatile liquid.
with three 50-mL portions of chloroform. Refractive index <2.45> n 20
D : about 1.411
Specific gravity <2.56> d 20
20: about 0.802
Methyl orange TS Dissolve 0.1 g of methyl orange in 100
Boiling point <2.57>: about 1329C
mL of water, and filter if necessary.
4-Methyl-2-pentanone CH3COCH2CH(CH3)2
Methyl orange-xylenecyanol FF TS Dissolve 1 g of
[K 8903, Special class]
methyl orange and 1.4 g of xylene cyanol FF in 500 mL of
dilute ethanol. 3-Methyl-1-phenyl-5-pyrazolone C10H10N20 [K 9548,
Special class]
Methyl palmitate for gas chromatography C17H34O2
White, crystals or waxy masses. Methyl prednisolone C22H30O3 [Same as the namesake
Congealing point <2.42>: 25 – 319
C monograph]
Methyl palmitoleate for gas chromatography C17H32O2 2-Methyl-1-propanol (CH3)2CHCH2OH [K 8811, Spe-
Specific gravity <2.56> d 20
20: 0.876 – 0.881 cial class]
Methyl parahydroxybenzoate HOC6H4COOCH3 N-Methylpyrrolidine C5H11N Colorless, clear liquid,
[Same as the namesake monograph] having a characteristic order.
Identification—Determine the spectrum of N-methylpyr-
Methyl parahydroxybenzoate for resolution check
rolidine in a solution of deuterated chloroform for nuclear
C8H8O3 Colorless crystals or a white crystalline powder.
magnetic resonance spectroscopy (2 in 25) as directed under
Freely soluble in methanol, in ethanol (95) and in acetone,
Nuclear Magnetic Resonance Spectroscopy <2.21> (1H): it ex-
and very slightly soluble in water. Melting point: 125 –
hibits a big signal, at around d 2.3 ppm.
1289C.
Content: not less than 95z. Assay—Put 30 mL of water
Identification—Determine the infrared absorption spec-
in a beaker, weigh accurately the beaker, add dropwise
trum of methyl parahydroxybenzoate for resolution check as
about 0.15 g of N-methylpyrrolidine, weigh accurately the
directed in the potassium bromide disk method under In-
beaker again, and titrate <2.50> with 0.05 mol/L sulfuric
frared Spectrophotometry <2.25>, and compare the spectrum
acid VS (potentiometric titration). Perform a blank determi-
with the Reference Spectrum of Methyl Parahydroxybenzo-
nation in the same manner and make any necessary correc-
ate or the spectrum of Methyl Parahydroxybenzoate RS:
tion.
both spectra exhibit similar intensities of absorption at the
same wave numbers. Each mL of 0.05 mol/L sulfuric acid VS
Purity Related substances—Dissolve 50 mg of methyl ＝ 8.515 mg of C5H11N
parahydroxybenzoate for resolution check in 2.5 mL of
Methyl red C15H15N3O2 [K 8896, Special class]
methanol, and add the mobile phase to make 50 mL. To 10
mL of this solution add the mobile phase to make 100 mL, Methyl red-methylene blue TS Dissolve 0.1 g of methyl
and use this solution as the sample solution. Pipet 1 mL of red and 0.1 g of methylene blue in ethanol (95) to make 100
the sample solution, add the mobile phase to make exactly 10 mL, and filter if necessary. Preserve in light-resistant con-
mL, and use this solution as the standard solution. Perform tainers.
the test with exactly 10 mL each of the sample solution and
Methyl red TS Dissolve 0.1 g of methyl red in 100 mL of
standard solution as directed under Liquid Chromatography
ethanol (95), and filter if necessary.
<2.01> according to the following conditions. Determine each
peak area by the automatic integration method: total area of Methyl red TS, dilute Dissolve 25 mg of methyl red in
the peaks other than methyl parahydroxybenzoate obtained 100 mL of ethanol (99.5), and filter if necessary. Prepare be-
from the sample solution is not larger than the peak area of fore use.
methyl parahydroxybenzoate obtained from the standard so-
Methyl red TS for acidity or alkalinity test To 0.1 g of
lution.
methyl red add 7.4 mL of 0.05 mol/L sodium hydroxide VS
Operating conditions
or 3.7 mL of 0.1 mol/L sodium hydroxide VS, triturate to
Detector, column, column temperature, mobile phase, and
dissolve in a mortar, and add freshly boiled and cooled water
flow rate: Proceed as directed in the operating conditions in
to make 200 mL. Preserve in light-resistant, glass-stoppered
the Assay under Methyl Parahydroxybenzoate.
bottles.
Time span of measurement: About 5 times as long as the
retention time of methyl parahydroxybenzoate. Methylrosaniline chloride See crystal violet.
System suitability
Methylrosaniline chloride TS See crystal violet TS.
Test for required detectability: Pipet 1 mL of the standard
solution, and add methanol to make exactly 20 mL. Confirm Methyl salicylate C8H8O3 [Same as the namesake
that the peak area of methyl parahydroxybenzoate obtained monograph]
with 10 mL of this solution is equivalent to 3.5 to 6.5z of
Methylsilicone polymer for gas chromatography Pre-
that obtained with 10 mL of the standard solution.
pared for gas chromatography.
System performance: When the procedure is run with 10
mL of the standard solution under the above operating con- Methyl stearate for gas chromatography C19H38O2
ditions, the number of theoretical plates and the symmetry White, crystals or crystalline masses.
factor of the peak of methyl parahydroxybenzoate are not Melting point <2.60>: 36 – 429C
less than 2500 and not more than 2.0, respectively.
Methyltestosterone C20H30O2 [Same as the namesake
System repeatability: When the test is repeated 6 times
monograph]
with 10 mL of the standard solution under the above operat-
ing conditions, the relative standard deviation of the peak 1-Methyl-1H-tetrazole-5-thiol C 2 H4 N 4 S White, crystals
area of methyl parahydroxybenzoate is not more than 5.0z. or crystalline powder.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
290 General Tests / 9.41 Reagents, Test Solutions JP XVII
Identification (1) Determine the ultraviolet-visible ab- requirement.]
sorption spectrum of a solution of 1-methyl-1H-tetrazole-5- Related substances—Weigh accurately about 25 mg of
thiol (1 in 200,000) as directed under Ultraviolet-visible Spec- metronidazole for assay, dissolve in 100 mL of a mixture of
trophotometry <2.24>: it exhibits a maximum between 222 water and methanol (4:1), and use this solution as the sample
nm and 226 nm. solution. Pipet 2 mL of the sample solution, and add the
(2) Determine the infrared absorption spectrum of 1- mixture of water and methanol (4:1) to make exactly 50 mL.
methyl-1H-tetrazole-5-thiol according to the potassium bro- Pipet 2.5 mL of this solution, add the mixture of water and
mide disk method under Infrared Spectrophotometry <2.25>: methanol (4:1) to make exactly 20 mL, and use this solution
it exhibits absorption at the wave numbers of about 3060 as the standard solution. Perform the test with exactly 10 mL
cm－1, 2920 cm－1, 2780 cm－1, 1500 cm－1, 1430 cm－1 and each of the sample solution and standard solution as directed
1410 cm－1. under Liquid Chromatography <2.01> according to the fol-
Melting point <2.60>: 125 – 1299 C lowing conditions, and determine each peak area by the au-
Purity Related substances—Dissolve 0.10 g of 1-methyl- tomatic integration method: the total area of the peaks other
1H-tetrazole-5-thiol in exactly 100 mL of water. Perform the than metronidazole with the sample solution is not more
test with 1 mL of this solution as directed in the Purity (4) than the peak area of metronidazole with the standard solu-
under Cefmetazole Sodium: any spot other than the princi- tion.
pal spot at the R f value of about 0.77 does not appear. Operating conditions
Detector, column, column temperature, mobile phase, and
1-Methyl-1H-tetrazole-5-thiol for liquid chromatography
flow rate: Proceed as directed in the operating conditions in
C2H4N4S White, crystals or crystalline powder. Very solu-
the Assay under Metronidazole Tablets.
ble in methanol, and freely soluble in water.
Time span of measurement: About 4 times as long as the
Melting point <2.60>: 123 – 1279C
retention time of metronidazole.
Loss on drying <2.41>: not more than 1.0z (1 g, in vacu-
System suitability
um, phosphorous (V) oxide, 2 hours).
Test for required detectability: Measure exactly 2 mL of
Content: not less than 99.0z. Assay—Weigh accurately
the standard solution, add a mixture of water and methanol
about 0.2 g of 1-methyl-1H-tetrazole-5-thiol, previously
(4:1) to make exactly 20 mL. Confirm that the peak area of
dried, dissolve in 80 mL of N, N-dimethylformamide, and
metronidazole obtained with 10 mL of this solution is equiva-
titrate <2.50> with 0.1 mol/L sodium methoxide VS (indica-
lent to 7 to 13z of that obtained with the standard solution.
tor: 3 drops of thymol blue-N, N-dimethylformamide TS).
System performance: When the procedure is run with 10
Perform a blank determination, and make any necessary
mL of the standard solution under the above operating con-
correction.
ditions, the number of theoretical plates and the symmetry
Each mL of 0.1 mol/L sodium methoxide VS factor of the peak of metronidazole are not less than 3000
＝ 11.61 mg of C2H4N4S and not more than 1.5, respectively.
System repeatability: When the test is repeated 6 times
Methylthymol blue C37H43N2NaO13S [K 9552, Special
with 10 mL of the standard solution under the above operat-
class]
ing conditions, the relative standard deviation of the peak
Methylthymol blue-potassium nitrate indicator Mix 0.1 g area of metronidazole is not more than 2.0z.
of methylthymol blue with 9.9 g of potassium nitrate, and
Miconazole nitrate C18H14Cl4N2O.HNO3 [Same as the
triturate until the mixture becomes homogeneous.
namesake monograph]
Sensitivity—When 20 mg of methylthymol blue-potassium
nitrate indicator is dissolved in 100 mL of 0.02 mol/L so- Microplate for antigen antibody reaction test A plate
dium hydroxide VS, the solution is slightly blue in color. On made from polystyrene, and prepared for antigen antibody
adding 0.05 mL of 0.01 mol/L barium chloride VS to this reaction test.
solution, the solution shows a blue color, then on the subse- Performance: Coefficient of variation of the binding
quent addition of 0.1 mL of 0.01 mol/L disodium dihydro- capacity of immunoglobulin G is not more than 5z, and the
gen ethylenediamineteraacetate VS, it becomes colorless. binding capacity of each well is within 10z of the mean
value.
Methylthymol blue-sodium chloride indicator Mix 0.25 g
of methylthymol blue and 10 g of sodium chloride, and grind Microplates Polystyrene plates with an inside diameter
to homogenize. of 7 (upper edge) to 6.4 (lower edge) mm, and 11.3 mm
thickness. Have 96 flat-bottomed truncated cone-shaped
Methyl yellow C14H15N3 [K 8494, Special class]
wells.
Methyl yellow TS Dissolve 0.1 g of methyl yellow in 200
Milk casein See casein, milk.
mL of ethanol (95).
Milk of lime Place 10 g of calcium oxide in a mortar,
Metoclopramide for assay C14H22ClN3O2 [Same as the
and add gradually 40 mL of water under grinding.
monograph Metoclopramide. When dried, it contains not
less than 99.0z of metoclopramide (C14H22ClN3O2).] Minocycline hydrochloride C23H27N3O7.HCl [Same as
the namesake monograph]
Metoprolol tartrate for assay (C15H25NO3)2.C4H6O6
[Same as the monograph Metoprolol Tartrate. When dried, Mitiglinide calcium hydrate C38H48CaN2O6.2H2O
it contains not less than 99.5z of metoprolol tartrate [Same as the namesake monograph]
((C15H25NO3)2.C4H6O6).]
Mixture of petroleum hexamethyl tetracosane branching
Metronidazole C6H9N3O3 [Same as the namesake hydrocarbons (L) for gas chromatography Prepared for
monograph] gas chromatography.
Metronidazole for assay C6H9N3O3 [Same as the Molecular mass marker for epoetin alfa A solution con-
monograph Metronidazole. It meets the following additional taining about 0.4 mg each of egg albumin, carbonic anhy-

drase, soybean trypsin inhibitor and lysozyme in 200 mL. Molybdenium-sulfuric acid TS Dissolve 2.5 g of hexaam-
monium heptamolybdate tetrahydrate in 20 mL of water by
Molecular mass marker for interferon alfa Molecular
heating. To this solution add a solution, prepared by careful
mass known marker proteins, which are adjusted for mo-
adding 28 mL of sulfuric acid to 50 mL of water, mixing and
lecular mass determination [4 components: egg albumin,
cooling, and add water to make 100 mL. Reserve in a poly-
carbonic anhydrase, soybean trypsin inhibitor, and a-lactal-
ethylene container.
bumin].
Identification Use a solution of the molecular mass Molybdenum trioxide See molybdenum (VI) oxide.
marker as the sample solution. Separately, add water to an
Molybdenum trioxide-citric acid TS See molybdenum
amount of egg albumin so that each mL contains 100 mg as
(VI) oxide-citric acid TS.
protein content of egg albumin, and use this solution as the
standard solution. Perform SDS-polyacrylamide gel elec- Monoammonium glycyrrhizinate for resolution check
trophoresis with the sample solution and standard solution C42H61O16NH4 Mainly composed with monoammonium
under the test conditions for molecular mass of Interferon glycyrrhizinate and its isomers. It is white, crystals or crys-
Alfa (NAMALWA). The sample solution indicates 4 main talline powder.
electrophoretic bands. In addition, the mobility of egg albu- Identification—Dissolve 1 mg of monoammonium glycyr-
min obtained from the sample solution corresponds with rhizinate for resolution check in 2 mL of diluted ethanol (2
that of the band obtained from the standard solution. in 5). Perform the test with 2 mL of this solution as directed
under Liquid Chromatography <2.01> according to the fol-
Molecular mass marker for nartograstim test A solution
lowing conditions: a peak having a relative retention time of
containing the following proteins. Egg albumin, carbonic
about 0.9 to glycyrrhizic acid is observed, and when per-
anhydrase, soybean trypsin inhibitor and lysozyme.
formed the test with these two peaks by liquid chromatogra-
Molecular mass standard stock solution Dissolve 1.2 g of phy-mass spectrometry (ESI method, positive mode) their
2-amino-2-hydroxymethyl-1,3-propanediol and 3.2 g of so- mass charge ratios (m/z values) are observed at 823 or 840 or
dium lauryl sulfate in a suitable amount of water, adjust to at the both of them, respectively.
pH 6.8 with 6 mol/L hydrochloric acid TS, 1 mol/L hydro- Operating conditions
chloric acid TS or 0.1 mol/L hydrochloric acid TS, add 32 Detector: An ultraviolet absorption photometer (wave-
mg of bromophenol blue and 16 mL of glycerin, and add length: 254 nm) and mass spectrometer.
water to make 40 mL. To 500 mL of this solution add 100 mL Column: A stainless steel column 2 mm in inside diameter
of molecular mass marker for epoetin alfa and 1400 mL of and 15 cm in length, packed with octadecylsilanized silica gel
water, and heat at 1009C for 5 minutes. It meets the follow- for liquid chromatography (3 mm in particle diameter).
ing requirement. Column temperature: A constant temperature of about
Identification—Dissolve 0.1 mg each of egg albumin, car- 409C.
bonic anhydrase, soybean trypsin inhibitor and lysozyme in Mobile phase: Dissolve 0.63 g of ammonium formate in
250 mL of buffer solution for epoetin alfa sample, add water water to make 1000 mL. To 800 mL of this solution add 200
to them to make 1 mL and heat at 1009C for 5 minutes, and mL of acetonitrile.
use these solutions as each standard solution. When perform Flow rate: About 0.5 mL per minute.
the test with the solution to be examined and each standard
Monobasic ammonium phosphate See ammonium dihy-
solution by the SDS-polyacrylamide gel electrophoresis as di-
drogenphosphate.
rected in the Molecular mass under Epoetin Alfa (Genetical
recombination), the each band in the chromatogram ob- 0.02 mol/L Monobasic ammonium phosphate TS See
tained from the solution to be examined shows the same 0.02 mol/L ammonium dihydrogenphosphate TS.
mobility as the band corresponding to egg albumin, carbonic
Monobasic potassium phosphate See potassium dihydro-
anhydrase, soybean trypsin inhibitor or lysozyme obtained
genphosphate.
from each standard solution.
Monobasic potassium phosphate for pH determination
Molecular mass markers for teceleukin Dissolve 0.4 mg
See potassium dihydrogenphosphate for pH determination.
each of lysozyme, soybean trypsin inhibitor, carbonic anhy-
drase, egg albumin, bovine serum albumin, and phosphory- 0.05 mol/L Monobasic potassium phosphate (pH 3.0)
lase b in 200 mL of diluted glycerin (1 in 2). See 0.05 mol/L potassium dihydrogenphosphate (pH 3.0).
Molybdenum (VI) oxide MoO3 A white to yellowish 0.05 mol/L Monobasic potassium phosphate TS (pH 4.7)
green powder. See 0.05 mol/L potassium dihydrogenphosphate TS (pH
Identification—Dissolve 0.5 g in 5 mL of ammonia solu- 4.7).
tion (28), acidify 1 mL of this solution with a suitable
0.02 mol/L Monobasic potassium phosphate TS See
amount of nitric acid, add 5 mL of sodium phosphate TS,
0.02 mol/L potassium dihydrogenphosphate TS.
and warm: yellow precipitates appear.
0.05 mol/L Monobasic potassium phosphate TS See
Molybdenum (VI) oxide-citric acid TS To 54 g of molyb-
0.05 mol/L potassium dihydrogenphosphate TS.
denum (VI) oxide and 11 g of sodium hydroxide add 200 mL
of water, and dissolve by heating while stirring. Separately, 0.2 mol/L Monobasic potassium phosphate TS See 0.2
dissolve 60 g of citric acid monohydrate in 250 mL of water, mol/L potassium dihydrogenphosphate TS.
and add 140 mL of hydrochloric acid. Mix these solutions,
0.2 mol/L Monobasic potassium phosphate TS for buffer
filter if necessary, add water to make 1000 mL, and add a so-
solution See 0.2 mol/L potassium dihydrogenphosphate
lution of potassium bromate (1 in 100) until a yellow-green
TS for buffer solution.
color appears.
Storage—Preserve in tightly stoppered containers, protect- Monobasic sodium phosphate See sodium dihydrogen-
ed from light. phosphate dihydrate.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
292 General Tests / 9.41 Reagents, Test Solutions JP XVII
0.05 mol/L Monobasic sodium phosphate TS (pH 2.6) sium chloride, 1.15 g of anhydrous disodium hydrogen
See 0.05 mol/L sodium dihydrogenphosphate TS (pH 2.6). phosphate and 0.2 g of potassium dihydrogen phosphate in
water to make 1000 mL, and sterilize in an autoclave for 15
0.05 mol/L Monobasic sodium phosphate TS (pH 3.0)
minutes at 1219C to make the PBS(－) solution. Dissolve
See 0.05 mol/L sodium dihydrogenphosphate TS (pH 3.0).
0.3 g of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetra-
0.1 mol/L Monobasic sodium phosphate TS (pH 3.0) zolium bromide in this PBS(－) solution to make 100 mL.
See 0.1 mol/L sodium dihydrogenphosphate TS (pH 3.0). Sterilize by membrane filtration (pore size, 0.45 mm), and
store in a cool place shielded from light.
0.05 mol/L Monobasic sodium phosphate TS See 0.05
mol/L sodium dihydrogenphosphate TS. Murexide C8H8N6O6 Red-purple powder. Practically
insoluble in water, in ethanol (95) and in diethyl ether.
0.1 mol/L Monobasic sodium phosphate TS See 0.1
Purity Clarity of solution—Dissolve 10 mg of murexide
mol/L sodium dihydrogenphosphate TS.
in 100 mL of water: the solution is clear.
2 mol/L Monobasic sodium phosphate TS See 2 mol/L Residue on ignition <2.44>: not more than 0.1z (1 g).
sodium dihydrogenphosphate TS. Sensitivity—Dissolve 10 mg of murexide in 2 mL of am-
monia-ammonium chloride buffer solution (pH 10.0), and
Monoethanolamine See 2-Aminoethanol.
add water to make 100 mL, and use this solution as the sam-
Morphine hydrochloride See morphine hydrochloride ple solution. Separately, add 2 mL of ammonia-ammonium
hydrate. chloride buffer solution (pH 10.0) to 5 mL of diluted Stand-
ard Calcium Solution (1 in 10), add water to make 25 mL,
Morphine hydrochloride for assay See morphine hydro-
and render the solution to pH 11.3 with sodium hydroxide
chloride hydrate for assay.
TS. Add 2 mL of the sample solution and water to this solu-
Morphine hydrochloride hydrate C17H19NO3.HCl.3H2O tion to make 50 mL: a red-purple color develops.
[Same as the namesake monograph]
Murexide-sodium chloride indicator Prepared by mixing
Morphine hydrochloride hydrate for assay 0.1 g of murexide and 10 g of sodium chloride and grinding
C17H19NO3.HCl.3H2O [Same as the monograph Morphine to get homogeneous.
Hydrochloride Hydrate. It contains not less than 99.0z of Storage—Preserve in light-resistant containers.
morphine hydrochloride (C17H19NO3.HCl), calculated on the
Myoglobin A hemoprotein obtained from horse heart
anhydrous basis.]
muscle. White crystalline powder. It contains not less than
3-(N-Morpholino)propanesulfonic acid C7H15NO4S 95z of myoglobin in the total protein.
White crystalline powder, freely soluble in water, and practi-
Myoinositol C6H6(OH)6 White, crystals or crystalline
cally insoluble in ethanol (99.5).
powder.
Melting point <2.60>: 275 – 2809 C
Myristicin for thin-layer chromatography C11H12O3
0.02 mol/L 3-(N-Morpholino)propanesulfonic acid buffer
Colorless, clear liquid, having a characteristic odor. Miscible
solution (pH 7.0) Dissolve 4.2 g of 3-(N-morpholino)pro-
with ethanol (95), and practically insoluble in water.
panesulfonic acid in 900 mL of water, adjust the pH to 7.0
Identification—Determine the infrared absorption spec-
with dilute sodium hydroxide TS, and add water to make
trum of myristicin for thin-layer chromatography as directed
1000 mL.
in the liquid film method under Infrared Spectrophotometry
0.1 mol/L 3-(N-Morpholino)propanesulfonic acid buffer <2.25>: it exhibits absorption at the wave numbers of about
solution (pH 7.0) Dissolve 20.92 g of 3-(N-morpholino) 3080 cm－1, 2890 cm－1, 1633 cm－1, 1508 cm－1, 1357 cm－1,
propanesulfonic acid in 900 mL of water, adjust the pH to 1318 cm－1, 1239 cm－1, 1194 cm－1, 1044 cm－1, 994 cm－1,
7.0 with sodium hydroxide TS, and add water to make 1000 918 cm－1, 828 cm－1 and 806 cm－1.
mL. Purity Related substances—Dissolve 20 mg of myristicin
for thin-layer chromatography in 1 mL of ethanol (95), and
0.02 mol/L 3-(N-Morpholino)propanesulfonic acid buffer
use this solution as the sample solution. Pipet 0.5 mL of this
solution (pH 8.0) Dissolve 4.2 g of 3-(N-morpholino)pro-
solution, add ethanol (95) to make exactly 25 mL, and use
panesulfonic acid in 700 mL of water, adjust the pH to 8.0
this solution as the standard solution. Perform the test with
with dilute sodium hydroxide TS, and add water to make
5 mL each of the sample solution and standard solution as di-
1000 mL.
rected in the Identification under Nutmeg: the spots other
Mosapride citrate for assay See mosapride citrate hy- than the principle spot at the R f value of about 0.4 obtained
drate for assay. from the sample solution are not more intense than the spot
obtained from the standard solution.
Mosapride citrate hydrate for assay
C21H25ClFN3O3.C6H8O7.2H2O [Same as the monograph NADH peroxidase One unit indicates an amount of the
Mosapride Citrate Hydrate. It contains not less than 99.0z enzyme which consumes 1 mmol of b-NADH in 1 minute at
of mosapride citrate (C21H25ClFN3O3.C6H8O7) calculated on 259C and pH 8.0 using b-nicotinamide adenine dinucleotide
the anhydrous basis.] (b-NADH) and hydrogen peroxide as the substrate.
Mouse anti-epoetin alfa monoclonal antibody A solution NADH peroxidase TS Suspend NADH peroxidase in
of the monoclonal antibody in phosphate-buffered sodium ammonium sulfate TS so that each mL contains 10 units of
chloride TS, which is obtained from mouse immunized with the activity.
a synthetic peptide having the amino acid sequence corre- Storage—Between 0 and 89C.
sponding to N-terminal 20 residues of epoetin alfa (genetical
Naftopidil for assay C24H28N2O3 [Same as the mono-
recombination). When perform the Western blotting against
graph Naftopidil. When dried, it contains not less than
Epoetin Alfa RS, it is reactable.
99.5z of naftopidil (C24H28N2O3).]
MTT TS Dissolve 8 g of sodium chloride, 0.2 g of potas-

Nalidixic acid C12H12N2O3 [Same as the namesake 1-Naphthol-sulfuric acid TS Dissolve 1.5 g of 1-naphthol
monograph] in 50 mL of ethanol (95), add 3 mL of water and 7 mL of
sulfuric acid, and mix well. Prepare before use.
Namalwa cell Human cell line derived from B lymphob-
lasts, taken from patients with Burkitt's lymphoma. 1-Naphthol TS Dissolve 6 g of sodium hydroxide and
16 g of anhydrous sodium carbonate in water to make 100
Naphazoline hydrochloride C14H14N2.HCl [Same as
mL. In this solution dissolve 1 g of 1-naphthol. Prepare be-
the namesake monograph]
fore use.
Naphazoline nitrate C14H14N2.HNO3 [Same as the
2-Naphthol TS Dissolve 1 g of 2-naphthol in sodium car-
namesake monograph]
bonate TS to make 100 mL. Prepare before use.
Naphazoline nitrate for assay C14H14N2.HNO3 [Same
a-Naphthol TS See 1-naphthol TS.
as the monograph Naphazoline Nitrate. When dried, it
contains not less than 99.0z of naphazoline nitrate b-Naphthol TS See 2-naphthol TS.
(C14H14N2.NHO3).]
Naphthoresorcin-phosphoric acid TS Dissolve 0.2 g of
Naphthalene C10H8 Colorless flake-like or lustrous 1,3-dihydroxynaphtharene in ethanol (99.5) to make 100
stick-like crystals, having a characteristic odor. mL. To this solution add 10 mL of phosphoric acid.
Melting point <2.60>: 78 – 829C
1-Naphthylamine C10H7NH2 [K 8692, Special class]
1,3-Naphthalenediol C10H8O2 Red-brown crystals or Preserve in light-resistant containers.
gray-brown powder. Freely soluble in water, in methanol
a-Naphthylamine See 1-naphthylamine.
and in ethanol (99.5). Melting point: about 1249C.
N-(1-Naphthyl)-N?-diethylethylenediamine oxalate See
1,3-Naphthalenediol TS Dissolve 50 mg of 1,3-naphtha-
N, N-diethyl-N?-1-naphthylethylenediamine oxalate.
lenediol in 25 mL of ethanol (99.5), and add 2.5 mL of phos-
phoric acid. N-(1-Naphthyl)-N?-diethylethylenediamine oxalate-ace-
tone TS See N, N-diethyl-N?-1-naphthylethylenediamine
2-Naphthalenesulfonic acid See 2-naphthalenesulfonic
oxalate-acetone TS.
acid monohydrate.
N-(1-Naphthyl)-N?-diethylethylenediamine oxalate TS
2-Naphthalenesulfonic acid monohydrate C10H8O3S.H2O
See N, N-diethyl-N?-1-naphthylethylenediamine oxalate TS.
White to pale yellowish white powder. Very soluble in water,
in methanol and in ethanol (95), and sparingly soluble in N-1-Naphthylethylenediamine dihydrochloride
diethyl ether and in chloroform. C10H7NHCH2CH2NH2.2HCl [K 8197, Special class]
Water <2.48>: 7.0 – 11.5z (0.5 g, volumetric titration,
Naphthylethylenediamine TS Dissolve 0.1 g of N-1-
direct titration).
naphthylethylenediamine dihydrochloride in water to make
Content: not less than 95.0z, calculated on the anhydrous
100 mL. Prepare before use.
basis. Assay—Weigh accurately about 0.5 g of 2-naphtha-
lenesulfonic acid hydrate, dissolve in 30 mL of water, and Naringin for thin-layer chromatography C27H32O14
titrate <2.50> with 0.1 mol/L sodium hydroxide VS (indica- White to light yellow crystalline powder. Freely soluble in
tor: 3 drops of bromothymol blue TS). Perform a blank de- ethanol (95) and in acetone, and slightly soluble in water.
termination, and make any necessary correction. Melting point: about 1709C (with decomposition).
Optical rotation <2.49> [a]20
D : －87 – －939(0.1 g, ethanol
Each mL of 0.1 mol/L soduim hydroxide VS
(95), 10 mL, 100 mm).
＝ 20.82 mg of C10H8O3S
Purity Related substances—Proceed with 10 mL of a so-
1-Naphthol C10H7OH [K 8698, Special class] Preserve lution, prepared by dissolving 10 mg of naringin for thin-
in light-resistant containers. layer chromatography in 10 mL of ethanol (95), as directed
in the Identification under Bitter Orange Peel: any spot
2-Naphthol C10H7OH [K 8699, Special class] Preserve
other than the principal spot with an R f value of about 0.4
in light-resistant containers.
does not appear.
a-Naphthol See 1-naphthol.
Neocarzinostatin C511H768N132O179S4 A white or pale
b-Naphthol See 2-naphthol. yellowish white powder.
Identification—Determine the absorption spectrum of a
p-Naphtholbenzein C27H18O2 [K 8693, Special class]
solution of the substance to be examined (1 in 3000) as di-
a-Naphtholbenzein See p-naphtholbenzein. rected under Ultraviolet-visible Spectrophotometry <2.24>: it
exhibits a maximum between 270 nm and 275 nm, and shoul-
p-Naphtholbenzein TS Dissolve 0.2 g of p-naphtholben-
ders between 288 nm and 292 nm and between 330 nm and
zein in acetic acid (100) to make 100 mL.
360 nm.
Purity Clarity and color of solution—Dissolve 0.10 g of
Purity—Dissolve 10 mg of the substance to be examined in
p-naphtholbenzein in 100 mL of ethanol (95): the solution is
the mobile phase to make 50 mL, and use this solution as the
red in color and clear.
sample solution. Perform the test with 0.25 mL of the sam-
Sensitivity—Add 100 mL of freshly boiled and cooled
ple solution as directed under Liquid Chromatography
water to 0.2 mL of a solution of p-naphtholbenzein in
<2.01>, determine each peak area by the automatic integra-
ethanol (95) (1 in 1000), and add 0.1 mL of 0.1 mol/L so-
tion method, and calculate the amount of neocarzinostatin
dium hydroxide VS: a green color develops. Add subse-
by the area percentage method: not less than 90.0z.
quently 0.2 mL of 0.1 mol/L hydrochloric acid VS: the color
Operating conditions
of the solution changes to yellow-red.
Detector: An ultraviolet absorption photometer (wave-
a-Naphtholbenzein TS See p-naphtholbenzein TS. length: 254 nm).
Column: Pre-column is a stainless steel column 7.5 mm in

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
294 General Tests / 9.41 Reagents, Test Solutions JP XVII
inside diameter and 75 mm in length, packed with silica gel based on the absorbance at 600 nm of the gel determined by
for liquid chromatography (10 mm in particle size). Separa- using a densitometer. Calculate the amount of neocar-
tion column is a stainless steel column 7.5 mm in inside di- zinostatin-styrene-maleic acid alternating copolymer partial
ameter and 60 cm in length, packed with silica gel for liquid butyl ester condensate (2:3) by the following formula: not
chromatography (10 mm in particle size), which is coupled to less than 90.0z.
the pre-column.
Amount (z) of neocarzinostatin-styrene-maleic acid
Column temperature: A constant temperature of about
alternating copolymer partial butyl ester condensate (2:3)
259 C.
＝ {AT/(AT ＋ A)} × 100
Mobile phase: Dissolve 3.78 g of potassium dihydrogen
phosphate and 5.52 g of anhydrous disodium hydrogen Water <2.48> Not more than 12.0z (10 mg, coulometric
phosphate in water to make 1000 mL. titration).
Flow rate: Adjust so that the retention time of neocar-
Neutral alumina containing 4z of water Take 50 g of
zinostatin is about 21 minutes.
neutral alumina for column chromatography, previously
Time span of measurement: About 2 times as long as the
dried at 1059C for 2 hours, in a tight container, add 2.0 mL
retention time of neocarzinostatin.
of water, shake well to make homogeneous, and allow to
System suitability
stand for more than 2 hours.
System performance: When the procedure is run with 0.25
mL of the sample solution under the above operating condi- Neutral detergent Synthetic detergent containing anionic
tions, the number of theoretical plates and the symmetry fac- or non-ionic surfactant, and pH of its 0.25z solution is be-
tor of the peak of neocarzinostatin are not less than 2000 tween 6.0 and 8.0. Dilute to a suitable concentration before
and not more than 2.5, respectively. use.
System repeatability: When the test is repeated 6 times
Neutralized ethanol See ethanol, neutralized.
with 0.25 mL of the sample solution under the above operat-
ing conditions, the relative standard deviation of the peak Neutral red C15H17N4Cl Slightly metallic, dark green
area of neocarzinostatin is not more than 2.0z. powder or masses.
Water <2.48> Not more than 10.0z (10 mg, coulometric Identification—Determine the infrared absorption spec-
titration). trum as directed in the potassium bromide disk method
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
Neocarzinostatin-styrene-maleic acid alternating copoly-
tion at the wave numbers of about 3310 cm－1, 3160 cm－1,
mer partial butyl ester condensate (2:3) An amido binding
1621 cm－1, 1503 cm－1, 1323 cm－1, 1199 cm－1 and 732 cm－1.
condensate of neocarzinostatin and styrene-maleic acid alter-
nating copolymer partial butyl ester in a rate of 2:3. Average Neutral red TS Dissolve 0.1 g of neutral red in acetic
molecular mass: about 28,400. A pale yellow powder. acid (100) to make 100 mL.
Identification—Dissolve 4 mg of the substance to be exa-
Neutral red-Eagle's minimum essential medium containing
mined in 0.05 mol/L phosphate buffer solution (pH 7.0) to
bovine serum To Eagle's minimum essential medium con-
make 10 mL. Determine the absorption spectrum of this so-
taining bovine serum, which contains N-2-hydroxyethyl-
lution as directed under Ultraviolet-visible Spectrophotome-
piperazine-N ?-2-ethanesulfonic acid but not sodium hydro-
try <2.24>: it exhibits a maximum between 266 nm and 270
gen carbonate, add a solution of neutral red (1 in 100), and
nm, and shoulders between 257 nm and 262 nm, between 286
adjust to pH 6.7 – 6.8 with sodium hydroxide TS.
nm and 291 nm and between 318 nm and 348 nm.
Absorbance <2.24> E 11zcm (268 nm): 13.0 – 17.5 [4 mg calcu- NFS-60 cell Prepared from leukemia mouse, infected
lated on the anhydrous basis, 0.05 mol/L phosphate buffer with retrovirus (Cas-Br-M). After conditioning with a sui-
solution (pH 7.0), 10 mL]. table medium, preserve the strain established by J. N. Ihle,
Purity—Proceed as directed in the Purity (3) under et al. (Proc. Natl. Acad. Sci. USA, 1985, 82, 6687) at not
Zinostatin Stimalamer, with the exception of without using exceeding －1509 C in conveniently sized packets.
of (iii) Standard solution, and changing (iv) Sample solution,
Nicardipine hydrochloride for assay C26H29N3O6.HCl
(v) Procedure and (vii) Determination as follows:
[Same as the monograph Nicardipine Hydrochloride. When
(iv) Sample solution Dissolve 3.0 mg of the substance
dried, it contains not less than 99.0z of nicardipine hydro-
to be examined in the buffer solution for sample to make 10
chloride (C26H29N3O6.HCl).]
mL.
(v) Procedure Mount the gel in the electrophoresis ap- Nicergoline for assay C24H26BrN3O3 [Same as the
paratus. Add a mixture of 200 mL of Solution F and 2 mL monograph Nicergoline, or Nicergoline purified according to
of bromophenol blue solution (1 in 100,000) to the top the method below. When dried, it contains not less than
reservoir (cathode) and 300 mL of Solution F to the lower 99.0z of nicergoline (C24H26BrN3O3), and when perform the
reservoir (anode). Introduce carefully exactly 100 mL of the test of the Purity (2) under Nicergoline, the total area of the
sample solution onto the surface of the gel, and allow elec- peaks other than nicergoline from the sample solution is not
trophoresis at room temperature to take place with a current more than 2.5 times the peak area of nicergoline from the
of 2 mA per tube as a bromophenol blue band is passing in standard solution.
the stacking gel and then increase the current to 4 mA per Method of purification: Dissolve 1 g of Nicergoline in 20
tube as the bromophenol blue band is passing in the resolv- mL of acetonitrile, allow to stand in a dark place for about
ing gel, and stop the current when the band reached at 5 cm 36 hours, filter, and dry the crystals so obtained at 609C for
from the upper end of the gel. 2 hours in vacuum.]
(vii) Determination Determine the peak area, AT, of
Nickel (II) sulfate hexahydrate NiSO4.6H2O [K 8989,
neocarzinostatin-styrene-maleic acid alternating copolymer
Special class]
partial butyl ester condensate (2:3) and the total area, A, of
the peaks other than neocarzinostatin-styrene-maleic acid Nicomol for assay C34H32N4O9 [Same as the mono-
alternating copolymer partial butyl ester condensate (2:3), graph Nicomol. When dried, it contains not less than 99.0z

of nicomol (C34H32N4O9).] matic integration method: the total area of the peaks other
than nifedipine obtained from the sample solution is not
Nicotinamide C 6 H6 N 2 O [Same as the namesake mono-
larger than the peak area of nifedipine obtained from the
graph]
standard solution.
b-Nicotinamide-adenine dinucleotide (b-NAD) Operating conditions
C21H27N7O14P2 [K 9802 b-NAD＋, and meets the following Detector: An ultraviolet absorption photometer (wave-
requirement.] length: 230 nm).
Content: not less than 94.5z. Assay—Weigh accurately Column: A stainless steel column 4.6 mm in inside diame-
about 25 mg of b-nicotinamide-adenine dinucleotide, and ter and 15 cm in length, packed with octadecylsilanized silica
dissolve in water to make exactly 25 mL. Pipet 0.2 mL of gel for liquid chromatography (5 mm in particle diameter).
this solution, add 0.1 mol/L phosphate buffer solution (pH Column temperature: A constant temperature of about
7.0) to make exactly 10 mL, and use this solution as the sam- 409C.
ple solution. Determine the absorbances, AT and AB, of the Mobile phase: Adjust to pH 6.1 of a mixture of methanol
sample solution and 0.1 mol/L phosphate buffer solution and diluted 0.05 mol/L disodium hydrogen phosphate TS (1
(pH 7.0) at 260 nm as directed under Ultraviolet-visible in 5) (11:9) with phosphoric acid.
Spectrophotometry <2.24>, using water as the blank. Flow rate: Adjust so that the retention time of nifedipine
is about 6 minutes.
Amount (mg) of b-nicotinamide-adenine dinucleotide
Time span of measurement: About 2 times as long as the
(C21H27N7O14P2)
retention time of nifedipine, beginning after the solvent
0.6634 × 10 peak.
＝ × (AT － AB) × 25
17.6 × 0.20 System suitability
Test for required detectability: To exactly 5 mL of the
b-Nicotinamide adenine dinucleotide TS Dissolve 40 mg
standard solution add the mobile phase to make exactly 20
of b-nicotinamide adenine dinucleotide ( b-NAD) in 10 mL
mL. Confirm that the peak area of nifedipine obtained with
of water. Prepare before use.
10 mL of this solution is equivalent to 18 to 32z of that ob-
b-Nicotinamide adenine dinucleotide reduced form (b- tained with 10 mL of the standard solution.
NADH) C21H27N7O14P2.Na2 A white to light yellowish System performance: When the procedure is run with 10
white powder. mL of the standard solution under the above operating con-
Absorbance ratio: Determine the absorbances at 260 nm ditions, the number of theoretical plates and the symmetry
and at 340 nm, A260 and A340, of a solution of b- factor of the peak of nifedipine are not less than 4000 and
nicotinamide adenine dinucleotide reduced form (b-NADH) not more than 1.2, respectively.
in phosphate buffer solution (pH 7.4) (1 in 50,000) as di- System repeatability: When the test is repeated 6 times
rected under Ultraviolet-visible Spectrophotometry <2.24>: with 10 mL of the standard solution under the above operat-
the result of A260/A340 is between 2.2 and 2.4. ing conditions, the relative standard deviation of the peak
Water <2.48>: not more than 8.0z (0.3 g, volumetric titra- area of nifedipine is not more than 2.0z.
tion, direct titration).
Nile blue C20H20ClN3O Blue-green powder.
b-Nicotinamide adenine dinucleotide reduced form TS
Ninhydrin C 9 H 6 O4 [K 8870, Special class]
Dissolve 0.4 mg of b-nicotinamide adenine dinucleotide
reduced form (b-NADH) in 1 mL of 0.6 mol/L 2,2?,2!- Ninhydrin TS Dissolve 0.2 g of ninhydrin in water to
nitrilotriethanol hydrochloride buffer solution (pH 8.0). Pre- make 10 mL. Prepare before use.
pare before use.
Ninhydrin-acetic acid TS Dissolve 1.0 g of ninhydrin in
Nicotinic acid C6H5NO2 White, crystals or crystalline 50 mL of ethanol (95), and add 10 mL of acetic acid (100).
powder.
Ninhydrin-L-ascorbic acid TS Dissolve 0.25 g of nin-
Identification—Determine the infrared absorption spec-
hydrin and 10 mg of L-ascorbic acid in water to make 50 mL.
trum of nicotinic acid as directed in the potassium bromide
Prepare before use.
disk method under Infrared Spectrophotometry <2.25>: it ex-
hibits absorption at the wave numbers of about 2440 cm－1, Ninhydrin-butanol TS Dissolve 0.3 g of ninhydrin in 100
1707 cm－1, 1418 cm－1, 811 cm－1, 747 cm－1 and 641 cm－1. mL of 1-butanol, and add 3 mL of acetic acid (100).
Nifedipine C17H18N2O6 [Same as the namesake mono- Ninhydrin-citric acid-acetic acid TS Dissolve 70 g of
graph] citric acid monohydrate in 500 mL of water, add 58 mL of
acetic acid (100), 70 mL of a solution of sodium hydroxide
Nifedipine for assay C17H18N2O6 [Same as the mono-
(21 in 50) and water to make 1000 mL. In 100 mL of this so-
graph Nifedipine. When dried, it contains not less than
lution dissolve 0.2 g of ninhydrin.
99.0z of nifedipine (C17H18N2O6) and meets the following
requirement.] Ninhydrin-ethanol TS for spraying Dissolve 1 g of nin-
Purity Related substances—Conduct this procedure hydrin in 50 mL of ethanol (95).
without exposure to light, using light-resistant vessels. Dis-
Ninhydrin-stannous chlorid TS See ninhydrin-tin (II)
solve 25 mg of nifedipine for assay in 25 mL of the mobile
chloride TS.
phase, and use this solution as the sample solution. Pipet 1
mL of the sample solution, add the mobile phase to make ex- Ninhydrin-sulfuric acid TS Dissolve 0.1 g of ninhydrin
actly 10 mL. Pipet 2 mL of this solution, add the mobile in 100 mL of sulfric acid. Prepare before use.
phase to make exactly 25 mL, and use this solution as the
Ninhydrin-tin (II) chloride TS Dissolve 21.0 g of citric
standard solution. Perform the test with exactly 10 mL each
acid monohydrate in water to make 200 mL, adjust the pH
of the sample solution and standard solution as directed
to 5.6 ± 0.2 with sodium hydroxide TS, add water to make
under Liquid Chromatography <2.01> according to the fol-
500 mL, and dissolve 1.3 g of tin (II) chloride dihydrate. To
lowing conditions, and determine the peak area by the auto-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
296 General Tests / 9.41 Reagents, Test Solutions JP XVII
50 mL of the solution, add 50 mL of a 2-methoxethanol so- 0.6 mol/L 2,2?,2!-Nitrilotriethanol hydrochloride buffer
lution of ninhydrin (1 in 25). Prepare before use. solution (pH 8.0) Dissolve 5.57 g of 2,2?,2!-nitrilotrie-
thanol hydrochloride in 40 mL of water, adjust to pH 8.0
0.2z Ninhydrin-water saturated 1-butanol TS Dissolve
with 5 mol/L sodium hydroxide TS, and add water to make
2 g of ninhydrin in 1-butanol saturated with water to make
50 mL.
1000 mL.
3-Nitroaniline C6H6N2O2 Yellow, crystals or crystalline
Nitrendipine for assay C18H20N2O6 [Same as the mono-
powder.
graph Nitrendipine. It, when dried, contains not less than
Melting point <2.60>: 112 – 1169C
99.0z of nitrendipine (C18H20N2O6), and meets the follow-
ing requirement. When perform the test as directed in the 4-Nitroaniline C6H4NO2NH2 Yellow to yellowish-red,
Purity (2) under Nitrendipine, the area of the peak of crystals or crystalline powder.
dimethyl ester, having the relative retention time of about Melting point <2.60>: 147 – 1509C.
0.8 to nitrendipine from the sample solution is not larger Preserve in a light-resistant tight container.
than 1/2 times the peak area of nitrendipine from the stand-
p-Nitroaniline See 4-nitroaniline.
ard solution, the area of the peak other than nitrendipine
and the dimethyl ester is not larger than 1/5 times the peak p-Nitroaniline-sodium nitrite TS See 4-nitroaniline-sodi-
area of nitrendipine from the standard solution, and the um nitrite TS.
total area of the peak other than nitrendipine is not larger
4-Nitroaniline-sodium nitrite TS To 90 mL of a solution
than 1/2 times the peak area of nitrendipine from the stand-
of 0.3 g of 4-nitroaniline in 100 mL of 10 mol/L hydrochlo-
ard solution.]
ric acid TS add 10 mL of a solution of sodium nitrite (1 in
Nitric acid HNO3 [K 8541, Special class, Concentra- 20), and mix well. Prepare before use.
tion: 69 – 70z, Density: about 1.42 g/mL]
o-Nitrobenzaldehyde See 2-nitrobenzaldehyde.
Nitric acid, dilute Dilute 10.5 mL of nitric acid with
2-Nitrobenzaldehyde O2NC6H4CHO Pale yellow, crys-
water to make 100 mL.
tals or crystalline powder.
Nitric acid, fuming [K 8739, Special class, Concentra- Melting point <2.60>: 42 – 449C
tion: not less than 97z, Density: about 1.52 g/mL]
Nitrobenzene C6H5NO2 [K 8723, Special class]
Nitric acid TS, 2 mol/L Dilute 12.9 mL of nitric acid
p-Nitrobenzenediazonium chloride TS See 4-nitroben-
with water to make 100 mL.
zenediazonium chloride TS.
Nitrilotriacetic acid C6H9NO6 A white crystalline pow-
4-Nitrobenzenediazonium chloride TS Dissolve 1.1 g of
der. Melting point: about 2409C (with decomposition).
4-nitroaniline in 1.5 mL of hydrochloric acid, add 1.5 mL of
Identification—Determine the infrared absorption spec-
water, and then add a solution prepared by dissolving 0.5 g
trum of nitrilotriacetic acid as directed in the paste method
of sodium nitrite in 5 mL of water, while cooling in an ice
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
bath. Prepare before use.
tion at the wave numbers of about 1718 cm－1, 1243 cm－1,
1205 cm－1, 968 cm－1, 903 cm－1, 746 cm－1 and 484 cm－1. p-Nitrobenzenediazonium chloride TS for spraying See
Loss on drying <2.41>: not more than 0.5z (1 g, 1059C, 4-nitrobenzenediazonium chloride TS for spraying.
3 hours).
4-Nitrobenzenediazonium chloride TS for spraying Dis-
Content: not less than 97.0z. Assay—Weigh accurately
solve 0.4 g of 4-nitroaniline in 60 mL of 1 mol/L hydrochlo-
about 0.2 g of nitrilotriacetic acid, dissolve in 50 mL of
ric acid TS, and add, while cooling in an ice bath, sodium ni-
water by heating, and titrate <2.50> after cooling with 0.1
trite TS until the mixture turns potassium iodide-starch
mol/L sodium hydroxide VS (potentiometric titration). Per-
paper to blue in color. Prepare before use.
form a blank determination in the same manner, and make
any necessary correction. p-Nitrobenzenediazonium fluoroborate See 4-nitroben-
zenediazonium fluoroborate.
Each mL of 0.1 mol/L sodium hydroxide VS
＝ 9.557 mg of C6H9NO6 4-Nitrobenzenediazonium fluoroborate
O2NC6H4N2BF4 Pale yellowish white, almost odorless pow-
2,2?,2!-Nitrilotriethanol (CH2CH2OH)3N [K 8663,
der. Freely soluble in dilute hydrochloric acid, slightly solu-
Special class]
ble in water, and very slightly solute in ethanol (95) and in
2,2?,2!-Nitrilotriethanol buffer solution (pH 7.8) chloroform. Melting point: about 1489 C (with decomposi-
Dissolve 149.2 g of 2,2?,2!-nitrilotriethanol in about 4500 tion).
mL of water, adjust to pH 7.8 with diluted 6 mol/L hydro- Identification—Add 1 mL each of a solution of phenol
chloric acid TS (2 in 3), and add water to make 5000 mL. (1 in 1000) and sodium hydroxide TS to 10 mL of a solution
of 4-nitrobenzenediazonium fluoroborate (1 in 1000): a red
2,2?,2!-Nitrilotriethanol hydrochloride
color develops.
(CH2CH2OH)3N.HCl White, crystals or powder.
Loss on drying <2.41>: not more than 1.0z (1 g, silica gel,
Purity Clarity of solution—A solution (1 in 20) is clear.
2 hours).
Content: not less than 98z. Assay—Dissolve 0.3 g of
2,2?,2!-nitrilotriethanol hydrochloride, accurately weighed, p-Nitrobenzoyl chloride See 4-nitrobenzoyl chloride.
in 50 mL of water, add 5 mL of diluted nitric acid (1 in 3),
4-Nitrobenzoyl chloride O2NC6H4COCl Light yellow
and titrate <2.50> with 0.1 mol/L silver nitrate VS (potentio-
crystals.
metric titration).
Melting point <2.60>: 70 – 749C
Each mL of 0.1 mol/L silver nitrate VS Content: not less than 98.0z. Assay—Weigh accurately
＝ 18.57 mg of (CH2CH2OH)3N.HCl about 0.5 g of 4-nitrobenzoyl chloride, add an excess of sil-
ver nitrate-ethanol TS, and boil under a reflux condenser for

1 hour. After cooling, filter the precipitate, wash with water, Amount (mg) of 2-nitrophenyl-b-D-galactopyranoside
dry at 1059C to constant mass, and weigh. The mass of A
＝ × 25,000
4-nitrobenzoyl chloride, multiplied by 1.107, represents the 133
mass of 4-nitrobenzoyl chloride (C7H4ClNO3).
1-Nitroso-2-naphthol C10H7NO2 A yellow-brawn to
p-Nitrobenzyl chloride See 4-nitrobenzyl chloride. red-brown crystalline powder.
Melting point <2.60>: 106 – 1109C
4-Nitrobenzyl chloride O2NC6H4CH2Cl Light yellow,
Preserve in a light-resistant tight container.
crystals or crystalline powder. Soluble in ethanol (95).
Melting point <2.60>: 71 – 739C 1-Nitroso-2-naphthol TS Dissolve 60 mg of 1-nitroso-2-
Content: not less than 98.0z. Assay—Weigh accurately naphthol in 80 mL of acetic acid (100), and add water to
about 0.5 g of 4-nitrobenzyl chloride, add 15 mL of a solu- make 100 mL.
tion prepared by dissolving 4 g of silver nitrate in 10 mL of
a-Nitroso-b-naphthol See 1-nitroso-2-naphthol.
water and adding ethanol (95) to make 100 mL, and heat on
a water bath under a reflex condensor for 1 hour. After cool- a-Nitroso-b-naphthol TS See 1-nitroso-2-naphthol TS.
ing, filter the precipitate with a glass filter, wash with water,
Nitrous oxide N2O Colorless and odorless gas. Use
dry at 1059C to constant mass, and weigh. The mass of the
nitrous oxide from a metal cylinder.
precipitate represents the amount of silver chloride (AgCl:
143.32). NK-7 cells Cells derived from mouse NK cells.
Amount (mg) of 4-nitrobenzyl chloride (C7H6ClNO2) NN Indicator Mix 0.5 g of 2-hydroxy-1-(2-hydroxy-4-
＝ amount (mg) of silver chloride (AgCl) × 1.1972 sulfo-1-naphthylazo)-3-naphthoic acid with 50 g of anhy-
drous sodium sulfate, and triturate until the mixture
4-(4-Nitrobenzyl)pyridine C12H10N2O2 Pale yellow, crystal-
becomes homogeneous.
line powder. Freely soluble in acetone, and soluble in
ethanol (95). Nodakenin for thin-layer chromatography C20H24O9
Melting point <2.60>: 69 – 719C White powder. Slightly soluble in water and in methanol,
and very slightly soluble in ethanol (99.5). Melting point:
Nitroethane C2H5NO2
about 2209C (with decomposition).
Density <2.56>: 1.048 – 1.053 g/cm3 (209C)
Identification—Determine the absorption spectrum of a
Water <2.48>: not more than 0.1z.
solution of nodakenin for thin-layer chromatography in
Nitrogen N2 [Same as the namesake monograph] methanol (1 in 100,000) as directed under Ultraviolet-visible
Spectrophotometry <2.24>: it exhibits a maximum between
Nitrogen monoxide NO A colorless gas. Prepare by
333 nm and 337 nm.
adding sodium nitrite TS to a solution of iron (II) sulfate
Optical rotation <2.49> [a]20
D : ＋50 – ＋689(5 mg, metha-
heptahydrate in dilute sulfuric acid. Nitrogen monoxide
nol, 10 mL, 100 mm).
from a metal cylinder may be used.
Purity Related substances—Dissolve 1 mg of nodakenin
Nitromethane CH3NO2 [K 9523, Special class] for thin-layer chromatography in 3 mL of methanol, and use
this solution as the sample solution. Pipet 1 mL of the sam-
2-Nitrophenol C6H5NO3 A yellow crystalline powder.
ple solution, add methanol to make exactly 100 mL, and use
Melting points <2.60>: 44.5 – 49.09C
this solution as the standard solution. Proceed with 5 mL
3-Nitrophenol C6H5NO3 A light yellow crystalline each of these solutions as directed in the Identification (2)
powder. under Peucedanum Root: the spot other than the principal
Melting point <2.60>: 96 – 999C spot of around R f value of 0.3 from the sample solution is
not more intense than the spot from the standard solution.
4-Nitrophenol C6H5NO3 [K 8721, p-nitrophenol, Spe-
cial class] Nonessential amino acid TS Dissolve 89 mg of L-alanine,
150 mg of L-asparagine monohydrate, 133 mg of L-aspartic
o-Nitrophenyl-b-D-galactopyranoside See 2-nitrophenyl-
acid, 147 mg of L-glutamic acid, 75 mg of glycine, 115 mg of
b-D-galactopyranoside.
L-proline and 105 mg of L-serine in 100 mL of water, and
2-Nitrophenyl-b-D-galactopyranoside C12H15NO8 sterilize by filtration through a membrane filter with a pore
White crystalline powder. Odorless. It is sparingly soluble in size not exceeding 0.22 mm.
water, slightly soluble in ethanol (95), and practically insolu-
Nonylphenoxypoly(ethyleneoxy)ethanol for gas chroma-
ble in diethyl ether.
tography Prepared for gas chromatography.
Melting point <2.60>: 193 – 1949 C
Purity Clarity and color of solution—A solution of 2- L-Norleucine C6H13NO2 White, crystals or powder.
nitrophenyl-b-D-galactopyranoside (1 in 100) is clear and Dissolves in water.
colorless.
Normal agar media for teceleukin Dissolve 5.0 g of meat
Loss on drying <2.41>: not more than 0.1z (0.5 g, 1059C,
extract, 10.0 g of peptone, 5.0 g of sodium chloride, and
2 hours).
15.0 to 20.0 g of agar in water to make 1000 mL, and steri-
Content: not less than 98.0z. Assay—Weigh accurately
lize. Adjust the pH to 6.9 to 7.1.
about 50 mg of 2-nitrophenyl-b-D-galactopyranoside, previ-
ously dried, dissolve in water to make exactly 100 mL. Pipet Nuclease-free water See water, nuclease-free.
20 mL of this solution, and add water to make exactly 50
n-Octadecane C18H38 Colorless or white solid at ordi-
mL. Determine the absorbance, A, of this solution at 262 nm
nary temperature.
as directed under Ultraviolet-visible Spectrophotometry
Purity Clarity of solution—A solution of n-octadecane
<2.24>.
in chloroform (1 in 25) is clear.
Octadecylsilanized silica gel for pretreatment Prepared

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
298 General Tests / 9.41 Reagents, Test Solutions JP XVII
for pretreatment. retention time of methyl oleate, beginning after the solvent
peak.
n-Octane C8H18
Specific gravity <2.56> d 20
4 : 0.700 – 0.705 Olive oil [Same as the namesake monograph]
Purity—Perform the test with 2 mL of n-octane as directed
Olopatadine hydrochloride for assay C21H23NO3.HCl
under Gas Chromatography <2.02> according to the operat-
[Same as the monograph Olopatadine Hydrochloride. When
ing conditions in the Assay under Hypromellose. Determine
dried, it contains not less than 99.5z of olopatadine hydro-
each peak area by the automatic integration method, and
chloride (C21H23NO3.HCl).]
calculate the amount of n-octane by the area percentage
method: not less than 99.0z. Omeprazole for assay C17H19N3O3S [Same as the
monograph Omeprazole]
Octane, iso A colorless liquid. Practically insoluble in
water. Miscible with diethyl ether and with chloroform. Ophiopogon root [Same as the namesake monograph]
Purity—Determine the absorbances of isooctane at 230
Orcine C7H3O2 White to light red-brown, crystals or
nm, 250 nm and 280 nm as directed under Ultraviolet-visible
crystalline powder, having an unpleasant, sweet taste. It
Spectrophotometry <2.24>, using water as the blank solution:
turns to red in color when oxidized in air. Soluble in water,
these values are not more than 0.050, 0.010 and 0.005, re-
in ethanol (95), and in diethyl ether.
spectively.
Meting point <2.60>: 107 – 1119C
1-Octanol CH3(CH2)6CH2OH [K 8213, Special class]
Orcine-ferric chloride TS See orcine-iron (III) chloride
Octyl alcohol See 1-octanol. TS.
n-Octylbenzene C14H22 Clear and colorless liquid, hav- Orcine-iron (III) chloride TS Dissolve 10 mg of orcine in
ing a characteristic odor. 1 mL of a solution of iron (III) chloride hexahydrate in hy-
Specific gravity <2.56> d 20
4 : 0.854 – 0.863 drochloric acid (1 in 1000). Prepare before use.
Distillation test <2.57>: 263 – 2659 C, not less than 95
Ordinary agar medium Dissolve 25 to 30 g of agar in
volz.
1000 mL of ordinary broth with the aid of heat, add water to
Ofloxacin C18H20FN3O4 [Same as the namesake mono- make up for the loss, adjust the pH to between 6.4 and 7.0,
graph] and filter. Dispense the filtrate, and sterilize by autoclaving.
When powdered agar is used, 15 to 20 g of it is dissolved.
Ofloxacin demethyl substance (±)-9-Fluoro-2,3-dihydro-
3-methyl-7-oxo-7H-10-(1-piperazinyl)-pirido[1,2,3-de] [1,4] Ordinary broth Dissolve 5 g of beef extract and 10 g of
benzoxazine-6-carboxylic acid C17H18FN3O4 White to peptone in 1000 mL of water by gentle heating. Adjust the
light green-yellowish white, crystals or crystalline powder. pH of the mixture between 6.4 and 7.0 after sterilization,
Identification—Determine the infrared absorption spec- cool, add water to make up for the loss, and filter. Sterilize
trum of ofloxacin demethyl substance as directed in the po- the filtrate by autoclaving for 30 minutes at 1219C.
tassium bromide disk method under Infrared Spectropho-
Osthole for thin-layer chromatography C15H16O3 A
tometry <2.25>: it exhibits absorption at the wave numbers of
white crystalline powder, having no odor. Freely soluble in
about 3050 cm－1, 2840 cm－1, 1619 cm－1, 1581 cm－1, 1466
methanol and in ethyl acetate, soluble in ethanol (99.5), and
cm－1, 1267 cm－1, 1090 cm－1, 1051 cm－1 and 816 cm－1.
practically insoluble in water. Melting point: 83 – 849
C.
Oleic acid C18H34O2 Occurs as a colorless or pale yel- Purity Related substances—Dissolve 1.0 mg of osthole
low transparent liquid and has a slightly distinct odor. It is for thin-layer chromatography in 1 mL of methanol. Per-
miscible with ethanol (95) and with diethyl ether, and practi- form the test with 10 mL of this solution as directed in the
cally insoluble in water. Identification under Cnidium Monnieri Fruit: on spot ap-
Specific gravity <2.56> d 20
20: about 0.9 pears other than the principal spot at around R f value of
Content: not less than 99.0z. Assay—To 40 mL of oleic 0.3.
acid to be examined add 1 mL of a solution of boron trifluo-
Oxalate pH standard solution See pH Determination
ride in methanol (3 in 20), mix, and heat on a water bath for
<2.54>.
3 minutes. After cooling, add 10 mL of petroleum ether and
10 mL of water, shake, collect the ether layer after allowing Oxalic acid See oxalic acid dihydrate.
to stand, and use this solution as the sample solution. Per-
Oxalic acid dihydrate H2C2O4.2H2O [K 8519, Special
form the test with 0.2 mL of the sample solution as directed
class]
under Gas Chromatography <2.02> according to the follow-
ing conditions, determine each peak area by the automatic Oxalic acid TS Dissolve 6.3 g of oxalic acid dihydrate in
integration method, and calculate the amount of methyl ole- water to make 100 mL (0.5 mol/L).
ate by the area percentage method.
Oxycodone hydrochloride for assay See oxycodone hy-
Operating conditions
drochloride hydrate for assay.
Detector: A hydrogen flame-ionization detector
Column: A glass column 3 mm in inside diameter and 2 m Oxycodone hydrochloride hydrate for assay
in length, packed with siliceous earth for gas chromatogra- C18H21NO4.HCl.3H2O [Same as the monograph Oxyco-
phy (149 – 177 mm) coated with methyl polyacrylate in a rate done Hydrochloride Hydrate. It contains not less than
of 5 – 10z. 99.0z of oxycodone hydrochloride (C18H21NO4.HCl), calcu-
Column temperature: A constant temperature of about lated on the anhydrous basis.]
2209C.
Oxygen O2 [K 1101]
Carrier gas: Helium.
Flow rate: Adjust so that the retention time of methyl 2-Oxy-1-(2?-oxy-4?-sulfo-1?-naphthylazo)-3-naphthoic acid
oleate is about 10 minutes. See 2-hydroxy-1-(2-hydroxy-4-sulfo-1-naphthylazo)-3-
Time span of measurement: About 2 times as long as the naphthoic acid.

8-Oxyquinoline See 8-quinolinol. Operating conditions

Column, column temperature, mobile phase, and flow
Oxytocin C43H66N12O12S2 [Same as the namesake
rate: Proceed as directed in the operating conditions in the
monograph]
Assay under Moutan Bark.
Paeoniflorin for thin-layer chromatography C23H28O11 Detector: A photodiode array detector (wavelength: 274
A white powder. Freely soluble in water, in methanol and in nm, measuring range of spectrum: 220 – 400 nm).
ethanol (99.5). System suitability
Identification Determine the infrared absorption spec- System performance: Proceed as directed in the system
trum of paeoniflorin to be examined as directed in the potas- suitability in the Assay under Moutan Bark.
sium bromide disk method under Infrared Spectrophotome- Assay—Weigh accurately 5 mg of paeonol for assay 2 and
try <2.25>: it exhibits absorption at the wave numbers of 1 mg of 1,4-BTMSB-d4 for nuclear magnetic resonance
about 3410 cm－1, 1711 cm－1, 1279 cm－1, 823 cm－1 and 714 spectroscopy using an ultramicrobalance, dissolve in 1 mL of
cm－1. deuterated methanol for nuclear magnetic resonance spec-
Purity Related substances—Dissolve 1 mg of paeoni- troscopy, and use this solution as the sample solution.
florin to be examined in exactly 1 mL of methanol. Perform Transfer the sample solution into an NMR tube 5 mm in
the test with 20 mL of this solution as directed in the Identifi- outer diameter, measure 1H-NMR as directed under Nuclear
cation (2) under Peony Root: any spot other than the princi- Magnetic Resonance Spectroscopy <2.21> and Crude Drugs
pal spot with an R f value of about 0.3 does not appear. Test <5.01> according to the following conditions, using 1,4-
BTMSB-d4 for nuclear magnetic resonance spectroscopy as
Paeonol for assay C9H10O3 Use paeonol for thin-layer
the internal reference compound. Calculate the resonance in-
chromatography meeting the following additional specifica-
tensities, A1 (equivalent to 2 hydrogen) and A2 (equivalent
tions, 1) Paeonol for assay 1 or 2) Paeonol for assay 2
to 1 hydrogen), of the signals around d 6.17 – 6.25 ppm and
(Purity value by quantitative NMR). The former is used
d 7.54 ppm assuming the signal of the internal reference
after drying in a desiccator (silica gel) for 1 hour, and the
compound as d 0 ppm.
latter is corrected the content based on the amount (z)
obtained in the Assay. Amount (z) of paeonol (C9H10O3)
1) Paeonol for assay 1 ＝ MS × I × P/(M × N) × 0.7336
Absorbance <2.24> E 11zcm (274 nm): 853 – 934 [5 mg after
M: Amount (mg) of paeonol for assay 2 taken
drying in a desiccator (silica gel) for 1 hour or more, metha-
MS: Amount (mg) of 1,4-BTMSB-d4 for nuclear magnetic
nol, 1000 mL].
resonance spectroscopy taken
Purity Related substances—Dissolve 5.0 mg of paeonol
I: Sum of the signal resonance intensities, A1 and A2,
for assay in 50 mL of the mobile phase, and use this solution
based on the signal resonance intensity of
as the sample solution. Pipet 1 mL of the sample solution,
1,4-BTMSB-d4 for nuclear magnetic resonance spectros-
add the mobile phase to make exactly 100 mL, and use this
copy as 18.000
solution as the standard solution (1). Perform the test with
N: Sum of numbers of the hydrogen derived from A1 and
exactly 10 mL each of the sample solution and standard solu-
A2
tion (1) as directed under Liquid Chromatography <2.01> ac-
P: Purity (z) of 1,4-BTMSB-d4 for nuclear magnetic
cording to the following conditions, and measure each peak
resonance spectroscopy
area of these solutions by the automatic integration method:
the total area of the peaks other than paeonol obtained from Operating conditions
the sample solution is not larger than the peak area of Apparatus: An apparatus of nuclear magnetic resonance
paeonol obtained from the standard solution (1). spectrum measurement having 1H resonance frequency of
Operating conditions not less than 400 MHz.
Proceed as directed in the operating conditions in the Target nucleus: 1H.
Assay under Moutan Bark except detection sensitivity and Digital resolution: 0.25 or lower.
time span of measurement. Measuring spectrum range: 20 ppm or upper, including
Detection sensitivity: Pipet 1 mL of the standard solution between －5 ppm and 15 ppm.
(1), add the mobile phase to make exactly 20 mL, and use Spinning: off.
this solution as the standard solution (2). Adjust the detec- Pulse angle: 909 .
tion sensitivity so that the peak area of paeonol obtained 13C decoupling: on.

with 10 mL of the standard solution (2) can be measured, and Delay time: Repeating pulse waiting time not less than 60
the peak height of paeonol obtained with 10 mL of the stand- seconds.
ard solution (1) is about 20z of the full scale. Integrating times: 8 or more times.
Time span of measurement: About 3 times as long as the Dummy scanning: 2 or more times.
retention time of paeonol, beginning after the solvent peak. Measuring temperature: A constant temperature between
2) Paeonol for assay 2 (Purity value by quantitative NMR) 209C and 309 C.
Unity of peak—Dissolve 5 mg of paeonol for assay 2 in 50 System suitability
mL of the mobile phase. To 1 mL of this solution add the Test for required detectability: When the procedure is run
mobile phase to make 50 mL, and use this solution as the with the sample solution under the above operating condi-
sample solution. Perform the test with 10 mL of the sample tions, S/N of the two signals of around d 6.17 – d 6.25 ppm
solution as directed under Liquid Chromatography <2.01> and d 7.54 ppm is not less than 100.
according to the following conditions, and compare the System performance: When the procedure is run with the
absorption spectra of at least 3 points including the top of sample solution under the above operating conditions, the
paeonol peak and around the two middle peak heights of two signals of around d 6.17 – d 6.25 ppm and d 7.54 ppm
before and after the top: no difference in form is observed are not overlapped with any signal of obvious foreign sub-
between their spectra. stance, and the ratios of the resonance intensities, (A1/2)/
A2, of each signal around d 6.17 – d 6.25 ppm and d 7.54

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
300 General Tests / 9.41 Reagents, Test Solutions JP XVII
ppm are between 0.99 and 1.01, respectively. Particle counter apparatus An apparatus that is able to
System repeatability: When the test is repeated 6 times count the fine particles derived from reticulocyte similar
with the sample solution under the above operating condi- cells.
tions, the relative standard deviation of the ratio of the reso-
PBS containing bovine serum To 100 mL of bovine se-
nance intensity, A1 or A2, to that of the internal reference is
rum add 900 mL of phosphate-buffered sodium chloride TS
not more than 1.0z.
containing thimerosal (0.1 g) to make 1000 mL.
Paeonol for component determination See paeonol for Storage—Store in a cool place shielded from light.
assay.
PBS containing bovine serum albumin Add phosphate-
Paeonol for thin-layer chromatography C9H10O3 buffered sodium chloride TS to 10 g of bovine serum albu-
White, crystals or crystalline powder, having a specific odor. min and 0.1 g of thimerosal to make 1000 mL.
Freely soluble in methanol and in diethyl ether, and slightly Storage—Store in a cool, dark place.
soluble in water. Melting point: about 509C
2-fold PCR reaction solution containing SYBR Green 2-
Purity Related substances—Dissolve 1.0 mg of paeonol
Fold reaction solution for real-time PCR, containing SYBR
for thin-layer chromatography in exactly 1 mL of methanol,
Green.
and perform with 10 mL of this solution as directed in the
Identification under Moutan Bark: any spot other than the Peanut oil [Same as the namesake monograph]
principal spot at the R f value of near 0.5 does not appear.
Pentane CH3(CH2)3CH3 Clear and colorless liquid.
Palladium chloride See palladium (II) chloride. Specific gravity <2.56> d 20
20: 0.620 – 0.650
Distilling range <2.57>: 35.5 – 379C, not less than 98
Palladium chloride TS See palladium (II) chloride TS.
volz.
Palladium (II) chloride PdCl2 [K 8154, Special class]
Pemirolast potassium C10H7KN6O [Same as the name-
Palladium (II) chloride TS Dissolve 0.2 g of palladium sake monograph]
(II) chloride in 500 mL of 0.25 mol/L sulfuric acid TS, by
Peptic digest of animal tissue See Peptone, animal
heating if necessary, cool, and add 0.25 mol/L sulfuric acid
tissue.
TS to make 1000 mL.
Peptone Prepared for microbial test.
Palmatin chloride C21H22ClNO4 A yellow-brown crys-
talline powder. Peptone, animal tissue Prepared for microbial test.
Purity Related substances—Dissolve 1 mg of palmatin
Peptone, casein Grayish yellow powder, having a char-
chloride in 10 mL of methanol, and use this solution as the
acteristic but not putrescent odor. It dissolves in water, but
sample solution. Proceed with 20 mL of the sample solution
not in ethanol (95) and in diethyl ether.
as directed in the Assay under Phellodendron Bark: when
Loss on drying <2.41>: not more than 7z (0.5 g, 1059C,
measure the peak areas for 2 times the retention time of ber-
constant mass).
berine, the total area of the peaks other than palmatin is not
Residue on ignition <2.44>: not more than 15z (0.5 g).
larger than 1/10 times the total area except the area of sol-
Degree of digestion—Dissolve 1 g of casein peptone in 10
vent peak.
mL of water, and perform the following test using this solu-
Palmitic acid for gas chromatography C16H32O2 tion as the sample solution:
[K 8756, Special class] (1) Overlay 1 mL of the sample solution with 0.5 mL of
a mixture of 1 mL of acetic acid (100) and 10 mL of dilute
Pancreatic digest of casein See Peptone, casein.
ethanol: no ring or precipitate forms at the junction of the
Pancreatic digest of gelatin See Peptone, gelatin. two liquids, and on shaking, no turbidity results.
(2) Mix 1 mL of the sample solution with 4 mL of a satu-
Papaic digest of soya bean See Peptone, soybean.
rated solution of zinc sulfate heptahydrate: a small quantity
Papaverine hydrochloride C20H21NO4.HCl [Same as of precipitate is produced (proteoses).
the namesake monograph] (3) Filter the mixture of (2), and to 1 mL of the filtrate
add 3 mL of water and 4 drops of bromine TS: a red-purple
Papaverine hydrochloride for assay C20H21NO4.HCl
color is produced.
[Same as the monograph Papaverine Hydrochloride. When
Nitrogen content <1.08>: not less than 10z (1059C, con-
dried, it contains not less than 99.0z of papaverine hydro-
stant mass, after drying).
chloride (C20H21NO4.HCl).]
Peptone, gelatin Prepared for microbial test.
Paraffin [Same as the namesake monograph]
Peptone, soybean Prepared for microbial test.
Paraffin, light liquid [Same as the namesake mono-
graph] Perchloric acid HClO4 [K 8223, Special class, Density:
about 1.67 g/mL. Concentration: 70.0 – 72.0z]
Parahydroxybenzoic acid C7H6O3 White crystals.
Melting point <2.60>: 212 – 2169
C Perchloric acid-dehydrated ethanol TS See perchloric
Content: not less than 98.0 z. Assay—Weigh accurately acid-ethanol TS.
about 0.7 g of parahydroxybenzoic acid, dissolve in 50 mL
Perchloric acid-ethanol TS Add cautiously 25.5 mL of
of acetone, add 100 mL of water, and titrate <2.50> with 0.5
perchloric acid to 50 mL of ethanol (99.5), cool, and add
mol/L sodium hydroxide VS (potentiometric titration). Per-
ethanol (99.5) to make 100 mL (3 mol/L).
form a blank determination, and make any necessary correc-
tion. Performic acid Mix 9 volumes of formic acid and 1
volume of hydrogen peroxide (30), and leave at room tem-
Each mL of 0.5 mol/L sodium hydroxide VS
perature for 2 hours.
＝ 69.06 mg of C7H6O3
Storage—Store in a cool place.

Perillaldehyde for assay C10H14O Perillaldehyde for TS (pH 7.4) so that the concentration of peroxidase-labeled
thin-layer chromatography meeting the following specifica- avidin is 0.3 mg/mL. Prepare before use.
tions.
Peroxidase labeled anti-rabbit antibody It is prepared as
Absorbance <2.24> E 11zcm (230 nm): 850 – 950 (10 mg,
follows: Immunize small animals with rabbit immunoglobu-
methanol, 2000 mL).
lin G to obtain the antiserum. From the obtained antiserum
Purity Related substances—Dissolve 10 mg of perillalde-
the specific antibody is separated by the affinity chromatog-
hyde for assay in 250 mL of methanol, and use this solution
raphy using a column coupled with rabbit immunoglobulin
as the sample solution. Pipet 1 mL of this solution, add
G, and the specific antibody is labeled with peroxidase by the
methanol to make exactly 50 mL, and use this solution as the
periodic acid method.
standard solution. Perform the test with exactly 10 mL each
of the sample solution and standard solution as directed Peroxidase labeled anti-rabbit antibody TS Dissolve
under Liquid Chromatography <2.01> according to the fol- 0.10 g of bovine serum albumin in phosphate-buffered so-
lowing conditions. Determine each peak area from both sodium chloride TS to make 100 mL. To 15 mL of this solution
lutions by the automatic integration method: the total area add 5 mL of peroxidase labeled anti-rabbit antibody. Prepare
of the peaks other than perillaldehyde from the sample solu- before use.
tion is not larger than perillaldehyde from the standard solu-
Peroxidase-labeled bradykinin A solution of horseradish
tion.
origin peroxidase-binding bradykinin in gelatin-phosphate
Operating conditions
buffer solution (pH 7.0). A colorless to light brown clear so-
Detector, column, column temperature, mobile phase and
lution.
flow rate: Proceed as directed in the operating conditions in
the Assay under Perilla Herb. Peroxidase-labeled bradykinin TS To 0.08 mL of perox-
Time span of measurement: About 3 times as long as the idase-labeled bradykinin, 8 mg of sodium tetraborate deca-
retention time of perillaldehyde, beginning after the solvent hydrate, 8 mg of bovine serum albumin and 0.8 mL of
peak. gelatin-phosphate buffer solution (pH 7.0) add water to
System suitability make 8 mL, and lyophilize. Dissolve this in 8 mL of water.
System performance and system repeatability: Proceed as Prepare before use.
directed in the system suitability in the Assay under Perilla
Peroxidase-labeled rabbit anti-ECP antibody Fab? TS
Herb.
Mix 1 volume of ECP standard substance (equivalent to
Test for required detectability: Pipet 1 mL of the standard
about 1 mg of protein) and 1 volume of Freund's complete
solution, and add methanol to make exactly 20 mL. Confirm
adjuvant, and then immunize rabbits subcutaneously in the
that the peak area of perillaldehyde obtained from 10 mL of
back region and intramuscularly in the hind leg muscle with
this solution is equivalent to 3.5 to 6.5z of that of perillal-
this solution 5 times at 2 week intervals. Harvest blood on
dehyde obtained from 10 mL of the standard solution.
the 10th day after completing the immunization to obtain
Perillaldehyde for component determination See perillal- rabbit antiserum. Rabbit anti-ECP antibody Fab? is obtained
dehyde for assay. by preparing an immobilized ECP column in which ECP
standard substance is bound to agarose gel and then
Perillaldehyde for thin-layer chromatography C10H14O
purifying by affinity column chromatography to obtain rab-
Colorless to light brown transparent liquid, having a charac-
bit anti-ECP antibody which undergoes pepsin digestion to
teristic odor. Miscible with methanol and with ethanol
yield F(ab?)2 which is reacted with 2-aminoethanethiol hy-
(99.5), and very slightly soluble in water.
drochloride.
Identification—Determine the infrared absorption spec-
Horseradish peroxidase is reacted with maleimido reagent
trum of perillaldehyde for thin-layer chromatography as di-
[4-( N -maleimidylmethyl)-cyclohexane-1-carboxylate- N -hy-
rected in the liquid film method under Infrared Spectropho-
droxysuccinimide ester] to yield maleimido peroxidase.
tometry <2.25>: it exhibits absorption at the wave numbers of
Perform a coupling reaction by mixing rabbit anti-ECP
about 3080 cm－1, 2930 cm－1, 1685 cm－1, 1644 cm－1, 1435
antibody Fab? and maleimido peroxidase at 49C to prepare
cm－1 and 890 cm－1.
peroxidase-labeled rabbit anti-ECP antibody Fab?. Take a
Purity Related substances—Dissolve 1.0 mg of perillal-
specific amount of peroxidase-labeled rabbit anti-ECP
dehyde for thin-layer chromatography in 10 mL of metha-
antibody Fab? and dilute using PBS containing bovine serum
nol, and perform the test with 10 mL of this solution as di-
albumin. The peroxidase-labeled rabbit anti-ECP antibody
rected in the Identification under Perilla Herb: no spot other
Fab? TS is a diluted solution with a concentration that gives
than the principal spot at around R f value of 0.5 appears.
a good calibration curve with assay characteristics.
Peroxidase Obtained from horse-radish. A red-brown Description: Clear and colorless solution
powder. It is freely soluble in water. It contains about 250 Identification: Pipet 100 mL of the TS to be examined into
units per mg. One unit indicates an amount of the enzyme flat-bottomed microtest plates. When substrate buffer solu-
which produces 1 mg of purpurogallin in 20 seconds at 209C tion for celmoleukin is added to this, it immediately exhibits
and pH 6.0, from pyrogallol and hydrogen peroxide (30) a dark violet color, which changes to yellowish-red with
used as the substrate. time.
Peroxidase-labeled antibody stock solution 1 w/vz bo- Perphenazine maleate for assay C21H26ClN3OS.2C4H4O4
vine serum albumin-phosphate buffer-sodium chloride TS [Same as the monograph Perphenazine Maleate. When
containing antibody fragment (Fab') bound to peroxidase. dried, it contains not less than 99.0z of perphenazine male-
ate (C21H26ClN3OS.2C4H4O4).]
Peroxidase-labeled avidin A solution of avidin conjugat-
ed with horseradish peroxidase in an appropriate buffer so- Pethidine hydrochloride for assay C15H21NO2.HCl
lution. [Same as the monograph Pethidine Hydrochloride. When
dried, it contains not less than 99.0z of pethidine hydro-
Peroxidase-labeled avidin TS Dilute peroxidase-labeled
chloride C15H21NO2.HCl.]
avidin with 0.01 mol/L tris buffer solution-sodium chloride

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
302 General Tests / 9.41 Reagents, Test Solutions JP XVII
Petrolatum [Same as the monograph Yellow Petrolatum Phenol-sodium pentacyanonitrosylferrate (III) TS Dis-
or White Petrolatum] solve 5 g of phenol and 25 mg of sodium pentacyanonitro-
sylferrate (III) dihydrate in sufficient water to make 500 mL.
Petroleum benzine [K 8594, Special class]
Preserve in a dark, cold place.
Petroleum ether [K 8593, Special class]
Phenolsulfonphthalein for assay C19H14O5S [Same as
Phenacetin C10H13NO2 White, crystals or crystalline the monograph Phenolsulfonphthalein. When dried, it con-
powder. Soluble in ethanol (95), and very slightly soluble in tains not less than 99.0z of phenolsulfonphthalein
water. (C19H14O5S).]
Melting point <2.60>: 134 – 1379C
Phenylalanine See L-phenylalanine.
Loss on drying <2.41>: not more than 0.5z (1 g, 1059C,
2 hours). L-Phenylalanine C9H11NO2 [Same as the namesake
monograph]
o-Phenanthroline See 1,10-phenanthroline monohydrate.
H-D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide di-
o-Phenanthroline hydrochloride See 1,10-phenanthroli-
hydrochloride A white powder. Slightly soluble in water.
um chloride monohydrate.
Absorbance <2.24> E 11zcm (316 nm): 192 – 214 (10 mg,
1,10-Phenanthroline monohydrate C12H8N2.H2O water, 300 mL).
[K 8789, Special class]
Phenyl benzoate C6H5COOC6H5 White, crystals or
o-Phenanthroline TS See 1,10-phenanthroline TS. crystalline powder, having a slight, characteristic odor.
Melting point <2.60>: 68 – 709C
1,10-Phenanthroline TS Dissolve 0.15 g of 1,10-phenan-
Purity Clarity of solution—Dissolve 1.0 g of phenyl ben-
throline monohydrate in 10 mL of a freshly prepared iron
zoate in 20 mL of methanol: the solution is clear.
(II) sulfate heptahydrate solution (37 in 2500) and 1 mL of
dilute sulfuric acid. Preserve in tightly stoppered containers. 25z Phenyl-25z cyanopropyl-methylsilicone polymer for
gas chromatography Prepared for gas chromatography.
1,10-Phenanthrolinium chloride monohydrate
C12H8N2.HCl.H2O [K 8202, Special class] o-Phenylenediamine H2NC6H4NH2 White to dark
brown, crystals or crystalline powder. Freely soluble in
Phenethylamine hydrochloride C6H5CH2CH2NH2.HCl
ethanol (95) and in acetone, and soluble in water.
White, crystals or crystlline powder.
Content: not less than 95.0z. Assay—Accurately weigh
Melting point: <2.60> 220 – 2259 C
about 0.15 g of o-phenylenediamine, add 50 mL of acetic
Phenobarbital for assay C12H12N2O3 [Same as the acid for nonaqueous titration to dissolve, and then titrate
monograph Phenobarbital] <2.50> with 0.1 mol/L of perchloric acid VS (potentiometric
titration). Correct by conducting a blank test using the same
Phenol C6H5OH [K 8798, Special class]
method.
Phenol for assay C6H5OH [K 8798, Phenol, Special
Each mL of 0.1 mol/L perchloric acid VS
class]
＝ 10.81 mg of H2NC6H4NH2
Phenol-hydrochloric acid TS Dissolve 0.2 g of phenol in
o-Phenylenediamine dihydrochloride
10 mL of 6 mol/L hydrochloric acid TS.
H2NC6H4NH2.2HCl White to pale yellow or pale red, crys-
Phenolphthalein C20H14O4 [K 8799, Special class] tals or crystalline powder.
Purity Clarity—a solution (1 in 20) is clear.
Phenolphthalein-thymol blue TS Solution A: Dissolve
Content: not less than 98.0z. Assay—Weigh accurately
0.1 g of phenolphthalein in 100 mL of diluted ethanol (4 in
about 0.15 g of o-phenylenediamine dihydrochloride, dis-
5). Solution B: Dissolve 0.1 g of thymol blue in 50 mL of a
solve in 50 mL of water, and titrate <2.50> with 0.1 mol/L
mixture of ethanol (95) and dilute sodium hydroxide TS
sodium hydroxide VS (potentiometric titration).
(250:11), add water to make 100 mL. Mix 2 volumes of solu-
tion A and 3 volumes of solution B before use. Each mL of 0.1 mol/L sodium hydroxide VS
＝ 9.053 mg of H2NC6H4NH2.2HCl
Phenolphthalein TS Dissolve 1 g of phenolphthalein in
100 mL of ethanol (95). 1,3-Phenylenediamine hydrochloride C6H8N2.2HCl A
white or faintly reddish crystalline powder. It is colored to
Phenolphthalein TS, alkaline See Alcohol Number De-
red or brown by light.
termination <1.01>.
Identification—To 3 mL of a solution of 1,3-pheny-
Phenol red C19H14O5S [K 8800, Special class] lenediamine hydrochloride (1 in 6000) add 0.5 mL of a solu-
tion of sodium nitrite (3 in 20,000), then add 2 to 3 drops of
Phenol red TS Dissolve 0.1 g of phenol red in 100 mL of
hydrochloric acid: a yellow color is produced.
ethanol (95), and filter if necessary.
(S )-1-Phenylethyl isocyanate C6H5CH(CH3)NCO
Phenol red TS, dilute To 235 mL of a solution of ammo-
Colorless to light yellow, clear liquid, having a characteristic
nium nitrate (1 in 9400) add 105 mL of 2 mol/L sodium hy-
odor.
droxide TS and 135 mL of a solution prepared by dissolving
Optical rotation <2.49> a 20
D : －8.5 – －11.59(100 mm).
24 g of acetic acid (100) in water to make 200 mL. To this so-
Specific gravity <2.56> d 20
4 : 1.040 – 1.050
lution add 25 mL of a solution prepared by dissolving 33 mg
of phenol red in 1.5 mL of 2 mol/L sodium hydroxide TS Phenylfluorone C19H12O5 [K 9547, Special class]
and adding water to make 100 mL. If necessary, adjust the
Phenylfluorone-ethanol TS Dissolve 50 mg of phenyl-
pH to 4.7.
fluorone in ethanol (95) and in 10 mL of diluted hydrochlo-
Phenol-sodium nitroprusside TS See phenol-sodium ric acid (1 in 3), and add thanol (95) to make exactly 500 mL.
pentacyanonitrosylferrate (III) TS.

D-Phenylglycine C8H9NO2 White, crystals or crystal- for assay in 50 mL of the mobile phase, and use this solution
line powder. Slightly soluble in water. as the sample solution. To exactly 1 mL of the sample solu-
Loss on drying <2.41>: not more than 0.5z (1 g, 1059C, tion add the mobile phase to make exactly 200 mL, and use
3 hours). this solution as the standard solution. Perform the test with
Content: not less than 98.5z. Assay—Weigh accurately exactly 10 mL each of the sample solution and standard solu-
about 0.3 g of D-phenylglycine, previously dried, dissolve in tion as directed under Liquid Chromatography <2.01> ac-
3 mL of formic acid, add 50 mL of acetic acid (100), and cording to the following conditions, and determine each
titrate <2.50> with 0.1 mol/L perchloric acid VS (potentio- peak area by the automatic integration method: the total
metric titration). Perform a blank determination in the same area of the peaks other than phenytoin obtained from the
manner, and make any necessary correction. sample solution is not larger than the peak area of phenytoin
obtained from the standard solution.
Each mL of 0.1 mol/L perchloric acid VS
Operating conditions
＝ 15.12 mg of C8H9NO2
Column, column temperature, and flow rate: Proceed as
Phenylhydrazine C6H5NHNH2 Colorless or light yel- directed in the operating conditions in the Assay under
low, clear liquid, having a faint aromatic odor. Phenytoin Tablets.
Content: not less than 99.0z. Assay—Weigh accurately Detector: An ultraviolet absorption photometer (wave-
about 1 g of phenylhydrazine, add 30 mL of diluted hydro- length: 220 nm).
chloric acid (1 in 100) and water to make exactly 100 mL. Mobile phase: A mixture of 0.02 mol/L phosphate buffer
Put exactly 20 mL of this solution in a glass-stoppered coni- solution (pH 3.5) and acetonitrile for liquid chromatography
cal flask, and add 40 mL of diluted hydrochloric acid (3 in (11:9).
4). After cooling, add 5 mL of chloroform, and titrate <2.50> Time span of measurement: About 5 times as long as the
with 0.05 mol/L potassium iodate VS while shaking vigor- retention time of phenytoin, beginning after the solvent
ously until the red color of the chloroform layer disappears. peak.
Perform a blank determination in the same manner, and System suitability
make any necessary correction. Test for required detectability: To exactly 2 mL of the
standard solution add the mobile phase to make exactly 20
Each mL of 0.05 mol/L potassium iodate VS
mL. Confirm that the peak area of phenytoin obtained with
＝ 5.407 mg of C6H5NHNH2
10 mL of this solution is equivalent to 8 to 12z of that ob-
Phenylhydrazine hydrochloride See phenylhydrazinium tained with 10 mL of the standard solution.
chloride. System performance: When the procedure is run with 10
mL of the standard solution under the above operating con-
Phenylhydrazine hydrochloride TS See phenylhydrazini-
ditions, the number of theoretical plates and the symmetry
um chloride TS.
factor of the peak of phenytoin are not less than 6000 and
Phenylhydrazinium chloride C6H5NHNH2.HCl not more than 2.0, respectively.
[K 8203, Special class] System repeatability: When the test is repeated 6 times
with 10 mL of the standard solution under the above operat-
Phenylhydrazinium chloride TS Dissolve 65 mg of
ing conditions, the relative standard deviation of the peak
phenylhydrazinium chloride recrystallized from dilute
area of phenytoin is not more than 2.0z.
ethanol, in 100 mL of a solution previously prepared by add-
ing cautiously 170 mL of sulfuric acid to 80 mL of water. Phloroglucin See phloroglucinol dihydrate.
Phenyl isothiocyanate C7H5NS Prepared for amino Phloroglucin dihydrate See phloroglucinol dihydrate.
acid analysis or biochemistry.
Phloroglucinol dihydrate C6H3(OH)3.2H2O White to
1-phenyl-3-methyl-5-pyrazolone See 3-methyl-1-phenyl- pale yellow, crystals or crystalline powder.
5-pyrazolone. Melting point <2.60>: 215 – 2199C (after drying).
Loss on drying <2.41>: 18.0 – 24.0z (1 g, 1059 C, 1 hour).
50z Phenyl-50z methylpolysiloxane for gas chromatog-
raphy Prepared for gas chromatography. Phosphatase, alkaline Obtained from bovine small intes-
tine. A white to grayish white or yellowish brown, lyophi-
5z Phenyl-methyl silicone polymer for gas chromatogra-
lized powder.
phy Prepared for gas chromatography.
It contains not less than 1 unit per mg, not containing any
35z Phenyl-methyl silicone polymer for gas chromato- saline. One unit indicates an amount of the enzyme which
graphy Prepared for gas chromatography. produces 1 mmol of 4-nitrophenol at 379 C and pH 9.8 in 1
minute from 4-nitrophenyl phosphate used as the substrate.
50z Phenyl-methyl silicone polymer for gas chromato-
graphy Prepared for gas chromatography. Phosphatase TS, alkaline Dissolve 0.1 g of alkaline
phosphatase in 10 mL of boric acid-magnesium chloride
65z Phenyl-methyl silicone polymer for gas chromato-
buffer solution (pH 9.0). Prepare before use.
graphy Prepared for gas chromatography.
Phosphate-buffered sodium chloride TS Dissolve 8.0 g
Phenylpiperazine hydrochloride See 1-phenylpiperazine
of sodium chloride, 0.2 g of potassium chloride, 2.9 g of dis-
monohydrochloride.
odium hydrogen phosphate dodecahydrate, and 0.2 g of po-
1-Phenylpiperazine monohydrochloride C10H14N2.HCl tassium dihydrogen phosphate in water to make 1000 mL.
A white powder. Melting point: about 2479
C (with decom-
0.01 mol/L Phosphate buffer-sodium chloride TS (pH
position).
7.4) Dissolve 2.93 g of disodium hydrogen phosphate
Phenytoin for assay C15H12N2O2 [Same as the mono- dodecahydrate, 0.25 g of potassium dihydrogen phosphate,
graph Phenytoin. It meets the following requirements.] and 9 g of sodium chloride in water to make 1000 mL.
Purity Related substances—Dissolve 25 mg of phenytoin
Phosphate buffer solution for assay of bupleurum root

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
304 General Tests / 9.41 Reagents, Test Solutions JP XVII
To 100 mL of 0.2 mol/L potassium dihydrogen phosphate 1/15 mol/L Phosphate buffer solution (pH 5.6) Dissolve
TS add 59 mL of 0.2 mol/L sodium hydroxide TS. 9.07 g of potassium dihydrogen phosphate in about 750 mL
of water, adjust the pH to 5.6 with potassium hydroxide TS,
Phosphate buffer solution for component determination
and add water to make 1000 mL.
of bupleuram root See phosphate buffer solution for assay
of bupleuram root. Phosphate buffer solution (pH 5.9) Dissolve 6.8 g of po-
tassium dihydrogen phosphate in 800 mL of water, adjust
Phosphate buffer solution for cytotoxicity test Dissolve
the pH to 5.9 with diluted potassium hydroxide TS (1 in 10),
0.20 g of potassium chloride, 0.20 g of potassium dihydro-
and add water to make 1000 mL.
gen phosphate, 8.00 g of sodium chloride and 1.15 g of an-
hydrous disodium hydrogen phosphate in water to make Phosphate buffer solution (pH 6.0) Dissolve 8.63 g of
1000 mL, and sterilize in an autoclave at 1219C for 15 potassium dihydrogen phosphate and 1.37 g of anhydrous
minutes. disodium hydrogen phosphate in 750 mL of water, adjust
the pH to 6.0 with sodium hydroxide TS or diluted phos-
Phosphate buffer solution for epoetin alfa Dissolve
phoric acid (1 in 15), and add water to make 1000 mL.
0.247 g of sodium dihydrogen phosphate dihydrate, 0.151 g
of disodium hydrogen phosphate dodecahydrate and 8.77 g 0.05 mol/L Phosphate buffer solution (pH 6.0) To 50
of sodium chloride in water to make 1000 mL. mL of 0.2 mol/L potassium dihydrogen phosphate TS for
buffer solution add 5.70 mL of 0.2 mol/L sodium hydroxide
Phosphate buffer solution for microplate washing Dis-
TS and water to make 200 mL.
solve 0.62 g of sodium dihydrogen phosphate dihydrate,
9.48 g of disodium hydrogen phosphate dodecahydrate, Phosphate buffer solution (pH 6.2) Dissolve 9.08 g of
52.6 g of sodium chloride, 3.0 g of polysorbate 80 and 1.8 g potassium dihydrogen phosphate in 1000 mL of water (solu-
of polyoxyethylene (40) octylphenyl ether in water to make tion A). Dissolve 9.46 g of anhydrous disodium hydrogen
600 mL. Dilute this solution 10 times with water before use. phosphate in 1000 mL of water (solution B). Mix 800 mL of
the solution A and 200 mL of the solution B, and adjust the
Phosphate buffer solution for pancreatin Dissolve 3.3 g
pH to 6.2 with the solution A or the solution B if necessary.
of anhydrous disodium hydrogen phosphate, 1.4 g of potas-
sium dihydrogen phosphate and 0.33 g of sodium chloride in Phosphate buffer solution (pH 6.5) Mix 50 mL of 0.2
water to make 100 mL. mol/L potassium dihydrogen phosphate TS for buffer solu-
tion and 15.20 mL of 0.2 mol/L sodium hydroxide TS, and
Phosphate buffer solution for processed aconite root
add water to make 200 mL.
Dissolve 19.3 g of disodium hydrogen phosphate dodecahy-
drate in 3660 mL of water, and add 12.7 g of phosphoric Phosphate buffer solution for antibiotics (pH 6.5)
acid. Dissolve 10.5 g of disodium hydrogen phosphate dodecahy-
drate and 5.8 g of potassium dihydrogen phosphate in 750
Phosphate buffer solution (pH 3.0) Dissolve 136 g of po-
mL of water, adjust the pH to 6.5 with sodium hydroxide
tassium dihydrogen phosphate in water to make 1000 mL,
TS, and add water to make 1000 mL.
and adjust to pH 3.0 with phosphoric acid.
Phosphate buffer solution (pH 6.8) Dissolve 3.40 g of
0.02 mol/L Phosphate buffer solution (pH 3.0) Dissolve
potassium dihydrogen phosphate and 3.55 g of anhydrous
3.1 g of sodium dihydrogen phosphate dihydrate in 1000 mL
disodium hydrogen phosphate in water to make 1000 mL.
of water, and adjust the pH to 3.0 with diluted phosphoric
acid (1 in 10). 0.01 mol/L Phosphate buffer solution (pH 6.8) Dissolve
1.36 g of potassium dihydrogen phosphate in 900 mL of
Phosphate buffer solution (pH 3.1) Dissolve 136.1 g of
water, adjust the pH to 6.8 with 0.2 mol/L sodium hydrox-
potassium dihydrogen phosphate in 500 mL of water, and
ide TS, and add water to make 1000 mL.
add 6.3 mL of phosphoric acid and water to make 1000 mL.
0.1 mol/L Phosphate buffer solution (pH 6.8) Dissolve
0.02 mol/L Phosphate buffer solution (pH 3.5) Dissolve
6.4 g of potassium dihydrogen phosphate and 18.9 g of diso-
3.1 g of sodium dihydrogen phosphate dihydrate in 1000 mL
dium hydrogen phosphate dodecahydrate in about 750 mL
of water, and adjust the pH to 3.5 with diluted phosphoric
of water, adjust the pH to 6.8 with sodium hydroxide TS if
acid (1 in 10).
necessary, and add water to make 1000 mL.
0.05 mol/L Phosphate buffer solution (pH 3.5) To 1000
Phosphate buffer solution (pH 7.0) Mix 50 mL of 0.2
mL of 0.05 mol/L potassium dihydrogen phosphate TS add
mol/L potassium dihydrogen phosphate TS for buffer solu-
a suitable amount of a solution of phosphoric acid (49 in
tion and 29.54 mL of 0.2 mol/L sodium hydroxide TS, and
10,000) to make a solution having (pH 3.5).
add water to make 200 mL.
Phosphate buffer solution (pH 4.0) Adjust the pH of
0.05 mol/L Phosphate buffer solution (pH 7.0) Dissolve
0.05 mol/L potassium dihydrogen phosphate TS to 4.0 with
4.83 g of dipotassium hydrogen phosphate and 3.02 g of po-
diluted phosphoric acid (1 in 10).
tassium dihydrogen phosphate in 1000 mL of water, and
0.1 mol/L Phosphate buffer solution (pH 4.5) Dissolve adjust to (pH 7.0) with phosphoric acid or potassium hy-
13.61 g of potassium dihydrogen phosphate in 750 mL of droxide TS.
water, adjust to pH 4.5 with potassium hydroxide TS, and
0.1 mol/L Phosphate buffer solution (pH 7.0) Dissolve
add water to make 1000 mL.
17.9 g of disodium hydrogen phosphate dodecahydrate in
0.1 mol/L Phosphate buffer solution (pH 5.3) Dissolve water to make 500 mL (solution A). Dissolve 6.8 g of potas-
0.44 g of disodium hydrogen phosphate dodecahydrate and sium dihydrogen phosphate in water to make 500 mL (solu-
13.32 g of potassium dihydrogen phosphate in 750 mL of tion B). To a volume of solution A add solution B until the
water, adjust the pH to 5.3 with sodium hydroxide TS or mixture is adjusted to pH 7.0 (about 2:1 by volume of solu-
phosphoric acid, and add water to make 1000 mL. tions A and B).

Phosphate buffer solution (pH 7.2) Mix 50 mL of 0.2 mediately stoppered, gently shake, and allow to stand for 3
mol/L potassium dihydrogen phosphate TS for buffer solu- hours. Then add 20 mL of potassium iodide TS, stopper im-
tion and 34.7 mL of 0.2 mol/L sodium hydroxide TS, and mediately, shake vigorously, and titrate <2.50> liberated
add water to make 200 mL. iodine with 0.1 mol/L sodium thiosulfate VS (indicator: 1
mL of starch TS). Perform a blank determination in the
Phosphate buffer solution (pH 7.4) Mix 50 mL of 0.2
same manner.
mol/L potassium dihydrogen phosphate TS for buffer solu-
tion and 39.50 mL of 0.2 mol/L sodium hydroxide TS, and Each mL of 0.05 mol/L bromine VS
add water to make 200 mL. ＝ 1.650 mg of H3PO2
0.02 mol/L Phosphate buffer solution (pH 7.5) Dissolve Phosphate TS Dissolve 2.0 g of dipotassium hydrogen
2.72 g of potassium dihydrogen phosphate in 900 mL of phosphate and 8.0 g of potassium dihydrogen phosphate in
water, adjust to pH 7.5 with 0.2 mol/L sodium hydroxide water to make 1000 mL.
TS, and add water to make 1000 mL.
Phosphomolybdic acid See phosphomolybdic acid n-
0.03 mol/L Phosphate buffer solution (pH 7.5) Dissolve hydrate.
4.083 g of potassium dihydrogen phosphate in 800 mL of
Phosphomolybdic acid n-hydrate P2O5.24MoO3.xH2O
water, adjust the pH to 7.5 with 0.2 mol/L sodium hydrox-
Yellow, crystals or crystalline powder.
ide TS, and add water to make 1000 mL.
Identification (1) To 10 mL of a solution (1 in 10) add
Phosphate buffer solution (pH 8.0) Mix 50 mL of 0.2 0.5 mL of ammonia TS: yellow precipitates appear, which
mol/L potassium dihydrogen phosphate TS for buffer solu- disappear by the addition of 2 mL of ammonia TS, and yel-
tion and 46.1 mL of 0.2 mol/L sodium hydroxide TS, and low precipitates appear by further addition of 5 mL of
add water to make 200 mL. diluted nitric acid (1 in 2).
(2) To 5 mL of a solution (1 in 10) add 1 mL of ammonia
0.02 mol/L Phosphate buffer solution (pH 8.0) To 50
TS and 1 mL of magnesia TS: white precipitates appear.
mL of 0.2 mol/L potassium dihydrogen phosphate TS add
300 mL of water, adjust the pH to 8.0 with sodium hydrox- Phosphoric acid H3PO4 [K 9005, Special class]
ide TS, and add water to make 500 mL.
Phosphoric acid-acetic acid-boric acid buffer solution (pH
0.1 mol/L Phosphate buffer solution (pH 8.0) Dissolve 2.0) Dissolve 6.77 mL of phosphoric acid, 5.72 mL of
13.2 g of anhydrous disodium hydrogen phosphate and acetic acid (100) and 6.18 g of boric acid in water to make
0.91 g of potassium dihydrogen phosphate in about 750 mL 1000 mL. Adjust the pH of this solution to 2.0 with 0.5
of water, adjust to pH 8.0 with phosphoric acid, and add mol/L sodium hydroxide VS.
water to make 1000 mL.
Phosphoric acid-sodium sulfate buffer solution (pH 2.3)
0.1 mol/L Phosphate buffer solution for antibiotics (pH Dissolve 28.4 g of anhydrous sodium sulfate in 1000 mL of
8.0) Dissolve 16.73 g of dipotassium hydrogen phosphate water, and add 2.7 mL of phosphoric acid. If necessary,
and 0.523 g of potassium dihydrogen phosphate in about 750 adjust to pH 2.3 with 2-aminoethanol.
mL of water, adjust the pH to 8.0 with phosphoric acid, and
Phosphorus pentoxide See phosphorus (V) oxide.
add water to make 1000 mL.
Phosphorus, red P An odorless dark red powder. Practi-
0.2 mol/L Phosphate buffer solution (pH 10.5) Dissolve
cally insoluble in carbon disulfide and in water.
34.8 g of dipotassium hydrogen phosphate in 750 mL of
Free phosphoric acid: Not more than 0.5z.
water, adjust to pH 10.5 with 8 mol/L sodium hydroxide
To 5 g add 10 mL of a solution of sodium chloride (1 in 5),
TS, and add water to make 1000 mL.
mix, then add 50 mL of the solution of sodium chloride (1 in
Phosphate buffer solution (pH 12) To 5.44 g of anhy- 5), allow to stand for 1 hour, and filter. Wash the residue
drous disodium hydrogen phosphate add 36.5 mL of sodium with three 10-mL portions of the solution of sodium chloride
hydroxide TS and about 40 mL of water, dissolve by shaking (1 in 5), combine the filtrate and the washings, and titrate
well, and add water to make 100 mL. <2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 3
drops of thymol blue TS). Perform a blank determination in
0.01 mol/L Phosphate buffer solution Dissolve 1.15 g of
the same manner, and make any necessary correction.
anhydrous disodium hydrogen phosphate, 0.2 g of potas-
sium dihydrogen phosphate, 8.0 g of sodium chloride and Each mL of 0.1 mol/L sodium hydroxide VS
0.2 g of potassium chloride in water to make 1000 mL. ＝ 4.90 mg of H3PO4
Phosphinic acid H3PO2 Colorless or pale yellow vis- Phosphorus (V) oxide P2O5 [K 8342, Special class]
cous liquid.
Phosphotungstic acid See phosphotungstic acid n-
Identification—(1) To 0.5 mL of phosphinic acid add
hydrate.
0.5 mL of hydrogen peroxide (30) and 0.5 mL of diluted sul-
furic acid (1 in 6), and evaporate to nearly dryness on a Phosphotungstic acid n-hydrate P2O5.24WO3.xH2O
water bath. After cooling, add 10 mL of water and 5 mL of White to yellowish green, crystals or crystalline powder.
ammonia TS, and add 5 mL of magnesia TS: a white precipi- Identification—To 5 mL of a solution (1 in 10) add 1 mL
tate is produced. of acidic tin (II) chloride TS, and heat: blue precipitates
(2) To 1 mL of phosphinic acid add the mixture of iodine appear.
TS (1 mL) and water (20 mL): the iodine color disappears.
Phosphotungstic acid TS Dissolve 1 g of phospho-
Content: 30.0 – 32.0z. Assay—Weigh accurately about
tungstic acid n-hydrate in water to make 100 mL.
1.5 g of phosphinic acid, and dissolve in water to make ex-
actly 250 mL. Pipet 25 mL of this solution into an iodine o-Phthalaldehyde C6H4(CHO)2 Light yellow to yellow
bottle, add exactly 50 mL of 0.05 mol/L bromine VS, 100 crystals.
mL of water and 10 mL of diluted sulfuric acid (1 in 6), im- Content: not less than 99z. Assay—Dissolve 1 g of o-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
306 General Tests / 9.41 Reagents, Test Solutions JP XVII
phthalaldehyde in 10 mL of ethanol (95). Perform the test Picric acid-ethanol TS See 2,4,6-trinitrophenol-ethanol
with 2 mL of this solution as directed in Gas Chromatogra- TS.
phy <2.02> according to the following conditions, and deter-
Picric acid TS See 2,4,6-trinitrophenol TS.
mine each peak area by the automatic integration method.
Content (z) ＝ peak area of o-phthalaldehyed/total area Picric acid TS, alkaline See 2,4,6-trinitrophenol TS,
of all peaks × 100 alkaline.
Operating conditions
Pig bile powder for thin-layer chromatography A yel-
Detector: A thermal conductivity detector.
low-gray to yellow-brown powder, having a characteristic
Column: A glass column 3 mm in inside diameter and 2 m
odor and a bitter taste. It is practically insoluble in water, in
in length, packed with siliceous earth for gas chromatogra-
methanol and in ethanol (99.5).
phy treated with acid and silane (177 – 250 mm), coated with
Identification—To 0.1 g of pig bile powder for thin-layer
methyl silicon polymer for gas chromatography in 10z.
chromatography in a screw-capped test tube, add 1 mL of
Column temperature: A constant temperature of about
sodium hydroxide solution (3 in 25), and shake. Heat the
1809C.
tube in an oil bath at 1209C for 4 hours, allow to cool to a
Carrier gas: Helium.
lukewarm temperature, add 2 mL of 3 mol/L hydrochloric
Flow rate: Adjust so that the retention time of o-phthalal-
acid TS and 2 mL of ethyl acetate, shake at 509 C for 30
dehyde is 3 – 4 minutes.
minutes, and separate ethyl acetate layer as the sample solu-
Time span of measurement: About 7 times as long as the
tion. Separately, dissolve 10 mg of hyodeoxycholic acid for
retention time of o-phthalaldehyde, beginning after the sol-
thin-layer chromatography in 5 mL of methanol, and use
vent peak.
this solution as the standard solution. Perform the test with
Phthalein purple C32H32N2O12.x H2O Yellowish white these solutions as directed under Thin-layer Chromatogra-
to brown power. Soluble in ethanol (95), and practically in- phy <2.03>. Spot 2 mL each of the sample solution and stand-
soluble in water. ard solution on a plate of silica gel for thin-layer chromatog-
Sensitivity test—Dissolve 10 mg of phthalein purple in 1 raphy. Develop the plate with a mixture of chloroform, ace-
mL of ammonia solution (28), and add water to make 100 tone and acetic acid (100) (7:2:1) to a distance of about 10
mL. To 5 mL of this solution add 95 mL of water, 4 mL of cm, and air-dry the plate. Spray evenly dilute sulfuric acid
ammonia solution (28), 50 mL of ethanol (95) and 0.1 mL of on the plate, and heat the plate at 1059C for 10 minutes: one
diluted barium chloride TS (1 in 5): the solution shows a of the several spots obtained from the sample solution shows
blue-purple color which disappears on the addition of 0.15 the same color tone and the same R f value as the spot ob-
mL of 0.1 mol/L disodium dihydrogen ethylenediamine tained from the standard solution.
tetraacetate TS.
Pilocarpine hydrochloride for assay C11H16N2O2.HCl
Phthalic acid C8H6O4 Colorless or white crystalline [Same as the monograph Pilocarpine Hydrochloride. It
powder. Soluble in methanol and in ethanol (95), sparingly meets the following additional requirements.]
soluble in water, and practically insoluble in chloroform. Purity Related substances—Dissolve 40 mg of pilocar-
Melting point: about 2009 C (with decomposition). pine hydrochloride for assay in 100 mL of phosphate buffer
Content: not less than 98z. Assay—Weigh accurately solution (pH 4.0) and use this solution as the sample solu-
about 2.8 g of phthalic acid, add exactly 50 mL of 1 mol/L tion. Pipet 1 mL of the sample solution, add phosphate
sodium hydroxide VS and 25 mL of water, and dissolve by buffer solution (pH 4.0) to make exactly 100 mL, and use
heating on a hot plate. After cooling, add 5 drops of phenol- this solution as the standard solution. Perform the test with
phthalein TS, and titrate the excess sodium hydroxide with exactly 10 mL each of the sample solution and standard solu-
0.5 mol/L sulfuric acid VS. Perform a blank determination, tion as directed under Liquid Chromatography <2.01> ac-
and make any necessary correction. cording to the following conditions, and determine each
peak area by the automatic integration method: the area of
Each mL of 1 mol/L sodium hydroxide VS
the peaks, having the relative retention time of about 0.78
＝ 83.07 mg of C8H6O4
and about 0.92 to pilocarpine obtained from the sample so-
Phthalic anhydride C8H4O3 White, crystals or crystal- lution, is not larger than 1/2 times the peak area of pilocar-
line powder. pine obtained from the standard solution, the area of the
Melting point <2.60>: 131 – 1349
C. peak, other than pilocarpine and the peaks mentioned
above, is not larger than 1/5 times the peak area of pilocar-
Phthalimide C8H5NO2 White to pale brown, crystals or
pine from the standard solution, and the total area of the
powder.
peaks other than pilocarpine is not larger than the peak area
Melting point <2.60>: 232 – 2379
C
of pilocarpine from the standard solution.
Purity Clarity—1.0 g of phthalimide dissolves in 20 mL
Operating conditions
of sodium hydroxide TS as a slight turbid solution.
Detector, column, column temperature, mobile phase, and
Content: not less than 98.0z. Assay—Weigh accurately
flow rate: Proceed as directed in the operating conditions in
about 0.3 g of the substance to be tested, dissolve in 40 mL
the Assay under Pilocarpine Hydrochloride Tablets.
of N, N-dimethylformamide, and titrate <2.50> with 0.1
Time span of measurement: About 1.3 times as long as the
mol/L sodium methoxide VS (potentiometric titration). Per-
retention time of pilocarpine, beginning after the solvent
form a blank determination, and make any necessary correc-
peak.
tion.
System suitability
Each mL of 0.1 mol/L sodium methoxide VS Proceed as directed in the system suitability in the Purity
＝ 14.71 mg of C8H5NO2 under Pilocarpine Hydrochloride Tablets.
Phytonadione C31H46O2 [Same as the namesake mono- Pilsicainide hydrochloride hydrate for assay
graph] C17H24N2O.HCl.1/2H2O [Same as the monograph Pil-
sicainide Hydrochloride Hydrate. It contains not less than
Picric acid See 2,4,6-trinitrophenol.

99.3z of pilsicainide hydrochloride hydrate (C17H24N2O.

HCl.1/2H2O).] R f value Color and shape of the spot

Piperacillin hydrate C23H27N5O7S.H2O [Same as the Around 0 A yellow-greenish dark gray spot
namesake monograph] Around 0.05 A weak spot, dark grayish yellow-
green
Piperidine hydrochloride C5H11N.HCl A white crystal-
Around 0.2 A weak spot, dark green
line powder. Dissolves in water and in methanol. The pH of
Around 0.25 A strong spot, dark reddish purple
a solution of 1.0 g of piperidine hydrochloride in 20 mL of
(corresponding to geniposidic acid)
water is between 3.0 and 5.0.
Around 0.35 A weak spot, bright blue
Melting point <2.60>: 247 – 2529 C
Around 0.4 – 0.45 A weak tailing spot, dull greenish
Purity Clarity and color of solution—Dissolve 1.0 g of
blue
piperidine hydrochloride in 20 mL of water: the solution is
Around 0.45 A strong spot, deep yellow-green
clear and colorless.
(corresponding to acteoside)
Residue on ignition <2.44>: not more than 0.1z (1 g).
Around 0.5 A strong spot, deep blue (correspond-
Content: not less than 99.0z. Assay—Dissolve about
ing to plantagoguanidinic acid)
0.25 g of piperidine hydrochloride, accurately weighed, in
Around 0.95 A strong spot, dark grayish blue-
50 mL of water, add 5 mL of diluted nitric acid (1 in 3), and
green
titrate <2.50> with 0.1 mol/L silver nitrate VS (potentiomet-
Around 0.97 A dark grayish blue-green spot
ric titration). Perform a blank determination in the same
manner, and make any necessary correction.
Platycodon Root [Same as the namesake monograph.]
Each mL of 0.1 mol/L silver nitrate VS
＝ 12.16 mg of C5H11N.HCl Polyacrylamide gel for epoetin alfa A polyacrylamide gel
composed with the resolving gel 12.5z in acrylamide con-
Plantago seed for thin-layer chromatography [Same as centration.
the monograph Plantago Seed meeting the following addi-
tional specifications.] Polyacrylamide gel for filgrastim A polyacrylamide gel
Identification (1) To 1 g of pulverized plantago seed composed with the resolving gel 15z in acrylamide concen-
for thin-layer chromatography add 3 mL of methanol, and tration.
warm on a water bath for 3 minutes. After cooling, cen- Polyacrylamide gel for nartograstim A polyacrylamide
trifuge, and use the supernatant liquid as the sample solu- gel composed with the resolving gel 14z in acrylamide con-
tion. Perform the test with this solution as directed under centration.
Thin-layer Chromatography <2.03>. Spot 10 mL of the sam-
ple solution on a plate of silica gel for thin-layer chromatog- Polyalkylene glycol for gas chromatography Prepared
raphy. Develop the plate with a mixture of acetone, ethyl for gas chromatography.
acetate, water and acetic acid (100) (10:10:3:1) to a distance Polyalkylene glycol monoether for gas chromatography
of about 10 cm, and air-dry the plate. Splay evenly 4- Prepared for gas chromatography.
methoxybenzaldehyde-sulfuric acid TS on the plate, heat at
1059C for 10 minutes: spots equivalent to those described Polyethylene glycol 15000-diepoxide for gas chromatogra-
below appear. phy Prepared for gas chromatography.
Polyethylene glycol ester for gas chromatography Pre-
R f value Color and shape of the spot pared for gas chromatography.

Around 0 A strong spot, very dark blue Polyethylene glycol 20 M for gas chromatography Pre-
Around 0.08 A very dark blue spot pared for gas chromatography.
Around 0.1 – 0.2 A leading spot, very dark blue Polyethylene glycol 400 for gas chromatography Pre-
Around 0.25 A strong spot, deep blue (correspond- pared for gas chromatography.
ing to plantagoguanidinic acid)
Around 0.35 A strong spot, dark grayish blue (cor- Polyethylene glycol 600 for gas chromatography Pre-
responding to geniposidic acid) pared for gas chromatography.
Around 0.45 A weak spot, grayish yellowish green Polyethylene glycol 1500 for gas chromatography Pre-
Around 0.50 A strong spot, deep yellow-green pared for gas chromatography.
(corresponding to acteoside)
Around 0.6 A weak spot, light blue Polyethylene glycol 6000 for gas chromatography Pre-
Around 0.85 A deep blue spot pared for gas chromatography.
Around 0.9 – 0.95 A tailing spot, grayish blue Polyethylene glycol 2-nitroterephthalate for gas chroma-
tography Prepared for gas chromatography.
(2) Proceed as directed in the operating conditions under
Polygala root [Same as the namesake monograph]
(1), except using a mixture of ethyl acetate, water and formic
acid (6:1:1) as developing solvent: spots equivalent to those Polymethyl acrylate for gas chromatography Prepared
described below appear. for gas chromatography.
Polymethylsiloxane for gas chromatography Prepared
for gas chromatography.
Polyoxyethylene hydrogenated castor oil 60 A nonionic
surfactant prepared by addition polymerization of ethylene
oxide with hydrogenated castor oil. Average molar number

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
308 General Tests / 9.41 Reagents, Test Solutions JP XVII
of ethylene oxide added is about 60. A white or pale yellow mL of saturated sodium chloride solution, shake for about
petrolatum-like or waxy substance, having a faint, character- 15 seconds, then add sufficient saturated sodium chloride so-
istic odor and a slight bitter taste. Very soluble in ethyl ace- lution such that the upper layer of the content reaches the
tate and in chloroform, freely soluble in ethanol (95), neck of the flask. Take 2 mL of the upper layer, wash 3
slightly soluble in water, and practically insoluble in diethyl times with each 2-mL portion of water, dry with anhydrous
ether. sodium sulfate, and use this solution as the sample solution.
Identification (1) To 0.5 g of polyoxyethylene Separately, dissolve 50 mg of methyl laurate for gas chroma-
hydrogenated castor oil 60 add 10 mL of water and 5 mL of tography, 50 mg of methyl palmitate for gas chromatogra-
ammonium thiocyanate-cobalt (II) nitrate TS, and shake phy, 80 mg of methyl stearate and 100 mg of methyl oleate
thoroughly. Add 5 mL of chloroform, shake, and allow to for gas chromatography in heptane to make 50 mL, and use
stand: a blue color develops in the chloroform layer. this solution as the standard solution. Perform the test with
(2) To 0.2 g of polyoxyethylene hydrogenated castor oil 1 mL each of the sample solution and standard solution as di-
60 add 0.5 g of potassium bisulfate, and heat: an acrolein- rected under Gas Chromatography <2.02> according to the
like, irritating odor is perceptible. following conditions: the retention time of the principal peak
(3) To 0.5 g of polyoxyethylene hydrogenated castor oil obtained from the sample solution is the same with that of
60 add 10 mL of water, shake, and add 5 drops of bromine the peak of methyl laurate obtained from the standard
TS: the color of the test solution does not disappear. solution.
Congealing point <2.42>: 30 – 349 C Operating conditions
pH <2.54>—To 1.0 g of polyoxyethylene hydrogenated Detector: A hydrogen flame-ionization detector.
castor oil 60 add 20 mL of water, and dissolve by heating: Column: A fused silica column 0.25 mm in inside diameter
the pH of the solution is between 3.6 and 6.0. and 30 m in length, coated the inside surface with polyethy-
Acid value <1.13>: not more than 1.0. lene glycol 20 M for gas chromatography 0.5 mm in thick-
Saponification value <1.13>: 41 – 51 ness.
Hydroxyl value <1.13>: 39 – 49 Column temperature: Inject at a constant temperature of
Purity (1) Clarity and color of solution—Dissolve 1.0 g 809C, raise the temperature at the rate of 109C per minute to
of polyoxyethylene hydrogenated castor oil 60 in 20 mL of 2209C, and maintain the temperature at 2209C for 40
ethanol: the solution is clear and colorless. minutes.
(2) Heavy metals <1.07>—Proceed with 1.0 g of polyox- Injection port temperature: A constant temperature of
yethylene hydrogenated castor oil 60 according to Method 2, about 2509C.
and perform the test. Prepare the control solution with 2.0 Detector temperature: A constant temperature of about
mL of Standard Lead Solution (not more than 20 ppm). 2509C.
(3) Arsenic <1.11>—Prepare the test solution with 1.0 g Carrier gas: Helium.
of polyoxyethylene hydrogenated castor oil 60 according to Flow rate: Adjust so that the retention time of the peak of
Method 3, and perform the test (not more than 2 ppm). methyl laurate is about 10 minutes.
Water <2.48>: not more than 2.0z (1 g). Split ratio: 1:50.
Residue on ignition <2.44>: not more than 0.1z (1 g). System suitability
Storage—Preserve in tight containers. System performance: When the procedure is run with 1 mL
of the standard solution under the above operating condi-
Polyoxyethylene (23) lauryl ether
tions, methyl laurate, methyl palmitate, methyl stearate and
C12H25(OCH2CH2)nOH White masses. Melting point:
methyl oleate are eluted in this order, and the resolution be-
about 409C.
tween the peaks of methyl stearate and methyl oleate is not
Polyoxyethylene (40) octylphenyl ether Obtained by the less than 2.0.
addition polymerization with ethylene oxide to octylphenol. Acid value <1.13>: not more than 4.0.
A colorless or white to pale yellow, liquid, vaseline-like or Saponification value <1.13>: 43 – 55
waxy, having slightly a characteristic odor. Loss on drying <2.41>: not more than 3.0z (5 g, 1059C,
pH <2.54>: 7.0 – 9.5 (5 w/vz, 259C). 1 hour).
Specific gravity <2.56> d 20
4 : 1.10 – 1.11 Residue on ignition—Weigh accurately about 3 g of poly-
Purity Clarity of solution—Dissolve 1.0 g of polyox- sorbate 20, heat gently at first, and ignite gradually (800 –
yethylene (40) octylphenyl ether in 20 mL of water: the solu- 12009C) until the residue is completely incinerated. If any
tion is clear. carbonized substance remains, extract with hot water, filter
through a filter paper for quantitative analysis (5C), and
Polysorbate 20 Chiefly consists of addition polymer of
ignite the residue with the filter paper. Add the filtrate to it,
sorbitan monolaurate and ethylene oxide. Pale yellow to
evaporate to dryness, and ignite carefully until the car-
yellow liquid, having a faint, characteristic odor.
bonized substance does not remain. If any carbonized sub-
Identification (1) To 0.5 g of polysorbate 20 add 10 mL
stance still remains, add 15 mL of ethanol (95), crush the
of water and 10 mL of sodium hydroxide TS, boil for 5
carbonized substance with a glass rod, burn the ethanol, and
minutes, and acidify with dilute hydrochloric acid: an oily
ignite carefully. Cool in a desiccator (silica gel), and weigh
fraction is separated.
the residue accurately: not more than 1.0z.
(2) To 0.5 g of polysorbate 20 add 10 mL of water,
shake, and add 5 drops of bromine TS: the red color of the Polysorbate 20 for epoetin beta A clear to slightly tur-
test solution does not disappear. bid, yellow-brown liquid.
(3) Place 0.1 g of polysorbate 20 in a flask, dissolve in 2 Viscosity <2.53>: 300 – 500 mPa･s
mL of a solution of sodium hydroxide in methanol (1 in 50), Acid value <1.13>: not more than 3.
and heat under a reflux condenser for 30 minutes. Add 2 mL Saponification value <1.13>: 40 – 50
of boron trifluoride-methanol TS through the condenser, Hydroxyl value <1.13>: 95 – 110
and heat for 30 minutes. Then, add 4 mL of heptane through Water <2.48>: not more than 5.0z.
the condenser, and heat for 5 minutes. After cooling, add 10
Polysorbate 80 [Same as the namesake monograph].

Polyvinyl alcohol (–CH2CHOH–)n [K 9550, Special about 2.0 g of polyvinyl alcohol II, previously dried, transfer
class] to a glass-stoppered, conical flask, add 100 mL of water, and
warm while stirring for 2 hours. After cooling, add exactly
Polyvinyl alcohol I Colorless to white, or pale yellow,
25 mL of 0.5 mol/L sodium hydroxide VS, stopper tightly,
granules or powder. It is odorless, or has a faint odor of
and allow to stand for 2 hours. Then add exactly 30 mL of
acetic acid. It is tasteless. Practically insoluble in ethanol
0.25 mol/L sulfuric acid VS, shake well, and titrate <2.50>
(95) or in diethyl ether. To polyvinyl alcohol I add water,
with 0.5 mol/L sodium hydroxide VS (indicator: 3 drops of
and heat: A clear, viscous solution is obtained. It is
phenolphthalein TS). Perform a blank determination in the
hygroscopic.
same manner, and make any necessary correction.
Viscosity <2.53>: 25.0 – 31.0 mm2/s. Weigh 4.000 g of
polyvinyl alcohol I, previously dried, add 95 mL of water, Saponification value (molz)
allow to stand for 30 minutes, and heat to dissolve on a
44.05A
water bath under a reflux condenser for 2 hours while stir- ＝ 100 －
60.05 － 0.42A
ring. After cooling, add water to make 100.0 g, and mix.
Allow to stand still to remove bubbles, and perform the test 3.0025 × (a － b) f
A＝
at 20 ± 0.19C as directed in Method 1. amount (g) of polyvinyl alcohol II taken
pH <2.54>—The pH of a solution of 1.0 g of polyvinyl
a: Volume (mL) of 0.5 mol/L sodium hydroxide VS con-
alcohol I in 25 mL of water is between 5.0 and 8.0.
sumed in the test
Purity Clarity and color of solution—To 1.0 g of poly-
b: Volume (mL) of 0.5 mol/L sodium hydroxide VS con-
vinyl alcohol I add 20 mL of water, disperse by well stirring,
sumed in the blank test
warm between 609C and 809C for 2 hours, and cool: the so-
f: Molarity factor of 0.5 mol/L sodium hydroxide VS
lution is colorless and clear.
Saponification value: 98.0 – 99.0 molz. Weigh accurately Polyvinyl alcohol TS Weigh exactly 0.50 g of polyvinyl
about 3.0 g of polyvinyl alcohol I, previously dried, transfer alcohol, and add water to make exactly 100 mL.
to a glass-stoppered conical flask, add 100 mL of water, and
Polyvinylidene fluoride membrane For Western blotting.
dissolve by heating on a water bath. After cooling, add ex-
actly 25 mL of 0.1 mol/L sodium hydroxide VS, stopper Potassium acetate CH3COOK [K 8363, Special class]
tightly, and allow to stand for 2 hours. Then add exactly 30
Potassium acetate TS Dissolve 10 g of potassium acetate
mL of 0.05 mol/L sulfuric acid VS, shake well, and titrate
in water to make 100 mL (1 mol/L).
<2.50> with 0.1 mol/L sodium hydroxide VS (indicator: 3
drops of phenolphthalein TS). Perform a blank determina- Potassium aluminum sulfate See aluminum potassium
tion in the same manner, and make any necessary correction. sulfate dodecahydrate.
However, when the volume of 0.1 mol/L sodium hydroxide
Potassium bicarbonate See potassium hydrogen carbon-
VS consumed in the test is 25 mL or more, use about 2.0 g of
ate.
the sample.
Potassium biphthalate See potassium hydrogen phtha-
Saponification value (molz)
late.
44.05A
＝ 100 － Potassium biphthalate buffer solution (pH 3.5) See po-
60.05 － 0.42A
tassium hydrogen phthalate buffer solution (pH 3.5).
0.6005 × (a － b) f
A＝ Potassium biphthalate buffer solution (pH 4.6) See po-
amount (g) of polyvinyl alcohol I taken
tassium hydrogen phthalate buffer solution (pH 4.6).
a: Volume (mL) of 0.1 mol/L sodium hydroxide VS con-
Potassium biphthalate buffer solution (pH 5.6) See po-
sumed in the test
tassium hydrogen phthalate buffer solution (pH 5.6).
b: Volume (mL) of 0.1 mol/L sodium hydroxide VS con-
sumed in the blank test Potassium biphthalate for pH determination See potas-
f: Molarity factor of 0.1 mol/L sodium hydroxide VS sium hydrogen phthalate for pH determination.
Polyvinyl alcohol II Colorless to white or pale yellow, Potassium biphthalate (standard reagent) See potassium
granules or powder. It is odorless, or has a faint odor of hydrogen phthalate (standard reagent).
acetic acid. It is tasteless. Practically insoluble in ethanol
0.2 mol/L Potassium biphthalate TS for buffer solution
(95) or in diethyl ether. To polyvinyl alcohol II add water,
See 0.2 mol/L potassium hydrogen phthalate TS for buffer
and heat: A clear, viscous solution is obtained. It is
solution.
hygroscopic.
Viscosity <2.53>: 4.6 – 5.4 mm2/s. Weigh 4.000 g of poly- Potassium bisulfate See potassium hydrogen sulfate.
vinyl alcohol II, previously dried, add 95 mL of water, allow
Potassium bromate KBrO3 [K 8530, Special class]
to stand for 30 minutes, and dissolve by stirring on a water
bath between 609C and 809C for 2 hours. After cooling, add Potassium bromide KBr [K 8506, Special class]
water to make 100.0 g, and mix. Allow to stand still to re-
Potassium bromide for infrared spectrophotometry
move bubbles, and perform the test at 20 ± 0.19 C as di-
Crush homocrystals of potassium bromide or potassium bro-
rected in Method 1.
mide, collect a powder passed through a No. 200 (75 mm)
pH <2.54>—The pH of a solution of 1.0 g of polyvinyl
sieve, and dry at 1209C for 10 hours or at 5009
C for 5 hours.
alcohol II in 25 mL of water is between 5.0 and 8.0.
Prepare tablets with this powder, and determine the infrared
Purity Clarity and color of solution—To 1.0 g of poly-
absorption spectrum <2.25>: any abnormal absorption does
vinyl alcohol II add 20 mL of water, disperse by well stir-
not appear.
ring, heat on a water bath for 2 hours, and cool: the solution
is clear and colorless. Potassium carbonate K2CO3 [K 8615, Special class]
Saponification value: 86.5 – 89.5 molz. Weigh accurately

White, granules or crystalline powder. before use.

Identification—To 1 g add 100 mL of water, and dissolve
0.05 mol/L Potassium hydroxide TS Dilute 5 mL of po-
by warming. To 20 mL of this solution add 0.2 mL of so-
tassium hydroxide TS with water to make 100 mL. Prepare
dium chloride TS: white precipitates appear. Rubbing the in-
before use.
side wall of the vessel with a glass rod accelerates the form-
ing of the precipitates. 8 mol/L Potassium hydroxide TS Dissolve 52 g of potas-
sium hydroxide in water to make 100 mL. Preserve in poly-
Potassium hexahydroxoantimonate (V) TS To 2 g of po-
ethylene bottles.
tassium hexahydroxoantimonate (V) add 100 mL of water.
Boil the solution for about 5 minutes, cool quickly, add 10 Potassium iodate KIO3 [K 8922, Special class]
mL of a solution of potassium hydroxide (3 in 20), allow to
Potassium iodate (standard reagent) KIO3 In addition
stand for 1 day, and filter.
to JIS K 8005 standard reagent for volumetric analysis, cer-
Potassium hydrogen carbonate KHCO3 [K 8621, Spe- tified reference material which can be used for volumetric
cial class] analysis may be used.
Potassium hydrogen phthalate C6H4(COOK)(COOH) Potassium iodide KI [K 8913, Special class]
[K 8809, Special class]
Potassium iodide for assay [Same as the monograph
Potassium hydrogen phthalate (standard reagent) Potassium Iodide]
C6H4(COOK)(COOH) In addition to JIS K 8005 standard
Potassium iodide-starch TS Dissolve 0.5 g of potassium
reagent for volumetric analysis, certified reference material
iodide in 100 mL of freshly prepared starch TS. Prepare be-
which can be used for volumetric analysis may be used.
fore use.
Potassium hydrogen phthalate buffer solution (pH 3.5)
Potassium iodide TS Dissolve 16.5 g of potassium iodide
Dilute 50 mL of 0.2 mol/L potassium hydrogen phthalate
in water to make 100 mL. Preserve in light-resistant contain-
TS for buffer solution and 7.97 mL of 0.2 mol/L hydrochlo-
ers. Prepare before use (1 mol/L).
ric acid VS with water to make 200 mL.
Potassium iodide TS, concentrated Dissolve 30 g of po-
Potassium hydrogen phthalate buffer solution (pH 4.6)
tassium iodide in 70 mL of water. Prepare before use.
Dilute 50 mL of 0.2 mol/L potassium hydrogen phthalate
Storage—Preserve in light-resistant containers.
TS for buffer solution and 12.0 mL of 0.2 mol/L sodium hy-
droxide VS with water to make 200 mL. Potassium iodide TS, saturated Saturate 20 g of potas-
sium iodide in 10 mL of fleshly boiled and cooled water.
0.3 mol/L Potassium hydrogen phthalate buffer solution
Prepare before use.
(pH 4.6) Dissolve 61.26 g of potassium hydrogen phthalate
in about 800 mL of water, adjust the pH to 4.6 with sodium Potassium iodide-zinc sulfate TS Dissolve 5 g of potas-
hydroxide TS, and add water to make 1000 mL. sium iodide, 10 g of zinc sulfate heptahydrate, and 50 g of
sodium chloride in water to make 200 mL.
Potassium hydrogen phthalate buffer solution (pH 5.6)
Dilute 50 mL of 0.2 mol/L potassium hydrogen phthalate Potassium methanesulfonate CH3SO3K White, crystals
TS for buffer solution and 39.7 mL of 0.2 mol/L sodium hy- or crystalline powder.
droxide VS with water to make 200 mL. Purity Clarity and color of solution—Dissolve 1.0 g of
potassium methanesulfonate in 20 mL of water: the solution
Potassium hydrogen phthalate for pH determination
is transparent and colorless.
C6H4(COOK)(COOH) [K 8809, For pH determination]
Content: not less than 98.0z. Assay—Dissolve about
0.2 mol/L Potassium hydrogen phthalate TS for buffer 0.1 g of potassium methanesulfonate, accurately weighed, in
solution Dissolve 40.843 g of potassium hydrogen phtha- 10 mL of acetic acid (100), add 20 mL of acetic anhydride,
late for pH determination in water to make 1000 mL. and titrate <2.50> with 0.1 mol/L perchloric acid VS (poten-
tiometric titration). Perform a blank determination, and
Potassium hydrogen sulfate KHSO4 [K 8972, Special
make any necessary correction.
class]
Each mL of 0.1 mol/L perchloric acid VS
Potassium hydroxide KOH [K 8574, Special class]
＝ 13.42 mg of CH3SO3K
Potassium hydroxide-ethanol TS Dissolve 10 g of potas-
Potassium naphthoquinone sulfonate See potassium 1,2-
sium hydroxide in ethanol (95) to make 100 mL. Prepare be-
naphthoquinone-4-sulfonate.
fore use.
Potassium 1,2-naphthoquinone-4-sulfonate C10H5O2SO3K
0.1 mol/L Potassium hydroxide-ethanol TS To 1 mL of
[K 8696, Special class]
dilute potassium hydroxide-ethanol TS add ethanol (95) to
make 5 mL. Prepare before use. Potassium 1,2-naphthoquinone-4-sulfonate TS Dissolve
0.5 g of potassium 1,2-naphthoquinone-4-sulfonate in water
Potassium hydroxide-ethanol TS, dilute Dissolve 35 g of
to make 100 mL. Prepare before use.
potassium hydroxide in 20 mL of water, and add ethanol
(95) to make 1000 mL (0.5 mol/L). Preserve in tightly stop- Potassium nitrate KNO3 [K 8548, Special class]
pered bottles.
Potassium nitrite KNO2 A white to pale yellow crystal-
Potassium hydroxide TS Dissolve 6.5 g of potassium hy- line powder. It is deliquescent.
droxide in water to make 100 mL (1 mol/L). Preserve in Identification—(1) Dissolve 1 g of potassium nitrite in 20
polyethylene bottles. mL of water, and use this solution as the sample solution.
To 5 mL of the sample solution add 1 mL of sulfuric acid: a
0.02 mol/L Potassium hydroxide TS Dilute 2 mL of po-
yellowish brown gas is evolved.
tassium hydroxide TS with water to make 100 mL. Prepare
(2) The sample solution obtained in (1) responds to the

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
312 General Tests / 9.41 Reagents, Test Solutions JP XVII
Qualitative Tests <1.09> (1) for potassium salt. Potassium tetraphenylborate TS Add 1 mL of acetic acid
Preserve in a light-resistant tight container. (31) to 50 mL of a solution of potassium biphthalate (1 in
500), then to this solution add 20 mL of a solution of sodium
Potassium periodate KIO4 [K 8249, Special class]
tetraphenylborate (7 in 1000), shake well, and allow to stand
Potassium periodate TS To 2.8 g of potassium periodate for 1 hour. Collect the produced precipitate on filter paper,
add 200 mL of water, dissolve by adding dropwise 20 mL of and wash it with water. To 1/3 quantity of the precipitate
sulfuric acid under shaking, cool, and add water to make add 100 mL of water, warm, with shaking, at about 509C
1000 mL. for 5 minutes, cool quickly, allow to stand for 2 hours with
occasional shaking, and filter, discarding the first 30 mL of
1.6z Potassium periodate-0.2z potassium permanganate
the filtrate.
TS, alkaline Dissolve 1 g of potassium permanganate, 8 g
of potassium periodate and 10 g of potassium carbonate in Potassium thiocyanate KSCN [K 9001, Special class]
500 mL of water. After allowing to stand for 16 hours, filter
Potassium thiocyanate TS Dissolve 1 g of potassium
through a filter paper.
thiocyanate in water to make 10 mL.
Potassium permanganate KMnO4 [K 8247, Special
Potassium trihydrogen dioxalate dihydrate for pH deter-
class]
mination KH3(C2O4)2.2H2O [K 8474, for pH determina-
Potassium permanganate TS Dissolve 3.3 g of potas- tion]
sium permanganate in water to make 1000 mL (0.02 mol/L).
Potato extract Prepared for microbial test.
Potassium permanganate TS, acidic To 100 mL of po-
Potato starch [Same as the namesake monograph]
tassium permanganate TS add 0.3 mL of sulfuric acid.
Potato starch TS Prepare as directed under starch TS
Potassium peroxodisulfate K 2 S2 O8 [K 8253, Special
with 1 g of potato starch.
class]
Potato starch TS for amylolytic activity test Dry about
Potassium persulfate See potassium peroxodisulfate.
1 g of potato starch, accurately weighed, at 1059C for 2
Potassium pyroantimonate See potassium hexahydrox- hours, and measure the loss. Weigh accurately an amount of
oantimonate (V). potato starch, equivalent to 1.000 g on the dried basis, place
into a conical flask, add 20 mL of water, and make it pasty
Potassium pyroantimonate TS See potassium hexa-
by gradually adding 5 mL of a solution of sodium hydroxide
hydroxoantimonate (V) TS.
(2 in 25) while shaking well. Heat in a water bath for 3
Potassium pyrophosphate K4O7P2 White crystalline minutes while shaking, add 25 mL of water, and cool. Neu-
powder, very soluble in water. Melting point: 11099C‚ tralize exactly with 2 mol/L hydrochloric acid TS, add 10
mL of 1 mol/L acetic acid-sodium acetate buffer solution
Potassium pyrosulfate See potassium disulfate.
(pH 5.0) and add water to make exactly 100 mL. Prepare be-
Potassium sodium tartarate See potassium sodium tar- fore use.
tarate tetrahydrate.
Potency measuring medium for nartograstim test Dis-
Potassium sodium tartarate tetrahydrate solve 10.4 g of RPMI-1640 medium in a suitable amount of
KNaC4H4O6.4H2O [K 8536, (＋)-Potassium sodium tar- water, add 16 mL of sodium hydrogen carbonate solution
trate tetrahydrate, Special class] (3 in 40), then add water to make 1000 mL, adjust to pH 7.0
by passing carbon dioxide through the solution, and sterilize
Potassium sulfate K2SO4 [K 8962, Special class]
by filtration. To 90 mL of this solution add 10 mL of fetal
Potassium sulfate TS Dissolve 1 g of potassium sulfate bovine serum, previously heat at 569C for 30 minutes, 1 mL
in water to make 100 mL. of a solution dissolved 1.0 × 105 units of potassium benzyl-
penicillin and 0.1 g (potency) of streptomycin sulfate in 10
Potassium tartrate 2C4H4K2O6.H2O [K 8535, (＋)-
mL of isotonic sodium chloride solution, and add 5 mL of 2-
Potassium Tartrate-Water (2/1), Special class]
mercaptoethanol solution (9 in 125). Sterilize this solution by
Potassium tellurite K2TeO3 White, powder or small filtration.
masses obtained by melting an equimolar mixture of telluri-
Potency measuring medium for teceleukin Add 100 mL
um dioxide and potassium carbonate in a stream of carbon
of fetal calf serum to 1000 mL of medium for float culture.
dioxide. Soluble in water.
Store at 49C.
Content: not less than 90.0z. Assay—Dissolve about
1.0 g of potassium tellurite, accurately weighed, in 100 mL Powdered tragacanth [Same as the namesake mono-
of water, add 5 mL of diluted acetic acid (31) (1 in 3), and graph]
boil. After cooling, filter by suction through a crucible glass
(±)-Praeruptorin A for thin-layer chromatography
filter (1G4) [previously dried at 105  29C for 1 hour to
C21H22O7 White, crystals or crystalline powder. Soluble in
constant mass (b (g)]. Wash the filtrate with water, dry the
methanol, sparingly slightly soluble in ethanol (99.5), and
C for 3 hours, and measure the mass a (g).
glass filter at 1109
practically insoluble in water.
Content (z) of potassium tellurite (K2TeO3) Identification—Determine the absorption spectrum of a
solution of (±)-praeruptorin A for thin-layer chromatogra-
(a － b) × 1.5902
＝ × 100 phy in methanol (1 in 100,000) as directed under Ultraviolet-
S
visible Spectrophotometry <2.24>: it exhibits a maximum be-
S: Mass (g) of potassium tellurite taken. tween 320 nm and 324 nm.
Melting point <2.60>: 152 – 1569C
Potassium tetraoxalate for pH determination See potas-
Purity Related substances—Dissolve 2 mg of (±)-
sium trihydrogen dioxalate dihydrate for pH determination.
praeruptorin A for thin-layer chromatography in 2 mL of

methanol, and use this solution as the sample solution. Pipet Procainamide hydrochloride for assay C13H21N3O.HCl
1 mL of the sample solution, add methanol to make exactly [Same as the monograph Procainamide Hydrochloride.
100 mL, and use this solution as the standard solution. Pro- When dried, it contains not less than 99.0z of procainamide
ceed with 5 mL each of these solutions as directed in the Iden- hydrochloride (C13H21N3O.HCl).]
tification (1) under Peucedanum Root: the spot other than
Procaine hydrochloride C13H20N2O2.HCl [Same as the
the principal spot of around R f value of 0.3 from the sample
namesake monograph]
solution is not more intense than the spot from the standard
solution. Procaine hydrochloride for assay See procaine hydro-
chloride.
Pravastatin sodium C23H35NaO7 [Same as the name-
sake monograph] Procaterol hydrochloride See procaterol hydrochloride
hydrate.
Prazepam for assay C19H17ClN2O [Same as the mono-
graph Prazepam. When dried, it contains not less than Procaterol hydrochloride hydrate C16H22N2O3.HCl.
99.0z of prazepam (C19H17ClN2O).] 1/2 H
2O [Same as the namesake monograph]
Prednisolone C21H28O5 [Same as the namesake mono- Progesterone C21H30O2 [Same as the namesake mono-
graph] graph]
Prednisolone acetate C23H30O6 [Same as the namesake L-Proline C5H9NO2 [K 9107, L(－)-proline, Special
monograph] class]
Prednisone C21H26O5 White crystalline powder. Slight- Propafenone hydrochloride for assay C21H27NO3.HCl
ly soluble in methanol, in ethanol (95) and in chloroform, [Same as the monograph Propafenone Hydrochloride. When
and very slightly soluble in water. dried, it contains not less than 99.0z of propafenone hydro-
Optical rotation <2.49> [a]20
D : ＋167 – ＋1759(after dry- chloride (C21H27NO3.HCl). When perform the test as di-
ing, 0.1 g, 1,4-dioxane, 10 mL, 100 mm). rected in the Purity (2), the total area of the peaks other than
Loss on drying <2.41>: not more than 1.0z (1 g, 1059C, propafenone is not larger than 3 times the peak area of
3 hours). propafenone from the standard solution.]
Content: 96.0 – 104.0z. Assay—Weigh accurately
n-Propanol See 1-propanol.
about 20 mg of prednisone, previously dried, and dissolve in
methanol to make exactly 100 mL. Pipet 5 mL of this solu- 1-Propanol CH3CH2CH2OH [K 8838, Special class]
tion, dilute with methanol to make exactly 100 mL. Perform
2-Propanol (CH3)2CHOH [K 8839, Special class]
the test with this solution as directed under Ultraviolet-
visible Spectrophotometry <2.24>, and read the absorbance 2-Propanol for vitamin A assay (CH3)2CHOH
A at the wavelength of maximum absorption at about 238 [K 8839, Special class] When the absorbances at 300 nm
nm. and between 320 nm and 350 nm are determined as directed
under Ultraviolet-visible Spectrophotometry <2.24>, using
A
Amount (mg) of prednisone (C21H26O5) ＝ × 20,000 water as the control, they are not more than 0.05 and not
440
more than 0.01, respectively. If necessary, purify by distilla-
Primary antibody TS To a mixture of 1.5 mL of block- tion.
ing TS for epoetin alfa and 13.5 mL of sodium azide-
2-Propanol for liquid chromatography (CH3)2CHOH
phosphate-buffered sodium chloride TS add a volume of
Clear, colorless and volatile liquid, having a characteristic
mouse anti-epoetin alfa monoclonal antibody corresponding
odor. Miscible with water, with ethanol (95) and with diethyl
to 100 mg of protein, 50 mL of a solution of aprotinin con-
ether. Boiling point: about 829C.
taining 1 × 105 units in 5 mL of water and 100 mL of
Refractive index <2.45> n 20
D : 1.376 – 1.378
phenylmethylsulfonyl fluoride solution containing 1.74 mg
Specific gravity <2.56> d 20
20: 0.785 – 0.788
in 100 mL of methanol.
Purity (1) Ultraviolet absorbing substances—Perform
Primer F A primer corresponding to the Alu sequence. the test with 2-propanol for liquid chromatography as di-
Synthesize an oligonucleotide which nucleotide sequence is rected under Ultraviolet-visible Spectrophotometry <2.24>,
represented by ``5?-CATCCTGGCYAACAYGGTGAAAC- using water as the blank: the absorbance at 230 nm is not
3?'', and use. more than 0.2; at 250 nm, not more than 0.03; and between
280 nm and 400 nm, not more than 0.01.
Primer F TS To primer F add TE buffer solution so that
(2) Peroxide—Mix 100 mL of water and 25 mL of dilute
primer F is 100 mmol/L. Then add tris-glycine buffer solu-
sulfuric acid, and add 25 mL of a solution of potassium
tion (pH 6.8) so that primer F is 25 mmol/L.
iodide (1 in 10). Add this solution to 20 g of 2-propanol for
Primer R A primer corresponding to the Alu sequence. liquid chromatography. Stopper tightly, shake, allow to
Synthesize an oligonucleotide which nucleotide sequence is stand for 15 minute in a dark place, and titrate <2.50> with
represented by ``5?-ATTCTCCTGCCTCAGCCTCC-3?'', 0.01 mol/L sodium thiosulfate VS (indicator: 1 mL of starch
and use. TS). Perform a blank determination in the same manner,
and make any necessary correction (not more than
Primer R TS To primer R add TE buffer solution so that
0.0005z).
primer R is 100 mmol/L. Then, add tris-glycine buffer solu-
tion (pH 6.8) so that primer R is 25 mmol/L. Propanol, iso See 2-propanol.
Probenecid C13H19NO4S [Same as the namesake mono- Propranolol hydrochloride for assay C16H21NO2.HCl
graph] [Same as the monograph Propranolol Hydrochloride. When
dried, it contains not less than 99.5z of propranolol hydro-
Procainamide hydrochloride C13H21N3O.HCl [Same as
chloride (C16H21NO2.HCl).]
the namesake monograph]

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
314 General Tests / 9.41 Reagents, Test Solutions JP XVII
Propantheline bromide C23H30BrNO3 [Same as the Operating conditions
namesake monograph] Detector, column, column temperature, mobile phase, and
flow rate: Proceed as directed in the operating conditions in
Propionic acid CH3CH2COOH Colorless liquid.
the Assay under Propyl Parahydroxybenzoate.
Purity—Clarity and color of solution—Dissolve 1.0 g of
Time span of measurement: About 2.5 times as long as the
propionic acid in 20 mL of ethanol (95): the solution is clear
retention time of propyl parahydroxybenzoate.
and colorless.
System suitability
Specific gravity <2.56> d 20
20: 0.998 – 1.004
Test for required detectability: Pipet 1 mL of the standard
Distilling range <2.57>: 139 – 1439C, not less than 95
solution, and add methanol to make exactly 20 mL. Confirm
volz.
that the peak area of propyl parahydroxybenzoate obtained
Propylamine, iso (CH3)2CHNH2 Colorless liquid, hav- with 10 mL of this solution is equivalent to 3.5 to 6.5z of
ing a characteristic, amine-like odor. Miscible with water, that obtained with 10 mL of the standard solution.
with ethanol (95) and with diethyl ether. System performance: When the procedure is run with 10
Refractive index <2.45> n 20
D : 1.374 – 1.376 mL of the standard solution under the above operating con-
Specific gravity <2.56> d 20
20: 0.685 – 0.690 ditions, the number of theoretical plates and the symmetry
Distilling range <2.57>: 31 – 339C, not less than 95 volz. factor of the peak of propyl parahydroxybenzoate are not
less than 2500 and not more than 2.0, respectively.
Propyl benzoate C6H5COOC3H7 Clear, colorless liq-
System repeatability: When the test is repeated 6 times
uid, having a characteristic odor.
with 10 mL of the standard solution under the above operat-
Refractive index <2.45> n 20
D : 1.498 – 1.503
ing conditions, the relative standard deviation of the peak
Specific gravity <2.56> d 20
20: 1.022 – 1.027
area of propyl parahydroxybenzoate is not more than 5.0z.
Propylene carbonate C4H6O3 Colorless liquid.
Propylene glycol for gas chromatography C3H8O2 [K
Boiling point <2.57>: 240 – 2429
C
8837, Special class] When perform the test as directed in
Water <2.48>: less than 0.1z
the Purity (7) under Propylene Glycol, it does not show any
Propylene carbonate for water determination See Water peak at the retention times corresponding to ethylene glycol
Determination <2.48>. and diethylene glycol.
Propylene glycol CH3CH(OH)CH2OH [K 8837, Spe- Propylthiouracil for assay C7H10N2OS [Same as the
cial class] monograph Propylthiouracil. When dried, it contains not
less than 99.0z of propylthiouracil (C7H10N2OS).]
Propylene glycol cefatrizine C18H18N6O5S2.C3H8O2
[Same as the namesake monograph] Prostaglandin A1 C20H32O4 White, crystals or crystal-
line powder. Very soluble in ethanol (95) and in ethyl ace-
Propylether, iso (CH3)2CHOCH(CH3)2 Clear, colorless
tate, and very slightly soluble in water.
liquid, having a characteristic odor. Not miscible with water.
Purity Related substances—Dissolve 5 mg of prostaglan-
Refractive index <2.45> n 20
D : 1.368 – 1.369
din A1 in 10 mL of ethanol (95), and use this solution as the
Specific gravity <2.56> d 20
4 : 0.723 – 0.725
sample solution. Pipet 3 mL of the sample solution, add
Propyl parahydroxybenzoate ethanol (95) to make exactly 100 mL, and use this solution as
HOC6H4COOCH2CH2CH3 [Same as the namesake mono- the standard solution. Perform the test with exactly 10 mL
graph] each of the sample solution and standard solution as directed
under Liquid Chromatography <2.01> according to the fol-
Propyl parahydroxybenzoate for resolution check
lowing operating conditions. Determine areas of all peaks of
C10H12O3 Colorless crystals or a white crystalline powder.
both solutions by the automatic integration method: the
Freely soluble in methanol, in ethanol (95) and in acetone,
total area of the peaks other than prostaglandin A1 from the
and very slightly soluble in water. Melting point: 96 – 999C.
sample solution is not larger than the peak area of
Identification—Determine the infrared absorption spec-
prostaglandin A1 from the standard solution.
trum of propyl parahydroxybenzoate for resolution check as
Operating conditions
directed in the potassium bromide disk method under In-
Detector, column, column temperature, mobile phase,
frared Spectrophotometry <2.25>, and compare the spectrum
flow rate, and selection of column: Proceed the operating
with the Reference Spectrum of Propyl Parahydroxybenzo-
conditions in the Assay of Alprostadil Alfadex.
ate or the spectrum of Propyl Parahydroxybenzoate RS:
Detection sensitivity: Adjust the detection sensitivity so
both spectra exhibit similar intensities of absorption at the
that the peak height of prostaglandin A1 obtained from 10
same wave numbers.
mL of the standard solution is 5 to 10z of the full scale.
Purity Related substances—Dissolve 50 mg of propyl
Time span of measurement: About twice as long as the
parahydroxybenzoate for resolution check in 2.5 mL of
retention time of prostaglandin A1, beginning after the sol-
methanol, and add the mobile phase to make 50 mL. To 10
vent peak.
mL of this solution add the mobile phase to make 100 mL,
and use this solution as the sample solution. Pipet 1 mL of Protein digestive enzyme TS A solution of lysyl en-
the sample solution, add the mobile phase to make exactly 10 dopeptidase in 0.05 mol/L tris buffer solution (pH 8.6) (1 in
mL, and use this solution as the standard solution. Perform 50,000).
the test with exactly 10 mL each of the sample solution and
Pseudoephedrine hydrochloride C10H15NO.HCl White,
standard solution as directed under Liquid Chromatography
crystals or crystalline powder. Freely soluble in water, in
<2.01> according to the following conditions. Determine each
methanol and in acetic acid (100), soluble in ethanol (99.5),
peak area by the automatic integration method: total area of
and practically insoluble in acetic anhydride. Melting point:
the peaks other than propyl parahydroxybenzoate obtained
182 – 1869 C.
from the sample solution is not larger than the peak area of
Purity Related substances—Dissolve 1 mg of pseud-
propyl parahydroxybenzoate obtained from the standard so-
oephedrine hydrochloride in 10 mL of diluted methanol (1 in
lution.

2), and use this solution as the sample solution. Perform the Pyridoxine hydrochloride C8H11NO3.HCl [Same as the
test with 10 mL of the sample solution for twice as long as as namesake monograph]
the retention time of ephedrine as directed in the Assay (1)
1-(2-Pyridylazo)-2-naphthol C15H11N3O Orange-yellow
under Kakkonto Extract: the total area of the peaks other
or orange-red powder.
than pseudoephedrine and the solvent is not larger than 1/10
Absorbance—Dissolve 25 mg of 1-(2-pyridylazo)-2-
times the total area of the peaks other than the solvent.
naphthol in methanol to make exactly 100 mL. Pipet 2.0 mL of
Puerarin for thin-layer chromatography C21H20O9 this solution, and add methanol to make exactly 50 mL. Per-
White, crystals or crystalline powder. Freely soluble in meth- form the test with this solution as directed under Ultraviolet-
anol, soluble in ethanol (99.5), and practically insoluble in visible Spectrophotometry <2.24>, using methanol as the
water. blank: absorbance at the wavelength of 470 nm is not less
Identification Determine the infrared absorption spec- than 0.55.
trum of puerarin to be examined as directed in the potassium Melting point <2.60>: 137 – 1409C
bromide disk method under Infrared Spectrophotometry Purity Clarity and color of solution—Dissolve 25 mg of
<2.25>: it exhibits absorption at the wave numbers of about 1-(2-pyridylazo)-2-naphthol in 100 mL of methanol: the so-
3370 cm－1, 1632 cm－1, 1447 cm－1, 1060 cm－1 and 836 cm－1. lution is clear and orange-yellow.
Purity Related substances—Dissolve 1 mg of puerarin to Residue on ignition <2.44>: not more than 1.0z.
be examined in methanol to make exactly 1 mL. Perform the Sensitivity—On adding 50 mL of water, 30 mL of metha-
test with 2 mL of this solution as directed in the Identifica- nol and 10 mL of acetic acid-sodium acetate buffer solution
tion under Pueraria Root: any spot other than the principal (pH 5.5) to 0.2 mL of a solution of 1-(2-pyridylazo)-2-
spot with an R f value of about 0.4 does not appear. naphthol in methanol (1 in 4000), the solution is yellow in
color. Add 1 drop of a solution of copper (II) chloride dihy-
Pullulanase An enzyme obtained from Klebsiella pneu-
drate (1 in 600) to this solution: the solution is red-purple in
moniae. White crystals. It contains not less than 30 units per
color. Add a subsequent 1 drop of diluted 0.1 mol/L disodi-
mg. One unit is an enzymatic activity to produce 1 mmol of
um dihydrogen ethylenediamine tetraacetate TS (1 in 10): the
maltotriose from pullulan per minute at pH 5.0 and 309C.
color of the solution changes to yellow again.
Pullulanase TS A solution of pullulanase containing 10
1-(4-Pyridyl)pyridinium chloride hydrochloride
units per mL.
C10H9ClN2.HCl White to yellowish white crystalline pow-
Purified hydrochloric acid See hydrochloric acid, puri- der. Very soluble in water, very slightly soluble in ethanol
fied. (95), and practically insoluble in diethyl ether.
Melting point <2.60>: 154 – 1569C
Purified sodium hyaluronate See sodium hyaluronate,
purified. Pyrogallol C6H3(OH)3 [K 8780, Special class]
Purified methanol See methanol, purified. L-Pyroglutamylglycyl-L-arginine-p-nitroaniline hydrochlo-
ride C19H26N8O6.HCl White to light powder. Freely solu-
Purified sulfuric acid See sulfuric acid, purified.
ble in water, in methanol and in acetic acid (100).
Purified water [Use the water prescribed by the mono- Absorbance <2.24> E 11zcm (316 nm): 242 – 268 (2 mg, water,
graphs of Purified Water or Purified Water in Containers. It 100 mL).
is not necessary to check the conformity to all the specifica- Optical rotation <2.49> [ a ]25
D : －51 – －569[0.1 g, diluted
tion items of the monograph, if it is confirmed that the water acetic acid (100) (1 in 2), 10 mL, 100 mm].
to be used is suitable for the purpose of the relevant test.] Purity Related substances—Dissolve 50 mg of L-
pyroglutamylglycyl-L-arginine-p-nitroaniline hydrochloride
Pyrazole C3H4N2 White to pale yellow, crystals or crys-
in 10 mL of methanol, and use this solution as the sample so-
talline powder.
lution. Pipet 1 mL of the sample solution, add methanol to
Melting point <2.60>: 67 – 719C
make exactly 50 mL, and use this solution as the standard
Pyridine C5H 5N [K 8777, Special class] solution. Perform the test with these solutions as directed
under Thin-layer Chromatography <2.03>. Spot 20 mL each
Pyridine-acetic acid TS Dilute 20 mL of pyridine with
of the sample solution and standard solution on a plate of
sufficient diluted acetic acid (100) (1 in 25) to make 100 mL.
silica gel with fluorescent indicator for thin-layer chromatog-
Prepare before use.
raphy. Develop the plate with a mixture of 1-butanol, water,
Pyridine, dehydrated C5H5N To 100 mL of pyridine pyridine and acetic acid (100) (15:12:10:3) to a distance of
add 10 g of sodium hydroxide, and allow to stand for 24 about 10 cm, and air-dry the plate. Examine under ultravio-
hours. Decant the supernatant liquid, and distill. let light (main wavelength: 254 nm): the spots other than the
principal spot from the sample solution are not more intense
Pyridine for water determination See Water Determina-
than the spot from the standard solution.
tion <2.48>.
L-Pyroglutamylglycyl-L-arginine-p-nitroaniline hydrochlo-
0.2 mol/L Pyridine-formic acid buffer solution (pH 3.0)
ride TS Dissolve 25 mg of L-pyroglutamylglycyl-L-arginine-
To 15.82 g of pyridine add 900 mL of water, mix well, adjust
p-nitroaniline hydrochloride and 40 mg of D-mannitol in 2 to
the pH to 3.0 with diluted formic acid (1 in 2), and add water
3 mL of water, lyophilize, and add 16.7 mL of water to dis-
to make 1000 mL.
solve. To 1 volume of this solution add 9 volumes of water
Pyridine-pyrazolone TS Dissolve, with thorough shak- before use.
ing, 0.1 g of 3-methyl-1-phenyl-5-pyrazolone in 100 mL of
Pyrole C4H5N Clear, colorless liquid, having a charac-
water by heating between 659 C and 709C, and cool below
teristic oder. Soluble in ethanol (95) and in diethyl ether, and
309C. Mix this solution with a solution prepared by dissolv-
practically insoluble in water.
ing 20 mg of bis-(1-phenyl-3-methyl-5-pyrazolone) in 20 mL
Specific gravity <2.56> d 20
20: 0.965 – 0.975
of pyridine. Prepare before use.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
316 General Tests / 9.41 Reagents, Test Solutions JP XVII
Pyrophosphate buffer solution (pH 9.0) Dissolve 3.3 g Quinoline C9H 7N [K 8279, Special class]
of potassium pyrophosphate, 15 mg of dithiothreitol and 40
Quinoline TS Mix 50 mL of quinoline with 360 mL of
mg of disodium dihydrogen ethylenediamine tetraacetate di-
diluted hydrochloric acid (1 in 6), previously heated, cool,
hydrate in 70 mL of water, adjust the pH with a solution of
and filter if necessary.
citric acid monohydrate (21 in 100) to exactly 9.0, and add
water to make 100 mL. 8-Quinolinol C9H7NO [K 8775, Special class]
2-Pyrrolidone C4H7NO Clear, colorless to pale yellow Rabbit anti-nartograstim antibody Dissolve the antibody
liquid, or white to pale yellow, masses or powder. Odorless. obtained from rabbit antiserum, prepared by immunizing
Congealing point <2.42>: 22 – 269C with Nartograstim (Genetical Recombination), in tris-acetic
Purity Weigh accurately about 1 g of 2-pyrrolidone, dis- acid buffer solution (pH 8.0) so that each mL contains 1 mg
solve in methanol to make exactly 10 mL, and use this solu- of rabbit anti-nartograstim antibody. Storage at －809C.
tion as the sample solution. Perform the test with 1 mL of the Performance test: When perform the test by Ouchterlony
sample solution as directed under Gas Chromatography method, a precipitation line is appeared against Narto-
<2.02> according to the following conditions. Determine each grastim (Genetical Recombination).
peak area by the automatic integration method, and calcu- Protein concentration: Determine the absorbance at 280
late the amount of 2-pyrrolidone by the area percentage nm as directed under Ultraviolet-visible Spectrophotometry
method: not less than 98.0z. <2.24>, and calculate the protein concentration using the spe-
Operating conditions cific absorbance E 11zcm 15.
Detector: Hydrogen flame-ionization detector.
Rabbit anti-nartograstim antibody TS To rabbit anti-
Column: A capillary glass column 0.53 mm in inside diam-
nartograstim antibody add bovine serum albumin TS for
eter and 30 m in length, coated with a 1.0-mm layer of poly-
nartograstim test so that each mL contains 0.2 mg of rabbit
ethylene glycol 20 M for gas chromatography on the inner
anti-nartograstim antibody. Prepare before use.
surface.
Column temperature: Maintain the temperature at 809C Raney nickel catalyst Grayish black powder. An alloy
for 1 minute, then raise at the rate of 109 C per minute to containing 40 to 50z of nickel and 50 to 60z of aluminum.
1909C, and maintain at this temperature for 20 minutes.
Ranitidinediamine (C10H18N2OS)2.C4H4O4 White to
Temperature of sample vaporization chamber: A constant
pale yellow crystalline powder.
temperature of about 2009 C.
Identification—Determine the infrared absorption spec-
Carrier gas: Helium.
trum of ranitidinediamine as directed in the paste method
Flow rate: Adjust so that the retention time of 2-pyrroli-
under Infrared Spectrophotometry <2.25>: it exhibits absorp-
done is about 10 minutes.
tion at the wave numbers of about 2780 cm－1, 1637 cm－1,
Split ratio: 1:20.
1015 cm－1 and 788 cm－1.
Time span of measurement: About 2 times as long as the
Content: not less than 95z. Assay—Weigh accurately
retention time of 2-pyrrolidone.
about 0.1 g of ranitidinediamine, dissolve in 50 mL of acetic
Water <2.48> Not more than 0.2z (5 g, volumetric titra-
acid (100), and titrate <2.50> with 0.1 mol/L perchloric acid
tion, direct titration).
VS until the color of the solution changes from purple to
0.05 mol/L Pyrophosphate buffer solution (pH 9.0) Dis- green through blue (indicator: crystal violet TS). Perform
solve 0.83 g of potassium pyrophosphate in 40 mL of water, the blank determination in the same manner, and make any
adjust the pH with 1 mol/L hydrochloric acid TS to 9.0, and necessary correction.
add water to make 50 mL. Adjust the temperature to 22 ±
Each mL of 0.1 mol/L perchloric acid VS
29C before use.
＝ 13.62 mg of (C10H18N2OS)2.C4H4O4
Quinapril hydrochloride for assay C25H30N2O5.HCl
Raponticin for thin-layer chromatography C21H24O9 A
[Same as the monograph Quinapril Hydrochloride. When
white to pale yellow-brown crystalline powder, having no
perform the purity test (2) of Quinapril Hydrochloride, the
odor. Slightly soluble in methanol, and practically insoluble
area of the peaks, having the relative retention time of about
in water and in ethanol (99.5).
0.5 and about 2.0 to quinapril obtained from the sample so-
Identification—Determine the infrared absorption spec-
lution, is not larger than the peak area of quinapril obtained
trum of raponticin for thin-layer chromatography as di-
from the standard solution, the area of peak other than
rected in the potassium bromide disk method under Infrared
quinapril and the peak mentioned above from the sample so-
Spectrophotometry <2.25>: it exhibits absorption at the wave
lution is not larger than 2/5 times the peak area of quinapril
numbers of about 1612 cm－1, 1577 cm－1, 1513 cm－1, 948
from the standard solution, and the total area of the peaks
cm－1, 831 cm－1 and 798 cm－1.
other than quinapril from the sample solution is not larger
Purity Related substances—Dissolve 4 mg of raponticin
than 2 times the peak area of quinapril from the standard so-
for thin-layer chromatography in 2 mL of methanol, and use
lution.]
this solution as the sample solution. Pipet 1 mL of the sam-
Quinhydrone C6H4(OH)2.C6H4O2 Green, crystals or ple solution, add methanol to make exactly 50 mL, and use
crystalline powder. this solution as the standard solution. Perform the test with
Melting point <2.60>: 169 – 1729
C 10 mL each of the sample solution and standard solution as
directed in the Purity (3) under Rhubarb: the spot other than
Quinidine sulfate See quinidine sulfate hydrate.
the principal spot that appears at an Rf value of about 0.3
Quinidine sulfate hydrate (C20H24N2O2)2.H2SO4.2H2O obtained with the sample solution is not more intense than
[Same as the namesake monograph] the spot obtained with the standard solution.
Quinine sulfate See quinine sulfate hydrate. Rebamipide for assay C19H15ClN2O4 [Same as the
monograph Rebamipide. When dried, it contains not less
Quinine sulfate hydrate (C20H24N2O2)2.H2SO4.2H2O
than 99.5z of rebamipide (C19H15ClN2O4).]
[Same as the namesake monograph]

Reduced iron See iron powder. rected under Liquid Chromatography <2.01> according to
the following conditions. Determine each peak area by the
Reduction buffer solution for nartograstim sample Mix
automatic integration method, and calculate the amount of
0.8 mL of sodium lauryl sulfate solution (1 in 10), 0.5 mL of
resibufogenin by the area percentage method.
0.5 mol/L tris buffer solution (pH 6.8), 0.4 mL of glycerin,
Operating conditions
0.3 mL of 2-mercaptoethanol and 0.1 mL of bromophenol
Detector: An ultraviolet absorption photometer (wave-
blue solution (1 in 200). Prepare before use.
length: 300 nm).
Reduction liquid for molecular mass determination Dis- Column: A stainless steel column about 4 to 6 mm in in-
solve 10.6 g of sodium lauryl sulfate and 3.9 g of 2-amino-2- side diameter and 15 to 30 cm in length, packed with oc-
hydroxymethyl-1,3-propanediol in 60 mL of water, adjust to tadecylsilanized silica gel for liquid chromatography (5 to 10
pH 6.8 with hydrochloric acid, dissolve 31 g of sucrose, and mm in particle diameter).
add water to make 100 mL. To 97 mL of this solution add 3 Column temperature: A constant temperature of about
mL of bromophenol blue solution (11 in 2500). To 0.4 mL of 409C.
this solution add 0.1 mL of 2-mercaptoethanol. Prepare be- Mobile phase: A mixture of water and acetonitrile (1:1).
fore use. Flow rate: Adjust so that the retention time of resibufoge-
nin is about 9 minutes.
Reference anti-interleukin-2 antibody for teceleukin
Selection of column: Dissolve 10 mg each of resibufogenin
Monoclonal antibody obtained from a fusion cell strain
for assay, bufalin for assay and cinobufagin for assay in
from mouse spleen cells sensitized to teceleukin and mouse
methanol to make 200 mL. Perform the test with 20 mL of
melanoma cells, or alternately, rabbit antiserum towards hu-
this solution according to the above operating conditions,
man interleukin-2, that is purified using affinity chromatog-
and calculate the resolution. Use a column giving elution of
raphy. When determining the neutralizing activity, taking 1
bufalin, cinobufagin and resibufogenin in this odor, and
neutralizing unit as the titer that neutralizes one unit of ac-
clearly dividing each peak.
tivity of teceleukin, contains at least 2000 neutralizing units
Detection sensitivity: Pipet 1 mL of the sample solution,
per mL.
add methanol to make exactly 100 mL, and use this solution
Reference anti-interleukin-2 antiserum TS Anti-interleu- as the standard solution (1). Pipet 1 mL of the standard solu-
kin-2 antiserum is diluted with culture media for celmoleu- tion (1), add methanol to make exactly 20 mL, and use this
kin, so that the diluted antiserum solution neutralizes the solution as the standard solution (2). Adjust the detection
same volume of about 800 unit/mL solution of Celmoleukin sensitivity so that the peak area of resibufogenin obtained
(Genetical Recombination). from 20 mL of standard solution (2) can be measured by the
automatic integration method and the peak height of
Reference suspension 1 To 5.0 mL of formazin opales-
resibufogenin from 20 mL of the standard solution (1) is
cence standard solution add 95.0 mL of water. Mix and
about 20z of the full scale.
shake before use.
Time span of measurement: About twice as long as the
Reinecke salt See reinecke salt monohydrate. retention time of resibufogenin, beginning after the peak of
solvent.
Reinecke salt monohydrate NH4[Cr(NH3)2(SCN)4].H2O
Dark red, crystals or crystalline powder. Resibufogenin for component determination See resibu-
Identification—Determine the infrared absorption spec- fogenin for assay.
trum of Reinecke salt monohydrate as directed in the potas-
Resibufogenin for thin-layer chromatography C24H32O4
sium bromide disk method as directed under Infrared Spec-
White crystalline powder having no odor. It is freely soluble
trophotometry <2.25>: it exhibits absorption at the wave
in acetone and in methanol.
numbers of about 3310 cm－1, 2130 cm－1, 1633 cm－1, 1400
Purity Related substances—Dissolve 5.0 mg of resibu-
cm－1, 1261 cm－1 and 711 cm－1.
fogenin for thin-layer chromatography in exactly 5 mL of
Reinecke salt TS To 20 mL of water add 0.5 g of acetone. Perform the test with 5 mL of this solution as di-
Reinecke salt monohydrate, shake frequently for 1 hour, rected in the Identification under Toad Cake: no spot other
then filter. Use within 48 hours. than the principal spot with an R f value of about 0.4 appear.
Resazurin C12H6NNaO4 Brownish purple powder. It Resolving gel for celmoleukin Prepare the resolving gel
dissolves in water and the solution is purple in color. in tris buffer solution (pH 8.8) using ammonium persulfate
Residue on ignition <2.44>: not less than 28.5z (1 g). and N, N, N?, N?-tetramethylethylenediamine so the concen-
trations of acrylamide and sodium lauryl sulfate are 13.5z
Resazurin solution Prepared for the test for meas-
and 0.1z, respectively.
urement of living cell.
Resorcin See resorcinol.
Resibufogenin for assay C24H32O4 Odorless white crys-
talline powder. Resorcinol C6H4(OH)2 [K 9032, Special class]
Absorbance <2.24> E 11zcm (300 nm): 131 – 145 (10 mg,
Resorcinol sulfuric acid TS Dissolve 0.1 g of resorcinol
methanol, 250 mL), dried in a desiccator (silica gel) for 24
in 10 mL of diluted sulfuric acid (1 in 10).
hours.
Purity Related substances—Weigh accurately 40 mg of Resorcinol TS Dissolve 0.1 g of resorcinol in 10 mL of
resibufogenin for assay and proceed as directed in the Purity hydrochloric acid. Prepare before use.
under bufalin for assay.
Resorcinol-copper (II) sulfate TS Dissolve 0.1 g of resor-
Content: not less than 98.0z. Assay—Weigh accurately
cinol in 5 mL of water, add 125 mL of 0.1 mol/L copper (II)
about 10 mg of resibufogenin for assay, previously dried in a
sulfate solution, 24 mL of hydrochloric acid, and add water
desiccator (silica gel) for 24 hours, add methanol to make ex-
to make 50 mL. Prepare this TS at least 4 hours before the
actly 10 mL, and use this solution as the sample solution.
time of use.
Perform the test with 20 mL of the sample solution as di-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
318 General Tests / 9.41 Reagents, Test Solutions JP XVII
Resorcin sulfuric acid TS See resorcinol sulfuric acid TS. N: Number of the hydrogen derived from A
P: Purity (z) of DSS-d6 for nuclear magnetic resonance
Resorcin TS See resorcinol TS.
spectroscopy
L-Rhamnose monohydrate C6H12O.H2O White crystal-
Operating conditions
line powder having sweet taste. Freely soluble in water, and
Apparatus: An apparatus of nuclear magnetic resonance
sparingly soluble in ethanol (95).
spectrum measurement having 1H resonance frequency of
Optical rotation <2.49> [a]20
D : ＋7.8 – ＋8.39(1 g, 20 mL of
not less than 400 MHz.
water, 2 drops of ammonia TS, 100 mm).
Target nucleus: 1H.
Melting point <2.60>: 87 – 919C
Digital resolution: 0.25 or lower.
Purity Related substances—Dissolve 1.0 mg of L-rham-
Measuring spectrum range: 20 ppm or upper, including
nose monohydrate in 1 mL of water, and add methanol to
between － 5 ppm and 15 ppm.
make exactly 10 mL. Proceed with 20 mL of this solution as
Spinning: off.
directed in the Identification under Acacia: any spot other
Pulse angle: 909 .
than the principal spot at the R f value of about 0.5 does not 13C decoupling: on.
appear.
Delay time: Repeating pulse waiting time is not less than
Rhein for assay C15H8O6 Use rhein for thin-layer chro- 60 seconds.
matography meeting the following additional specifications. Integrating times: 8 or more times.
Its content is corrected based on the amount (z) of rhein Dummy scanning: 2 or more times.
obtained in the Assay. Measuring temperature: A constant temperature between
Absorbance <2.24> E 11zcm (257 nm): 678 – 720 (3 mg, meth- 209C and 309 C.
anol, 500 mL). System suitability
Unity of peak—Dissolve 1 mg of rhein for assay in 100 Test for required detectability: When the procedure is run
mL of acetone, and use this solution as the sample solution. with the sample solution under the above operating condi-
Perform the test with 10 mL of the sample solution as ditions, the S/N of the signal of around d 8.16 ppm is not less
rected under Liquid Chromatography <2.01> according to than 100.
the following conditions, and compare the absorption spec- System performance: When the procedure is run with the
tra of at least 3 points including the top of rhein peak and sample solution under the above operating conditions, the
around the two middle peak heights of before and after the signal of around d 8.16 ppm is not overlapped with any sig-
top: no difference in form is observed between their spectra. nal of obvious foreign substance.
Operating conditions System repeatability: When the test is repeated 6 times
Detector: An ultraviolet absorption photometer (wave- with the sample solution under the above operating condi-
length: 257 nm). tions, the relative standard deviation of the resonance inten-
Column: A stainless steel column 4.6 mm in inside diame- sity, A, to that of the internal reference is not more than
ter and 15 cm in length, packed with octadecylsilanized silica 1.0z.
gel for liquid chromatography (5 mm in particle diameter).
Rhein for thin-layer chromatography C15H8O6 A yel-
Column temperature: A constant temperature of about
low to reddish yellow powder. Very slightly soluble in ace-
509 C.
tone, and practically insoluble in water, in methanol, and in
Mobile phase: A mixture of water, acetonitrile, and phos-
ethanol (99.5).
phoric acid (650:350:1).
Identification—Determine the absorption spectrum of a
Flow rate: Adjust so that the retention time of rhein is
solution in methanol (3 in 500,000) as directed under Ultravi-
about 14 minute.
olet-visible Spectrophotometry <2.24>: it exhibits maxima be-
System suitability
tween 228 nm and 232 nm, between 255 nm and 259 nm, and
System performance: Proceed as directed in the system
between 429 nm and 433 nm.
suitability in the Assay (5) under Otsujito Extract.
Purity Related substances—Dissolve 1 mg of rhein for
Assay—Weigh accurately 5 mg of rhein for assay and 1
thin-layer chromatography in 10 mL of acetone, and per-
mg of DSS-d6 for nuclear magnetic resonance spectroscopy
form the test with 2 mL of this solution as directed in the
using an ultramicrobalance, dissolve in 1 mL of deuterated
Identification (1) under Daiokanzoto Extract: no spot other
dimethyl sulfoxide for nuclear magnetic resonance spectros-
than the principal spot (Rf value is about 0.3) appears.
copy, and use this solution as the sample solution. Transfer
the sample solution into an NMR tube 5 mm in outer diame- Rhyncophylline for assay C22H28N2O4 Rhyncophylline
ter, measure 1H-NMR as directed under Nuclear Magnetic for thin-layer chromatography. It meets the following re-
Resonance Spectroscopy (<2.21> and <5.01>) according to the quirements.
following conditions, using DSS-d6 for nuclear magnetic Absorbance <2.24>: E 11zcm (245 nm): 473 – 502 (5 mg of the
resonance spectroscopy as the internal reference compound. dried substance in a desiccator (silica gel) for 24 hours, a
Calculate the resonance intensity, A (equivalent to 1 hydro- mixture of methanol and dilute acetic acid (7:3), 500 mL).
gen), of the signal around d 8.16 ppm assuming the signal of Purity Related substances—Dissolve 5 mg of rhyn-
the internal reference compound as d 0 ppm. cophylline for assay in 100 mL of a mixture of methanol and
dilute acetic acid (7:3), and use this solution as the sample
Amount (z) of rhein (C15H8O6)
solution. Pipet 1 mL of the sample solution, add a mixture
＝ MS × I × P/(M × N ) × 1.2668
of methanol and dilute acetic acid (7:3) to make exactly 100
M: Amount (mg) of rhein for assay taken mL, and use this solution as the standard solution. Perform
MS: Amount (mg) of DSS-d6 for nuclear magnetic reso- the test with exactly 20 mL each of the sample solution and
nance spectroscopy taken standard solution as directed under Liquid-chromatography
I: Signal resonance intensity based on the signal resonance <2.01> according to the following conditions. Determine the
intensity of DSS-d6 for nuclear magnetic resonance peak area of each solution by the automatic integration
spectroscopy as 9.000 method: the total area of the peaks other than rhyncophyl-

line obtained from the sample solution is not larger than the the following 1) Rosmarinic acid for assay 1 or 2) Rosmarin-
peak area of rhyncophylline obtained from the standard so- ic acid for assay 2 (Purity value by quantitative NMR). The
lution. former is used after drying in a desiccator (silica gel) for 24
Operating conditions hours, and the latter is used with correction for its amount
Detector, column, column temperature, mobile phase, and based on the result obtained in the Assay.
flow rate: Proceed as directed in the operating conditions in 1) Rosmarinic acid for assay 1
the Assay under Uncaria Hook. Absorbance <2.24> E 11zcm (325 nm): 502 – 534 (5 mg,
Time span of measurement: About 4 times as long as the water, 500 mL).
retention time of rhyncophylline, beginning after the solvent Purity Related substances—Conduct this procedure
peak. without exposure to light, using light-resistant vessels. Dis-
System suitability solve 5 mg of rosmarinic acid for assay 1 in 20 mL of the
System performance and system repeatability: Proceed as mobile phase, and use this solution as the sample solution.
directed in the system suitability in the Assay under Uncaria Pipet 1 mL of the sample solution, add methanol to make
Hook. exactly 50 mL, and use this solution as the standard solution.
Test for required detectability: Pipet 1 mL of the standard Perform the test with exactly 10 mL each of the sample solu-
solution, add a mixture of methanol and dilute acetic acid tion and standard solution as directed under Liquid Chroma-
(7:3) to make exactly 20 mL. Confirm that the peak area of tography <2.01> according to the following conditions, and
rhyncophylline obtained with 20 mL of this solution is determine each peak area by the automatic integration
equivalent to 3.5 to 6.5z of that obtained with 20 mL of the method: the total area of the peaks other than rosmarinic
standard solution. acid from the sample solution is not larger than the peak
area of rosmarinic acid from the standard solution.
Rhynchophylline for component determination See
Operating conditions
rhynchophylline for assay.
Detector: An ultraviolet absorption photometer (wave-
Rhyncophylline for thin-layer chromatography length: 240 nm).
C22H28N2O4 White, crystals or crystalline powder. Slightly Column: A stainless steel column 4.6 mm in inside diame-
soluble in ethanol (99.5) and in acetone, and practically inter and 15 cm in length, packed with octadecylsilanized silica
soluble in water. Melting point: 205 – 2099 C. gel for liquid chromatography (5 mm in particle diameter).
Identification—Determine the absorption spectrum of a Column temperature: A constant temperature of about
solution of rhyncophylline for thin-layer chromatography in 409C.
a mixture of methanol and dilute acetic acid (7:3) (1 in Mobile phase: A mixture of water, acetonitrile and phos-
100,000) as directed under Ultraviolet-visible Spectropho- phoric acid (800:200:1).
tometry <2.24>: it exhibits a maximum between 242 nm and Flow rate: Adjust so that the retention time of rosmarinic
246 nm. acid is about 14 minutes.
Purity Related substances—Dissolve 1.0 mg of rhyn- Time span of measurement: About 4 times as long as the
cophylline for thin-layer chromatography in 1 mL of ace- retention time of rosmarinic acid.
tone, and use this solution as the sample solution. Perform System suitability
the test with this solution as directed under Thin-layer Chro- Test for required detectability: Pipet 1 mL of the standard
matography <2.03>. Spot 10 mL of the sample solution on a solution, and add methanol to make exactly 20 mL. Confirm
plate of silica gel with fluorescent indicator for thin-layer that the peak area of rosmarinic acid obtained with 10 mL of
chromatography. Develop the plate with a mixture of 1- this solution is equivalent to 3.5 to 6.5z of that obtained
butanol, water and acetic acid (100) (7:2:1) to a distance of with 10 mL of the standard solution.
about 10 cm, and air-dry the plate. Examine under ultravio- System performance: When the procedure is run with 10
let light (main wavelength: 254 nm): no spot other than the mL of the standard solution under the above operating con-
principal spot at around R f value of 0.5 appears. ditions, the number of theoretical plates and the symmetry
factor of the peak of rosmarinic acid are not less than 5000
Ribavirin C8H12N4O5 [Same as the namesake mono-
and not more than 1.5, respectively.
graph]
System repeatability: When the test is repeated 6 times
Riboflavin C17H20N4O6 [Same as the namesake mono- with 10 mL of the standard solution under the above operat-
graph] ing conditions, the relative standard deviation of the peak
area of rosmarinic acid is not more than 1.5z.
Riboflavin sodium phosphate C17H20N4NaO9P [Same
2) Rosmarinic acid for assay 2 (Purity value by quantita-
as the namesake monograph]
tive NMR)
Ribonuclease A for gel filtration molecular mass marker Unity of peak—Conduct this procedure without exposure
Obtained from bovine pancreas, for gel filtration chroma- to light, using light-resistant vessels. Dissolve 1 mg of ros-
tography. marinic acid for assay 2 in 50 mL of ethanol, and use this so-
lution as the sample solution. Perform the test with 10 mL of
Risperidone for assay C23H27FN4O2 [Same as the
the sample solution as directed under Liquid Chromatogra-
monograph Risperidone. It contains not less than 99.5z of
phy <2.01> according to the following conditions, and com-
risperidone (C23H27FN4O2), calculated on the dried basis.]
pare the absorption spectra of at least 3 points including the
Ritodrine hydrochloride C17H21NO3.HCl [Same as the top of rosmarinic acid peak and around the two middle peak
namesake monograph] heights of before and after the top: no difference in form is
observed between their spectra.
Rose Bengal C20H2Cl4I4Na2O5 [Special class] A red-
Operating conditions
brown powder, and shows a purple red when dissolved in
Detector: A photodiode array detector (wavelength: 330
water.
nm, measuring range of spectrum: 220 – 400 nm).
Rosmarinic acid for assay C18H16O8 Rosmarinic acid Column: A stainless steel column 4.6 mm in inside diame-
for thin-layer chromatography. It meets the requirement of ter and 15 cm in length, packed with octadecylsilanized silica

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
320 General Tests / 9.41 Reagents, Test Solutions JP XVII
gel for liquid chromatography (5 mm in particle diameter). with the sample solution under the above operating condi-
Column temperature: A constant temperature of about tions, the relative standard deviation of the ratio of the reso-
409 C. nance intensity A to that of the internal reference is not more
Mobile phase: A mixture of diluted acetic acid (1 in 100) than 1.0z.
and methanol (13:7).
Rosmarinic acid for component determination See ros-
Flow rate: Adjust so that the retention time of rosmarinic
maric acid for assay.
acid is about 10 minutes.
System suitability Rosmarinic acid for thin-layer chromatography
System performance: When the procedure is run with 10 C18H16O8 White to pale yellow, crystals or crystalline pow-
mL of the sample solution, previously irradiated ultraviolet der. Freely soluble in ethanol (99.5), and slightly soluble in
light (main wavelength: 365 nm) for 30 minutes, under the water. Melting point: about 1709C (with decomposition).
above operating conditions, an obvious peak is observed just Identification—Determine the absorption spectrum of a
before the peak of rosmarinic acid, and the resolution be- solution of rosmarinic acid for thin-layer chromatography (1
tween these peaks is not less than 1.5. in 100,000) as directed under Ultraviolet-visible Spectropho-
Assay—Weigh accurately 5 mg of rosmarinic acid for tometry <2.24>: it exhibits maxima between 215 nm and 219
assay 2 and 1 mg of DSS-d6 for nuclear magnetic resonance nm and between 322 nm and 326 nm.
spectroscopy using an ultramicrobalance, dissolve in 1 mL of Purity Related substances—Conduct this procedure
deuterated dimethylsulfoxide for nuclear magnetic resonance using light-resistance vessels. Dissolve 10 mg of rosmarinic
spectroscopy, and use this solution as the sample solution. acid for thin-layer chromatography in 2 mL of ethanol
Transfer the sample solution into an NMR tube 5 mm in (99.5), and use this solution as the sample solution. Pipet 1
outer diameter, measure 1H-NMR as directed under Nuclear mL of the sample solution, add ethanol (99.5) to make ex-
Magnetic Resonance Spectroscopy <2.21> and Crude Drugs actly 50 mL, and use this solution as the standard solution.
Test <5.01> according to the following conditions, using Proceed with 10 mL each of the sample solution and standard
DSS-d6 for nuclear magnetic resonance spectroscopy as the solution as directed in the Identification (2) under Han-
internal reference compound. Calculate the resonance inten- gekobokuto Extract: the spot other than the principal spot
sity A (equivalent to 1 hydrogen) of the signal around d 6.27 of around R f value of 0.5 from the sample solution is not
ppm assuming the signal of the internal reference compound more intense than the spot from the standard solution.
as d 0 ppm.
Roxatidine acetate hydrochloride C19H28N2O4.HCl
Amount (z) of rosmarinic acid (C18H16O8) [Same as the namesake monograph]
＝ MS × I × P/(M × N ) × 1.6059
RPMI-1640 powdered medium Powdered medium for
M: Amount (mg) of rosmarinic acid for assay 2 taken cell culture containing 6 g of sodium chloride, 400 mg of po-
MS: Amount (mg) of DSS-d6 for nuclear magnetic reso- tassium chloride, 800 mg of anhydrous sodium dihydrogen
nance spectroscopy taken phosphate, 100 mg of anhydrous calcium nitrate, 49 mg of
I: Signal resonance intensity A based on the signal reso- anhydrous magnesium sulfate, 2 g of dextrose, 200 mg of L-
nance intensity of DSS-d6 for nuclear magnetic reso- arginine, 1 mg of glutathione, 50 mg of L-isoleucine, 15 mg
nance spectroscopy as 9.000 of L-phenylalanine, 5 mg of L-tryptophan, 0.2 mg of biotin,
N: Number of the hydrogen derived from A 1 mg of nicotinamide, 1 mg thiamine hydrochloride, 300 mg
P: Purity (z) of DSS-d6 for nuclear magnetic resonance of L-glutamine, 56.8 mg of L-asparagine, 10 mg of glycine,
spectroscopy 50 mg of L-leucine, 20 mg of L-proline, 20 mg of L-tyrosine,
0.25 mg of D-calcium pantothenate, 5 mg of cyanocobala-
Operating conditions
min, 1 mg of aminobenzoic acid, 20 mg of L-aspartic acid,
Apparatus: A nuclear magnetic resonance spectrometer
15 mg of L-histidine, 40 mg of L-lysine hydrochloride, 30 mg
having 1H resonance frequency of not less than 400 MHz.
of L-serine, 20 mg of L-valine, 1 mg of folic acid, 1 mg of
Target nucleus: 1H.
pyridoxine hydrochloride, 20 mg of L-glutamic acid, 20 mg
Digital resolution: 0.25 or lower.
of L-hydroxyproline, 15 mg of L-methionine, 20 mg of
Measuring spectrum range: 20 ppm or upper, including
L-threonine, 3 mg of choline chloride, 35 mg of i-inositol,
between －5 ppm and 15 ppm.
0.2 mg of riboflavin, 59 mg of L-cystine, and 5 mg of phenol
Spinning: off.
red per 1 L.
Pulse angle: 909.
13C decoupling: on. Rubidium chloride RbCl White, crystals or crystalline
Delay time: Repeating pulse waiting time not less than 60 powder.
seconds. Content: Not less than 99.0z. Assay—Weigh accurately
Integrating times: 8 or more times. about 0.2 g of rubidium chloride, dissolve in 100 mL of
Dummy scanning: 2 or more times. water, add 5 mL of diluted nitric acid (1 in 2), and titrate
Measuring temperature: A constant temperature between <2.50> with 0.1 mol/L silver nitrate VS (potentiometric titra-
209 C and 309C. tion).
System suitability
Each mL of 0.1 mol/L silver nitrate VS
Test for required detectability: When the procedure is run
＝ 12.09 mg of RbCl
with the sample solution under the above operating condi-
tions, the S/N of the signal of around d 6.27 ppm is not less Rutin for thin-layer chromatography C27H30O16 Pale
than 100. yellow to yellow-green, crystals or crystalline powder, having
System performance: When the procedure is run with the no odor. Soluble in methanol, slightly soluble in ethanol
sample solution under the above operating conditions, the (99.5), and practically insoluble in water.
signal of around d 6.27 ppm is not overlapped with any obvi- Identification (1) Determine the absorption spectrum
ous signal of foreign substance. of a solution of rutin for thin-layer chromatography in
System repeatability: When the test is repeated 6 times methanol (1 in 100,000) as directed under Ultraviolet-visible

Spectrophotometry <2.24>: it exhibits maxima between 255 lution between these peaks being not less than 1.5.
nm and 259 nm, and between 356 nm and 360 nm. System repeatability: When the test is repeated 6 times
(2) Determine the infrared absorption spectrum of rutin with 20 mL of the standard solution under the above operat-
for thin-layer chromatography as directed in the potassium ing conditions, the relative standard deviation of the peak
bromide disk method under Infrared Spectrophotometry area of saikosaponin a is not more than 1.0z.
<2.25>: it exhibits absorption at the wave numbers of about
Saikosaponin a for component determination See sai-
1655 cm－1, 1600 cm－1, 1507 cm－1 and 1363 cm－1.
kosaponin a for assay.
Purity Related substances—Dissolve 10 mg of rutin for
thin-layer chromatography in 2 mL of methanol, and use Saikosaponin a for thin-layer chromatography A white,
this solution as the sample solution. Pipet 1 mL of the sam- crystalline powder or powder. Freely soluble in methanol
ple solution, add methanol to make exactly 20 mL, and use and in ethanol (99.5), and practically insoluble in water.
this solution as the standard solution. Perform the test with Melting point: 225 – 2329C (with decomposition).
2 mL each of the sample solution and standard solution as di- Absorbance <2.24> E 11zcm (206 nm): 60–68 (15 mg, metha-
rected in Identification (1) under Crataegus Fruit: the spot nol, 200 mL). Previously dried in a desiccator (in vacuum,
other than the principal spot appeared at an R f value of silica gel) for 24 hours.
about 0.3 obtained with the sample solution is not more in- Purity Related substances—Dissolve 1.0 mg of sai-
tense than the spot obtained with the standard solution. kosaponin a for thin-layer chromatography in exactly 1 mL
of methanol, and perform the test with 10 mL of this solution
Saccharated pepsin [Same as the namesake monograph]
as directed in the Identification (2) under Bupleurum Root:
Saikosaponin a for assay Use saikosaponin a for thin- any spot other than the principal spot at the Rf value of
layer chromatography meeting the following additional spe- about 0.4 does not appear.
cifications.
Saikosaponin b2 for assay C42H68O13 Saikosaponin b2
Purity Related substances—
for thin-layer chromatography. It meets the requirement of
(1) Dissolve 2.0 mg of saikosaponin a for assay in 2 mL
the following 1) Saikosaponin b2 for assay 1 or 2) Saikosapo-
of methanol, and use this solution as the sample solution.
nin b2 for assay 2 (Purity value by quantitative NMR). The
Pipet 1 mL of the sample solution, add methanol to make
former is used after drying in a desiccator (silica gel) for 24
exactly 100 mL, and use this solution as the standard solu-
hours, and the latter is used with correction for its amount
tion. Perform the test with exactly 10 mL each of the sample
based on the result obtained in the Assay.
solution and standard solution as directed in the Purity (2)
1) Saikosaponin b2 for assay 1
under Bupleurum Root: the spot other than the principal
Absorbance <2.24> E 11zcm (252 nm): 352 – 424 [5 mg dried
spot around R f value of 0.4 obtained with the sample solu-
in a desiccator (in vacuum, silica gel) for 24 hours, metha-
tion is not larger and not more intense than the spot ob-
nol, 250 mL].
tained with the standard solution.
Purity Related substances—Dissolve 5 mg of saikosapo-
(2) Dissolve 10 mg of saikosaponin a for assay in 20 mL
nin b2 for assay in 5 mL of the mobile phase, and use this so-
of methanol, and use this solution as the sample solution.
lution as the sample solution. Pipet 1 mL of the sample solu-
Pipet 1 mL of the sample solution, add methanol to make
tion, add the mobile phase to make exactly 50 mL, and use
exactly 100 mL, and use this solution as the standard solu-
this solution as the standard solution. Perform the test with
tion. Perform the test with exactly 20 mL each of the sample
exactly 10 mL each of the sample solution and standard solu-
solution and standard solution as directed under Liquid
tion as directed under Liquid Chromatography <2.01> ac-
Chromatography <2.01> according to the following condi-
cording to the following conditions, and determine each
tions, and determine each peak area by the automatic in-
peak area by the automatic integration method: the total
tegration method: the total area of the peaks other than sai-
area of the peaks other than saikosaponin b2 obtained from
kosaponin a obtained with the sample solution is not more
the sample solution is not larger than the peak area of sai-
than the peak area of saikosaponin a obtained with the
kosaponin b2 obtained from the standard solution.
standard solution.
Operating conditions
Operating conditions
Detector, column, column temperature, mobile phase, and
Detector, and column: Proceed as directed in the operat-
flow rate: Proceed as directed in the operating conditions in
ing conditions in the Assay under Bupleurum Root.
the Assay (1) under Saireito Extract.
Column temperature: A constant temperature of about
Time span of measurement: About 6 times as long as the
409 C.
retention time of saikosaponin b2, beginning after the sol-
Mobile phase: A mixture of water and acetonitrile (13:7).
vent peak.
Flow rate: Adjust so that the retention time of saikosapo-
System suitability
nin a is about 16 minutes.
System performance, and system repeatability: Proceed as
Time span of measurement: About 6 times as long as the
directed in the system suitability in the Assay (1) under
retention time of saikosaponin a, beginning after the solvent
Saireito Extract.
peak.
Test for required detectability: To exactly 1 mL of the
System suitability
standard solution add the mobile phase to make exactly 20
Test for required detectability: Measure exactly 1 mL of
mL. Confirm that the peak area of saikosaponin b2 obtained
the standard solution, and add methanol to make exactly 20
from 10 mL of this solution is equivalent to 3.5 to 6.5z of
mL. Confirm that the peak area of saikosaponin a obtained
that obtained from 10 mL of the standard solution.
with 20 mL of this solution is equivalent to 3.5 to 6.5z of
2) Saikosaponin b2 for assay 2 (Purity value by quantita-
that obtained with 20 mL of the standard solution.
tive NMR)
System performance: Dissolve 6 mg each of saikosaponin
Unity of peak—Dissolve 1 mg of saikosaponin b2 for
a for assay and saikosaponin b2 for assay in methanol to
assay 2 in 50 mL of the mobile phase, and use this solution
make 100 mL. When the procedure is run with 20 mL of this
as the sample solution. Perform the test with 10 mL of the
solution under the above operating conditions, saikosaponin
sample solution as directed under Liquid Chromatography
a and saikosaponin b2 are eluted in this order with the reso-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
322 General Tests / 9.41 Reagents, Test Solutions JP XVII
<2.01> according to the following conditions, and compare with the sample solution under the above operating condi-
the absorption spectra of at least 3 points including the top tions, the relative standard deviation of the ratio of the reso-
of saikosaponin b2 peak and around the two middle peak nance intensity, A, to that of the internal reference is not
heights of before and after the top: no difference in form is more than 1.0z.
observed between their spectra.
Saikosaponin b2 for component determination See sai-
Operating conditions
kosaponin b2 for assay.
Column, column temperature, mobile phase, and flow
rate: Proceed as directed in the operating conditions in the Saikosaponin b2 standard TS for assay Prepare as de-
Assay (1) under Saireito Extract. scribed in the following 1), 2)-1 or 2)-2.
Detector: A photodiode array detector (wavelength: 252 1) Weigh accurately about 10 mg of saikosaponin b2 for
nm, measuring range of spectrum: 220 – 400 nm). assay (for assay 1), previously dried in a desiccator (silica
System suitability gel) for more than 24 hours, dissolve in 50 mL of methanol,
System performance: Proceed as directed in the system and add water to make exactly 100 mL. Pipet 10 mL of this
suitability in the Assay (1) under Saireito Extract. solution, add diluted methanol (1 in 2) to make exactly 100
Assay—Weigh accurately 5 mg of saikosaponin b2 for mL, and use this solution as saikosaponin b2 standard TS for
assay 2 and 1 mg of 1,4-BTMSB-d4 for nuclear magnetic assay.
resonance spectroscopy using an ultramicrobalance, dissolve 2)-1 Weigh accurately about 10 mg of saikosaponin b2
in 1 mL of deuterated methanol for nuclear magnetic reso- for assay (for assay 2), and dissolve in methanol to make ex-
nance spectroscopy, and use this solution as the sample solu- actly 250 mL. Pipet 500 mL of this solution, and evaporate
tion. Transfer the sample solution into an NMR tube 5 mm the solvent under reduced pressure. Before using, add ex-
in outer diameter, measure 1H-NMR as directed under actly 2 mL of a mixture of water and methanol (1:1), and use
Nuclear Magnetic Resonance Spectroscopy <2.21> and Crude this solution as the saikosaponin b2 standard TS for assay. It
Drugs Test <5.01> according to the following conditions, contains 10 mg of saikosaponin b2 for assay in 1000 mL of a
using 1,4-BTMSB-d4 for nuclear magnetic resonance spec- mixture of water and methanol (1:1). This standard TS is
troscopy as the internal reference compound. Calculate the corrected by the content obtained in the Assay for sai-
resonance intensity A (equivalent to 1 hydrogen) of the sig- kosaponin b2 for assay (for assay 2).
nal around d 6.20 ppm assuming the signal of the internal 2)-2 Weigh accurately about 10 mg of saikosaponin b2
reference compound as d 0 ppm. for assay (for assay 2), dissolve in 50 mL of methanol, and
add water to make exactly 100 mL. Pipet 10 mL of this solu-
Amount (z) of saikosaponin b2 (C42H68O13)
tion, add a mixture of water and methanol (1:1) to make
＝ MS × I × P/(M × N ) × 3.4480
exactly 100 mL, and use this solution as saikosaponin b2
M: Amount (mg) of saikosaponin b2 for assay 2 taken standard TS for assay. This standard TS is corrected by the
MS: Amount (mg) of 1,4-BTMSB-d4 for nuclear magnetic content obtained in the Assay for saikosaponin b2 for assay
resonance spectroscopy taken (for assay 2).
I: Signal resonance intensity A based on the signal reso-
Saikosaponin b2 for thin-layer chromatography
nance intensity of 1,4-BTMSB-d4 for nuclear magnetic
C42H68O13 White, crystals or crystalline powder. Freely
resonance spectroscopy as 18.000
soluble in ethanol (99.5), soluble in methanol, and practi-
N: Number of the hydrogen derived from A
cally insoluble in water. Melting point: about 2409C
P: Purity (z) of 1,4-BTMSB-d4 for nuclear magnetic
Identification—Determine the absorption spectrum of a
resonance spectroscopy
solution of saikosaponin b2 for thin-layer chromatography
Operating conditions in methanol (1 in 50,000) as directed under Ultraviolet-
Apparatus: A nuclear magnetic resonance spectrometer visible Spectrophotometry <2.24>: it exhibits maxima be-
having 1H resonance frequency of not less than 400 MHz. tween 241 nm and 245 nm, between 250 nm and 254 nm, and
Target nucleus: 1H. between 259 nm and 263 nm.
Digital resolution: 0.25 or lower. Purity Related substances—Dissolve 2 mg of saikosapo-
Measuring spectrum range: 20 ppm or upper, including nin b2 for thin-layer chromatography in 2 mL of methanol,
between －5 ppm and 15 ppm. and use this solution as the sample solution. Pipet 1 mL of
Spinning: off. the sample solution, add methanol to make exactly 50 mL,
Pulse angle: 909 and use this solution as the standard solution. Proceed the
13C Decoupling: on. test with 10 mL each of the sample solution and standard
Delay time: Repeating pulse waiting time not less than 60 solution as directed in the Identification (1) under Saireito
seconds. Extract: the spot other than the principle spot, having R f
Integrating times: 8 or more times. value of about 0.3, obtained from the sample solution is not
Dummy scanning: 2 or more times. more intense than the spot obtained from the standard solu-
Measuring temperature: A constant temperature between tion.
209 C and 309C.
Saikosaponin d for assay C42H68O13 A white, crystal-
System suitability
line powder or powder. Freely soluble in methanol and in
Test for required detectability: When the procedure is run
ethanol (99.5), and practically insoluble in water. Melting
with the sample solution under the above operating condi-
point: about 2409C.
tions, the S/N of the signal of around d 6.20 ppm is not less
Absorbance <2.24> E 11zcm (206 nm): 63 – 71 (15 mg, metha-
than 100.
nol, 200 mL). Previously dried in a desiccator (in vacuum,
System performance: When the procedure is run with the
silica gel) for 24 hours.
sample solution under the above operating conditions, the
Purity Related substances—
signal of around d 6.20 ppm is no overlapped with any obvi-
(1) Dissolve 2.0 mg of saikosaponin d for assay in 2 mL
ous signal of foreign substance.
of methanol, and use this solution as the sample solution.
System repeatability: When the test is repeated 6 times
Pipet 1 mL of this solution, add methanol to make exactly

100 mL, and use this as the standard solution. Proceed the Povidone: any spot other than the principal spot does not
test with 10 mL each of the sample solution and standard so- appear.
lution as directed in the Identification (2) under Bupleurum
Salicylaldehyde HOC6H4CHO [K 8390, Special class]
Root: the spot other than the principal spot around R f value
of 0.4 obtained with the sample solution is not larger and not Salicylamide C7H7NO2 White, crystals or crystalline
more intense than the spot obtained with the standard solu- powder, and it is odorless and tasteless. Very soluble in N, N-
tion. dimethylformamide, freely soluble in ethanol (95), soluble in
(2) Dissolve 10 mg of saikosaponin d for assay in 20 mL propylene glycol, sparingly soluble in diethyl ether, and
of methanol, and use this solution as the sample solution. slightly soluble in water and in chloroform. It dissolves in so-
Pipet 1 mL of the sample solution, add methanol to make dium hydroxide TS.
exactly 100 mL, and use this solution as the standard solu- Melting point <2.60>: 139 – 1439C
tion. Perform the test with exactly 20 mL each of the sample Purity Ammonium <1.02>—Shake 1.0 g of salicylamide
solution and standard solution as directed under Liquid with 40 mL of water, and filter through filter paper previ-
Chromatography <2.01> according to the following condi- ously washed well with water. Discard the first 10 mL of the
tions, and determine each peak area by the automatic in- filtrate, transfer the subsequent 20 mL to a Nessler tube, and
tegration method: the total area of the peaks other than sai- add water to make 30 mL. Perform the test using this solu-
kosaponin d obtained with the sample solution is not more tion as the test solution. Prepare the control solution as
than the peak area of saikosaponin d obtained with the follows: transfer 2.5 mL of Standard Ammonium Solution
standard solution. to a Nessler tube, and add water to make 30 mL.
Operating conditions Loss on drying <2.41>: not more than 0.5z (1 g, silica gel,
Detector, and column: Proceed as directed in the operat- 4 hours).
ing conditions in the Assay under Bupleurum Root. Residue on ignition <2.44>: not more than 0.1z (1 g).
Column temperature: A constant temperature of about Content: not less than 98.5z. Assay—Weigh accurately
409 C. about 0.2 g of salicylamide, previously dried, dissolve in 70
Mobile phase: A mixture of water and acetonitrile (11:9). mL of N, N-dimethylformamide, and titrate <2.50> with 0.1
Flow rate: Adjust so that the retention time of saikosapo- mol/L tetramethylammonium hydroxide VS (potentiometric
nin d is about 13 minutes. titration). Separately, perform a blank determination with a
Time span of measurement: About 4 times as long as the solution of 70 mL of N, N-dimethylformamide in 15 mL of
retention time of saikosaponin d, beginning after the solvent water, and make any necessary correction.
peak.
Each mL of 0.1 mol/L tetramethylammonium hydroxide VS
System suitability
＝ 13.71 mg of C7H7NO2
Test for required detectability: Measure exactly 1 mL of
the standard solution, and add methanol to make exactly 20 Salicylic acid HOC6H4COOH [K 8392, Special class]
mL. Confirm that the peak area of saikosaponin d obtained
Salicylic acid for assay HOC6H4COOH [K 8392, Spe-
with 20 mL of this solution is equivalent to 3.5 to 6.5z of
cial class]
that obtained with 20 mL of the standard solution.
System performance: Dissolve 6 mg each of saikosaponin Salicylic acid TS Dissolve 0.1 g of salicylic acid in 10 mL
d for assay and saikosaponin a for assay in methanol to of sulfuric acid. Prepare before use.
make 100 mL. When the procedure is run with 20 mL of this
Salmon sperm DNA Salmon sperm or nuclear fraction
solution under the above operating conditions, saikosaponin
extracted from salmon sperm, which is sonicated and dried.
a and saikosaponin d are eluted in this order with the resolu-
tion between these peaks being not less than 1.5. Santonin C15H18O3 [Same as the namesake mono-
System repeatability: When the test is repeated 6 times graph]
with 20 mL of the standard solution under the above operat-
Santonin for assay [Same as the monograph Santonin. It
ing conditions, the relative standard deviation of the peak
contains not less than 99.0z of santonin (C15H18O3).]
area of saikosaponin d is not more than 1.0z.
Sarpogrelate hydrochloride C24H31NO6.HCl [Same as
Saikosaponin d for component determination See sai-
the namesake monograph]
kosaponin d for assay.
Sarsasapogenin for thin-layer chromatography C27H44O3
Salicylaldazine C14H12N2O2 Dissolve 0.30 g of hydra-
It is a white or slightly greyish white, crystalline powder or
zinium sulfate in 5 mL of water. To this solution add 1 mL
powder. Slightly soluble in methanol and in ethanol (99.5),
of acetic acid (100) and 2 mL of a freshly prepared solution
and practically insoluble in water.
of salicylaldehyde in 2-propanol (1 in 5), shake well, and
Identification Determine the infrared absorption spec-
allow to stand until a yellow precipitate is produced. Extract
trum of the substance to be examined as directed in the po-
with two 15 mL portions of dichloromethane, to the com-
tassium bromide disk method under Infrared Spectropho-
bined dichloromethane extracts add 5 g of anhydrous so-
tometry <2.25>: it exhibits absorption at the wave numbers of
dium sulfate, shake, decant or filter, and evaporate the
about 2930 cm－1, 1448 cm－1, 1173 cm－1, 985 cm－1 and 850
dichloromethane in the supernatant liquid or filtrate. Dis-
cm－1.
solve the residue in a warmed mixture of toluene and metha-
Purity Dissolve 1 mg of sarsasapogenin for thin-layer
nol (3:2), and cool. Filter the crystals produced, and dry in a
chromatography in 1 mL of methanol, and use this solution
desiccator (in vacuum, silica gel) for 24 hours. It is a yellow,
as the sample solution. Pipet 0.5 mL of the sample solution,
crystalline powder.
add methanol to make exactly 25 mL, and use this solution
Melting point <2.60>: 213 – 2199 C
as the standard solution. Perform the test with these solu-
Purity Related substances—Dissolve 90 mg of salicylal-
tions as directed under Thin-layer Chromatography <2.03>.
dazine in toluene to make exactly 100 mL. Pipet 1 mL of this
Perform the test with 5 mL each of the sample solution and
solution, add toluene to make exactly 100 mL, and perform
standard solution as directed in the Identification (2) under
the test with this solution as directed in the Purity (6) under

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
324 General Tests / 9.41 Reagents, Test Solutions JP XVII
Anemarrhena Rhizome: any spot other than the principal potassium bromide disk method under Infrared Spectropho-
spot with an Rf value of about 0.4 obtained from the sample tometry <2.25>: it exhibits absorption at the wave numbers of
solution is not more intense than the spot obtained from the about 3340 cm－1, 1702 cm－1, 1566 cm－1, 1436 cm－1 and 923
standard solution. cm－1.
Purity Related substances－Dissolve 1.0 mg of scopole-
Saussurea root [Same as the namesake monograph]
tin for thin-layer chromatography in 10 mL of methanol,
Schisandrin for thin-layer chromatography C24H32O7 and use this solution as the sample solution. Pipet 1 mL of
White, crystals or crystalline powder. Freely soluble in meth- the sample solution, add methanol to make exactly 50 mL,
anol and in diethyl ether, and practically insoluble in water. and use this solution as the standard solution. Perform the
Melting point <2.60>: 130 – 1359 C test with 5 mL each of the sample solution and standard solu-
Purity Related substances—Dissolve 1.0 mg of schisan- tion as directed in the Identification under Artemisia Leaf:
drin for thin-layer chromatography in exactly 1 mL of meth- the spot other than the principal spot, having an R f value of
anol. Perform the test with 5 mL of this solution as directed about 0.4, obtained with the sample solution is not more in-
in the Identification under Schisandra Fruit: any spot other tense than the spot obtained with the standard solution.
than the principal spot at the R f value of about 0.4 does not
Sea sand A mixture of white, grey, brown or black
appear.
grains, 0.3 to 1.0 mm in particle size.
Scopolamine hydrobromide See scopolamine hydro-
Secondary antibody TS To a mixture of 1.5 mL of
bromide hydrate.
blocking TS for epoetin alfa and 13.5 mL of sodium azide-
Scopolamine hydrobromide for thin-layer chromato- phosphate-buffered sodium chloride TS, add 1 drop of
graphy See scopolamine hydrobromide hydrate for thin- biotinylated equine anti-mouse IgG antibody.
layer chromatography.
2nd Fluid for disintegration test To 250 mL of 0.2
Scopolamine hydrobromide hydrate mol/L potassium dihydrogen phosphate TS add 118 mL of
C17H21NO4.HBr.3H2O [Same as the namesake monograph] 0.2 mol/L sodium hydroxide TS and water to make 1000
mL. It is clear and colorless, and has a pH about 6.8.
Scopolamine hydrobromide hydrate for thin-layer chro-
matography C17H21NO4.HBr.3H2O [Same as the mono- 2nd Fluid for dissolution test A mixture of phosphate
graph Scopolamine Hydrobromide Hydrate], or scopola- buffer solution (pH 6.8) and water (1:1).
mine hydrobromide hydrate for thin-layer chromatography
Selenious acid H2SeO3 Colorless or white crystals. It is
meeting the following requirements. Colorless or white crys-
hygroscopic.
tals, or white, grains or powder. Freely soluble in water,
Identification—(1) Dissolve 0.2 g of selenious acid in 20
sparingly soluble in ethanol (95) and in acetic acid (100), and
mL of water, and use this solution as the sample solution.
practically insoluble in diethyl ether.
To 10 mL of the sample solution add 2 mL of tin (II) chlo-
Identification Determine the infrared absorption spec-
ride TS: a red precipitate is produced.
trum of Scopolamine Hydrobromide Hydrate as directed in
(2) To 10 mL of the sample solution obtained in (1) add
the potassium bromide disk method under Infrared Spectro-
1 mL of diluted hydrochloric acid (2 in 3) and 1 mL of potas-
photometry <2.25>: it exhibits absorption at the wave num-
sium iodide TS: a brown color is produced.
bers of about 1731 cm－1, 1204 cm－1, 1070 cm－1 and 735
Preserve in a light-resistant tight container.
cm－1.
Purity Dissolve 5 mg of Scopolamine Hydrobromide Selenious acid-sulfuric acid TS Dissolve 50 mg of seleni-
Hydrate in 1 mL of ethanol (95), and use this solution as the ous acid in 10 mL of sulfuric acid.
sample solution. Pipet 0.5 mL of the sample solution, add
Selenium Se [K 8598, Special class]
ethanol (95) to make exactly 25 mL, and use this solution as
the standard solution. Perform the test with these solutions Selenium dioxide SeO2 White, crystals or crystalline
as directed under Thin-layer Chromatography <2.03>. Spot powder.
20 mL each of the sample solution and standard solution on a Identification—(1) To 10 mL of a solution of selenium
plate of silica gel for thin-layer chromatography. Develop dioxide (1 in 100) add 2 mL of tin (II) chloride TS: a red pre-
the plate with a mixture of acetone, water and ammonia so- cipitate is produced.
lution (28) (90:7:3) to a distance of about 10 cm, and dry the (2) To 10 mL of a solution of selenium dioxide (1 in 100)
plate at 809C for 10 minutes. After cooling, spray evenly add 1 mL of diluted hydrochloric acid (2 in 3) and 1 mL of
Dragendorff's TS for spraying on the plate: any spot other potassium iodide TS: a brown color is produced.
than the principal spot with an Rf value of about 0.6 ob- Content: not less than 97.0z. Assay—Weigh accurately
tained from the sample solution is not more intense than the about 0.6 g of selenium dioxide, and dissolve in water to
spot obtained from the standard solution. make exactly 200 mL. Pipet 20 mL of this solution into an
iodine bottle, add 80 mL of water, 3 g of potassium iodide
Scopoletin for thin-layer chromatography C10H8O4
and 5 mL of diluted hydrochloric acid (2 in 3), allow to stand
White or light brown, crystalline powder or powder. Spar-
at a dark place for 5 minutes, and titrate <2.50> with 0.1
ingly soluble in methanol and in ethanol (99.5), and practi-
mol/L sodium thiosulfate VS (indicator: starch TS). Per-
cally insoluble in water. Melting point: about 2069C.
form a blank determination in the same manner, and make
Identification—(1) Determine the absorption spectrum
any necessary correction.
of a solution of scopoletin for thin-layer chromatography in
methanol (1 in 250,000) as directed under Ultraviolet-visible Each mL of 0.1 mol/L sodium thiosulfate VS
Spectrophotometry <2.24>: it exhibits maxima between 226 ＝ 2.774 mg of SeO2
nm and 230 nm, between 295 nm and 299 nm and between
Semicarbazide acetate TS Place 2.5 g of semicarbazide
343 nm and 347 nm.
hydrochloride, 2.5 g of anhydrous sodium acetate and 30
(2) Determine the infrared absorption spectrum of
mL of methanol in a flask, heat on a water bath for 2 hours,
scopoletin for thin-layer chromatography as directed in the
cool to 209C, and filter. To the filtrate add methanol to

make 100 mL. Preserve in a cold place. Do not use the solu- Absorbance <2.24>: E 11zcm (225 nm): 727 – 781 (5 mg,
tion showing a yellow color. ethanol (99.5), 500 mL).
Purity Related substances—Dissolve 5 mg of [6]-shogaol
Semicarbazide hydrochloride H2NNHCONH2.HCl
for assay in 10 mL of a mixture of acetonitrile and water
White to light yellow crystals.
(2:1), and use this solution as the sample solution. Pipet 1
Identification (1) To 10 mL of a solution of semicar-
mL of the sample solution, add a mixture of acetonitrile and
bazide hydrochloride (1 in 100) add 1 mL of silver nitrate
water (2:1) to make exactly 100 mL, and use this solution as
TS: white precipitates appear.
the standard solution. Perform the test with 10 mL each of
(2) Determine the infrared absorption spectrum as di-
the sample solution and standard solution as directed under
rected in the potassium bromide disk method under Infrared
Liquid Chromatography <2.01> according to the following
Spectrophotometry <2.25>: it exhibits absorption at the wave
conditions. Determine the peak area of each solution by the
numbers of about 3420 cm－1, 3260 cm－1, 2670 cm－1, 1684
automatic integration method: the total area of the peaks
cm－1, 1582 cm－1, 1474 cm－1, 1386 cm－1, 1210 cm－1, 1181
other than [6]-shogaol obtained form the sample solution is
cm－1, 770 cm－1 and 719 cm－1.
not larger than the peak area of [6]-shogaol obtained from
Sendai virus RNA virus of Paramyxoviridae, which is the standard solution.
grown in the allantoic cavity of embryonated chicken eggs. Operating conditions
Measure hemagglutination titer (HA titer) with chicken red Detector, column, column temperature, mobile phase, and
blood cells, and use it with 800 to 3200 HA titer/mL. flow rate: Proceed as directed in the operating conditions
under Assay (2) of Mukoi-Daikenchuto Extract.
Sennoside A for thin-layer chromatography C42H38O20
Time span of measurement: 3 times as long as the reten-
A yellow powder. Practically insoluble in water and in
tion time of [6]-shogaol, beginning after the solvent peak.
ethanol (99.5).
System suitability
Identification Determine the infrared absorption spec-
Test for required detectability: Pipet 1 mL of the standard
trum of sennoside A to be examined as directed in the potas-
solution, and add a mixture of acetonitorile and water (2:1)
sium bromide disk method under Infrared Spectrophotome-
to make exactly 20 mL. Confirm that the peak area of [6]-
try <2.25>: it exhibits absorption at the wave numbers of
shogaol obtained with 10 mL of this solution is equivalent to
about 3420 cm－1, 1712 cm－1, 1637 cm－1, 1597 cm－1 and
3.5 to 6.5z of that obtained with 10 mL of the standard so-
1074 cm－1.
lution.
Purity Related substances—Conduct this procedure
System performance: When the procedure is run with 10
without exposure to light, using light-resistant vessels. Dis-
mL of the standard solution under the above operating con-
solve 1 mg of sennoside A to be examined in 1 mL of a mix-
ditions, the number of theoretical plates and the symmetry
ture of tetrahydrofuran and water (7:3), and use this solu-
factor of the peak of [6]-shogaol are not less than 5000 and
tion as the sample solution. Pipet 0.5 mL of the sample solu-
not more than 1.5z, respectively.
tion, add a mixture of tetrahydrofuran and water (7:3) to
System repeatability: When the test is repeated 6 times
make exactly 25 mL, and use this solution as the standard
with 10 mL of the standard solution under the above operat-
solution. Then, perform the test with 5 mL each of the sam-
ing conditions, the relative standard deviation of the peak
ple solution and standard solution as directed in the identifi-
area of [6]-shogaol is not more than 1.5z.
cation (2) under Senna Leaf: any spot other than the princi-
pal spot with an Rf value of about 0.3 obtained from the [6]-Shogaol for thin-layer chromatography C17H24O3 A
sample solution is not more intense than the spot obtained pale yellow oil. Miscible with methanol, ethanol (99.5) and
from the standard solution. with diethyl ether, and practically insoluble in water.
Purity Related substances—Dissolve 1.0 mg of [6]-
L-Serine C3H7NO3 [K 9105, Special class]
shogaol for thin-layer chromatography in 2 mL of methanol,
Sesame oil [Same as the namesake monograph] and use this solution as the sample solution. Perform the test
with this solution as directed under Thin-layer Chromatogra-
Sesamin for thin-layer chromatography C20H18O6
phy <2.03>. Spot 10 mL of the sample solution on a plate of
White, crystals or crystalline powder. Slightly soluble in
silica gel for thin-layer chromatography, develop the plate
methanol and in ethanol (99.5), and practically insoluble in
with a mixture of ethyl acetate and hexane (1:1) to a distance
water.
of about 10 cm, and air-dry the plate. Spray evenly 4-
Melting point <2.60>: 122 – 1249 C
dimethylaminobenzaldehyde TS on the plate, heat at 1059C
Identification Determine the absorption spectrum of a
for 5 minutes, and allow to cool: no spot other than the prin-
solution of Sesame in methanol (3 in 200,000) as directed
cipal spot at around R f value of 0.5 appears.
under Ultraviolet-visible Spectrophotometry <2.24>: it exhib-
its maxima between 235 nm and 239 nm and between 285 nm Silica gel An amorphous, partly hydrated silicic acid oc-
and 289 nm. curring in glassy granules of various sizes. When used as a
Purity Related substances—Dissolve 2.0 mg of sesamin desiccant, it is frequently coated with a substance that
for thin-layer chromatography in 2 mL of methanol, and use changes color when the capacity to absorb water is exhaust-
this solution as the sample solution. Pipet 1 mL of the sam- ed. Such colored products may be regenerated by being heat-
ple solution, add methanol to make exactly 50 mL, and use ed at 1109 C until the gel assumes the original color.
this solution as the standard solution. Perform the test with Loss on ignition <2.43>: not more than 6z (2 g, 950 ±
5 mL each of the sample solution and standard solution as 509C).
directed in the Identification under Sesame: the spot other Water absorption: not less than 31z. Weigh accurately
than the principal spot obtained from the sample solution is about 10 g of silica gel, and allow to stand for 24 hours in a
not more intense than the spot obtained from the standard closed container in which the atmosphere is maintained at
solution. 80z relative humidity with sulfuric acid having a specific
gravity of 1.19. Weigh again, and calculate the increase in
[6]-Shogaol for assay C17H24O3 [6]-Shogaol for thin-
mass.
layer chromatography. It meets the following requirements.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
326 General Tests / 9.41 Reagents, Test Solutions JP XVII
Siliceous earth [K 8330, Diatomaceous earth, First class] the peaks other than sinomenine and the solvent peak ob-
tained from the sample solution is not larger than the peak
Silicone oil Colorless clear liquid, having no odor.
area of sinomenine obtained from the standard solution.
Viscosity <2.53>: 50 – 100 mm2/s.
Operation conditions
Silicone resin Light gray, half-clear, viscous liquid or a Column, column temperature, mobile phase and flow
pasty material. It is almost odorless. rate: Proceed as directed in the operating conditions in the
Viscosity and refractive index—Place 15 g of silicone resin Assay (1) under Boiogito Extract.
in a Soxhlet extractor, then extract with 150 mL of carbon Detector: An ultraviolet absorption photometer (wave-
tetrachloride for 3 hours. The kinematic viscosity of the length: 261 nm).
residual liquid, obtained by evaporating carbon tetrachloride Time span of measurement: About 3 times as long as the
from the extract on a water bath, is 100 to 1100 mm2/s retention time of sinomenine.
(259C). Its refractive index is 1.400 to 1.410 (259
C). System suitability
Specific gravity <2.56> d: 0.98 – 1.02 System performance: When the procedure is run with 10
Loss on drying <2.41>: 0.45 – 2.25 g with the extracted mL of the standard solution under the above operating con-
residue obtained in the Viscosity and refractive index ditions, the number of theoretical plates and the symmetry
(1009C, 1 hour). factor of the peak of sinomenine are not less than 5000 and
not more than 1.5, respectively.
Silicotungstic acid 26-water SiO2.12WO3.26H2O
White to slightly yellowish, crystals. Deliquescent. Very Sinomenine for thin-layer chromatography C19H23NO4
soluble in water and in ethanol (95). A white or pale brown crystalline powder. Freely soluble in
Purity Clarity and color of solution—a solution (1 in 20) methanol, soluble in ethanol (99.5), and very slightly soluble
is clear and colorless. in water.
Loss on ignition <2.43>: 14 – 15z (2 g, dry at 1109C for Identification Determine the infrared absorption spec-
2 hours then 700 – 7509C, constant mass). trum of sinomenine for thin-layer chromatography as di-
rected in the potassium bromide disk method under Infrared
Silodosin C25H32F3N3O4 [Same as the namesake mono-
Spectrophotometry <2.25>: it exhibits absorption at the wave
graph]
numbers of about 2830 cm－1, 1687 cm－1, 1630 cm－1, 1441
Silver chromate-saturated potassium chromate TS Dis- cm－1 and 1279 cm－1.
solve 5 g of potassium chromate in 50 mL of water, add sil- Purity Related substances—Dissolve 5 mg of sinomenine
ver nitrate TS until a pale red precipitate is produced, and for thin-layer chromatography in 2 mL of methanol, and use
filter. To the filtrate add water to make 100 mL. this solution as the sample solution. Pipet 1 mL of the sam-
ple solution, add methanol to make exactly 100 mL, and use
Silver diethyldithiocarbamate See silver N, N-diethyl-
this solution as the standard solution. Perform the test with
dithiocarbamate.
10 mL each of the sample solution and standard solution as
Silver nitrate AgNO3 [K 8550, Special class] directed in the Identification (1) under Boiogito Extract: the
spot other than the principal spot, which appears at an Rf
Silver nitrate-ammonia TS Dissolve 1 g of silver nitrate
value of about 0.2, obtained from the sample solution is not
in 20 mL of water, and add ammonia TS dropwise with stir-
more colored than the spot obtained from the standard solu-
ring until the precipitate is almost entirely dissolved.
tion.
Storage—Preserve in tight, light-resistant containers.
Sivelestat sodium hydrate C20H21N2NaO7S.4H2O
Silver nitrate TS Dissolve 17.5 g of silver nitrate in water
[Same as the namesake monograph]
to make 1000 mL (0.1 mol/L). Preserve in light-resistant
containers. Soda lime [K 8603, For carbon dioxide absorption]
Silver N, N-diethyldithiocarbamate C5H10AgNS2 Sodium Na [K 8687, special class]
[K 9512, Special class]
Sodium acetate See sodium acetate trihydrate.
Sindbis virus RNA virus of Togaviridae, proliferated by
Sodium acetate-acetone TS Dissolve 8.15 g of sodium
chick embryo cell primary culture. Determine the number of
acetate trihydrate and 42 g of sodium chloride in 100 mL of
plaques on the cell culture, and use the virus with not less
water, and add 68 mL of 0.1 mol/L hydrochloric acid VS,
than 1 × 108 PFU/mL.
150 mL of acetone and water to make 500 mL.
Sinomenine for assay C19H23NO4 See sinomenine for
Sodium acetate, anhydrous CH3COONa [K 8372, Spe-
thin-layer chromatography. However, it meets the following
cial class]
additional requirements.
Identification Determine the absorption spectrum of a Sodium acetate trihydrate CH3COONa.3H2O [K 8371,
solution of sinomenine for assay in methanol (1 in 25,000) as Special class]
directed under Ultraviolet-visible Spectrophotometry <2.24>:
Sodium acetate TS Dissolve 13.6 g of sodium acetate tri-
it exhibits the maximum between 259 nm and 263 nm.
hydrate in water to make 100 mL (1 mol/L).
Purity Related substances—Dissolve 5 mg of sinomenine
for assay in 10 mL of a mixture of water and acetonitrile Sodium azide NaN3 [K 9501, Special class]
(7:3), and use this solution as the sample solution. Pipet 1
Sodium azide-phosphate-buffered sodium chloride TS
mL of the sample solution, add the mixture of water and
Dissolve 8.0 g of sodium chloride, 0.2 g of potassium chlo-
acetonitrile (7:3) methanol to make exactly 100 mL, and use
ride, 2.9 g of disodium hydrogen phosphate dodecahydrate
this solution as the standard solution. Perform the test with
and 0.2 g of potassium dihydrogen phosphate in water to
10 mL each of the sample solution and standard solution as
make 1000 mL. Dissolve 0.25 g of sodium azide in this solu-
directed under Liquid Chromatography <2.01> according to
tion.
the following operating conditions, and determine each peak
area by the automatic integration method: the total area of Sodium benzoate C7H5NaO2 [Same as the namesake

monograph] graph, Sodium Chloride]

Sodium bicarbonate See sodium hydrogen carbonate. Sodium chloride TS Dissolve 10 g of sodium chloride in
water to make 100 mL.
Sodium bicarbonate for pH determination See sodium
hydrogen carbonate for pH determination. 0.1 mol/L Sodium chloride TS Dissolve 6 g of sodium
chloride in water to make 1000 mL.
Sodium bicarbonate TS See sodium hydrogen carbonate
TS. 0.2 mol/L Sodium chloride TS Dissolve 11.7 g of so-
dium chloride in water to make 1000 mL.
7z Sodium bicarbonate injection [Same as the mono-
graph Sodium Bicarbonate Injection. However, labeled 1 mol/L Sodium chloride TS Dissolve 29.22 g of sodium
amount should be 7 w/vz.] chloride in water to make 500 mL.
Sodium bismuthate See bismuth sodium trioxide. Sodium cholate hydrate C24H39O5Na.H2O A white
powder.
Sodium bisulfite See sodium hydrogen sulfite.
Identification—Determine the infrared absorption spec-
Sodium bisulfite TS See sodium hydrogen sulfite TS. trum of sodium cholate hydrate as directed in the potassium
bromide disk method under Infrared Spectrophotometry
Sodium bitartrate See sodium hydrogen tartrate mono-
<2.25>: it exhibits absorption at the wave numbers of about
hydrate.
3400 cm－1, 2940 cm－1, 1579 cm－1, 1408 cm－1 and 1082
Sodium bitartrate TS See sodium hydrogen tartrate TS. cm－1.
Water <2.48>: 3.5 – 5.0z (40 mg, coulometric titration).
Sodium borate See sodium tetraborate decahydrate.
Content: not less than 99.0z of sodium cholate
Sodium borate for pH determination See sodium (C24H39O5Na), calculated on the anhydrous basis.
tetraborate decahydrate for pH determination. Assay—Weigh accurately about 0.35 g of sodium cholate hy-
drate, dissolve in 60 mL of acetic acid (100), and titrate
Sodium borohydride NaBH4 White to grayish white,
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
crystals, powder or masses. Freely soluble in water.
titration). Perform a blank determination in the same man-
Content: not less than 95z. Assay—Weigh accurately
ner, and make any necessary correction.
0.25 g of sodium borohydride, dissolve in 20 mL of diluted
sodium hydroxide TS (3 in 10), and add water to make Each mL of 0.1 mol/L perchloric acid VS
exactly 500 mL. Pipet 20 mL of this solution, put in a ＝ 43.06 mg of C24H39O5Na
glassstoppered iodine flask, and cool in ice. Add exactly 40
Sodium citrate See sodium citrate hydrate.
mL of iodine TS, allow to stand at a dark place for 10
minutes, add exactly 10 mL of diluted sulfuric acid (1 in 6), Sodium citrate hydrate C6H5Na3O7.2H2O [K 8288,
and titrate <2.50> with 0.1 mol/L sodium thiosulfate VS Trisodium citrate dihydrate, or same as the namesake mono-
(back titration) (indicator: starch solution). Perform a blank graph]
determination in the same manner, and make any necessary
Sodium cobaltinitrite See sodium hexanitrocobaltate
correction.
(III).
Each mL of 0.1 mol/L sodium thiosulfate VS
Sodium cobaltinitrite TS See sodium hexanitrocobaltate
＝ 0.4729 mg of NaBH4
(III) TS.
Sodium bromide NaBr [K 8514, Special class]
Sodium 1-decanesulfonate C10H21NaO3S A white pow-
Sodium carbonate See sodium carbonate decahydrate. der.
Purity Clarity and color of solution—Dissolve 1.0 g of
Sodium carbonate, anhydrous Na2CO3 [K 8625, So-
sodium decanesulfonate in 20 mL of water: the solution is
dium carbonate, Special class]
clear and colorless.
Sodium carbonate decahydrate Na2CO3.10H2O Loss on drying <2.41>: not more than 3.0z (1 g, 1059C,
[K 8624, Special class] 3 hours).
Content: not less than 98.0z. Assay—Weigh accurately
Sodium carbonate for pH determination Na2CO3
about 0.45 g of sodium 1-decanesulfonate, dissolve in 50 mL
[K 8625, for pH determination]
of water, and pass through a column, about 1.2 cm in inside
Sodium carbonate (standard reagent) Na2CO3 In addi- diameter and about 25 cm in length, packed with about
tion to JIS K 8005 standard reagent for volumetric analysis, 20 mL of strongly acidic ion-exchange resin (0.3 to 1.0 mm,
certified reference material which can be used for volumetric H type) for column chromatography at a flow rate of about
analysis may be used. 4 mL per minute. Wash with 150 mL of water at a flow rate
of about 4 mL per minute. Combine the washing and the
Sodium carbonate TS Dissolve 10.5 g of anhydrous so-
elute, and titrate <2.50> with 0.1 mol/L sodium hydroxide
dium carbonate in water to make 100 mL (1 mol/L).
VS (potentiometric titration). Perform a blank determina-
0.55 mol/L Sodium carbonate TS Dissolve 5.83 g of an- tion in the same manner, and make any necessary correction.
hydrous sodium carbonate in water to make 100 mL.
Each mL of 0.1 mol/L sodium hydroxide VS
Sodium chloride NaCl [K 8150, Special class] ＝ 24.43 mg of C10H21NaO3S
Sodium chloride (standard reagent) NaCl In addition 0.0375 mol/L Sodium 1-decanesulfonate TS Dissolve
to JIS K 8005 standard reagent for volumetric analysis, cer- 3.665 g of sodium 1-decanesulfonate in 400 mL of water.
tified reference material which can be used for volumetric
Sodium desoxycholate C24H39NaO4 White, odorless,
analysis may be used.
crystalline powder.
Sodium chloride for assay NaCl [Same as the mono- Identification—Determine the infrared absorption spec-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
328 General Tests / 9.41 Reagents, Test Solutions JP XVII
trum of sodium desoxycholate, previously dried, according in 500 mL of water (solution A). Dilute 2.45 g of phosphoric
to the potassium bromide disk method under Infrared Spec- acid with water to make 500 mL (solution B). To a volume
trophotometry <2.25>: it exhibits absorption at the wave of solution A add solution B until the mixture is adjusted to
numbers of about 3400 cm－1, 2940 cm－1, 1562 cm－1 and (pH 3.0).
1408 cm－1.
0.1 mol/L Sodium dihydrogen phosphate TS (pH 3.0)
Purity Related substances—Dissolve 0.10 g of sodium
Dissolve 15.60 g of sodium dihydrogen phosphate dihydrate
desoxycholate in 10 mL of methanol, and use this solution as
in 900 mL of water, adjust the pH to 3.0 with phosphoric
the sample solution. Pipet 1 mL of this solution, add metha-
acid, and add water to make 1000 mL.
nol to make exactly 100 mL, and use this solution as the
standard solution. Perform the test with these solutions as 0.05 mol/L Sodium dihydrogen phosphate TS (pH 5.5)
directed under Thin-layer Chromatography <2.03>. Spot 10 Dissolve 7.80 g of sodium dihydrogen phosphate dihydrate
mL each of the sample solution and standard solution on a in 900 mL of water, adjust to (pH 5.5) with sodium hydrox-
plate of silica gel for thin-layer chromatography. Develop ide TS, and add water to make 1000 mL.
the plate with a mixture of 1-butanol, methanol and acetic
0.05 mol/L Sodium dihydrogen phosphate TS Dissolve
acid (100) (80:40:1) to a distance of about 10 cm, and air-dry
7.80 g of sodium dihydrogen phosphate dihydrate in water
the plate. Spray evenly concentrated sulfuric acid on the
to make 1000 mL.
plate, and heat at 1059 C for 10 minutes: the spots other than
the principal spot from the sample solution are not more in- 0.1 mol/L Sodium dihydrogen phosphate TS Dissolve
tense than the spot from the standard solution. 7.80 g of sodium dihydrogen phosphate dihydrate in 450 mL
of water, adjust to a pH of 5.8 exactly with sodium hydrox-
Sodium 2,6-dichloroindophenol-sodium acetate TS Mix
ide TS, and add water to make 500 mL.
before use an equal volume of sodium 2,6-dichloroin-
dophenol dihydrate solution (1 in 20) and acetic acid-sodium 2 mol/L Sodium dihydrogen phosphate TS Dissolve
acetate TS (pH 7.0). 312.02 g of sodium dihydrogen phosphate dihydrate in water
to make 1000 mL.
Sodium diethyldithiocarbamate See sodium N, N-diethyl-
dithiocarbamate trihydrate. Sodium disulfite Na2S2O5 [K 8501, First class]
Sodium N, N-diethyldithiocarbamate trihydrate Sodium disulfite TS Dissolve 0.10 g of sodium disulfite
(C2H5)2NCS2Na.3H2O [K 8454, Special class] in 10 mL of 1 mol/L hydrochloric acid TS, and add acetone
to make 100 mL.
Sodium di-2-ethylhexyl sulfosuccinate
C8H17COOCH2(C8H17COO)CHSO3Na White or translu- Sodium dithionite Na2S2O4 A white to grayish white
cent white mucilaginous soft masses. Sparingly soluble in crystalline powder, having a strong irritating odor. It is
water. decomposed with moisture or atmospheric oxygen.
Purity Clarity and color of solution—A solution pre- Identification—(1) Dissolve 0.5 g of sodium dithionite in
pared by dissolving 1.0 g of sodium di-2-ethylhexyl sulfosuc- 50 mL of water, and use this solution as the sample solution.
cinate in 100 mL of water is clear and colorless. To 10 mL of the sample solution add 1 mL of copper (II)
Loss on drying <2.41>: not more than 5.0z (1 g, 1059C, sulfate TS: a grayish brown color is produced.
2 hours). (2) The sample solution obtained in (1) responds to the
Qualitative Tests <1.09> (1) for sodium salt.
Sodium dihydrogen phosphate See sodium dihydrogen
Preserve in a light-resistant tight container.
phosphate dihydrate.
Sodium dodecylbenzene sulfonate C18H29SO3Na
Sodium dihydrogen phosphate anhydrous NaH2PO4 A
White, crystalline powder or mass.
white, powder or crystalline powder. Freely soluble in water,
pH <2.54>—The pH of a solution of 0.5 g of sodium
and very slightly soluble in ethanol (99.5). It has a
dodecylbenzene sulfonate in 50 mL of freshly boiled and
hygroscopic property.
cooled water is between 5.0 and 7.0. Measure the pH at 259 C
A solution is acidic.
passing nitrogen with stirring.
Sodium dihydrogen phosphate dihydrate Loss on drying <2.41>: not more than 0.5z (1 g, 1059C,
NaH2PO4.2H2O [K 9009, Special class] 2 hours).
Content: not less than 99.0z. Assay—Weigh accurately
Sodium dihydrogen phosphate-ethanol TS To 500 mL of
about 40 mg of sodium dodecylbenzene sulfonate, previ-
sodium dihydrogen phosphate solution (39 in 2500) add 200
ously dried, and perform the test as directed in (4) Sulfur in
mL of water, and add 300 mL of ethanol (99.5).
the Procedure of determination under Oxygen Flask Com-
Sodium dihydrogen phosphate TS (pH 2.2) Dissolve bustion Method <1.06>, using a mixture of 20 mL of water
1.56 g of sodium dihydrogen phosphate dihydrate in 800 mL and 2 mL of strong hydrogen peroxide water as absorbing
of water, adjust the pH to 2.2 with phosphoric acid, and add solution.
water to make 1000 mL.
Each mL of 0.005 mol/L barium perchlorate VS
Sodium dihydrogen phosphate TS (pH 2.5) Dissolve ＝ 1.742 mg of C18H29SO3Na
2.7 g of sodium dihydrogen phosphate dihydrate in 1000 mL
Sodium fluoride NaF [K 8821, Special class]
of water, and adjust the pH to 2.5 with phosphoric acid.
Sodium fluoride (standard reagent) NaF In addition to
0.05 mol/L Sodium dihydrogen phosphate TS (pH 2.6)
JIS K 8005 standard reagent for volumetric analysis, certi-
Dissolve 7.80 g of sodium dihydrogen phosphate dihydrate
fied reference material which can be used for volumetric
in 900 mL of water, adjust the pH to 2.6 with phosphoric
analysis may be used.
acid and add water to make 1000 mL.
Sodium fluoride-hydrochloric acid TS Dissolve 0.5 g of
0.05 mol/L Sodium dihydrogen phosphate TS (pH 3.0)
sodium fluoride in 100 mL of 0.5 mol/L hydrochloric acid
Dissolve 3.45 g of sodium dihydrogen phosphate dihydrate

TS. Prepare before use. and dissolve in 25 mL of water. Transfer 15–20 mL of this
solution into a chromatographic column about 11 mm in di-
Sodium fluoride TS Dissolve 0.5 g of sodium fluoride in
ameter and about 500 mm in height of strongly acidic ion ex-
100 mL of 0.1 mol/L hydrochloric acid TS. Prepare before
change resin for column chromatography (246 mm to 833 mm
use.
in particle diameter, type H), and elute at the rate of about
Sodium gluconate C6H11NaO7 A white or pale yellow- 5 – 10 mL per minute, then wash the column with five 50-mL
ish brown crystalline powder. portions of water at the rate of about 5 – 10 mL per minute.
Purity Clarity and color of solution—A solution ob- Combine the washings to the eluate, and titrate <2.50> with
tained by dissolving 1.0 g of sodium gluconate in 10 mL of 0.1 mol/L sodium hydroxide VS (indicator: 3 drops of phe-
water is clear and colorless or pale yellow. nolphthalein TS).
Sodium glycocholate for thin-layer chromatography Each mL of 0.1 mol/L sodium hydroxide VS
C26H42NNaO6 White to pale brown, crystalline powder or ＝ 18.82 mg of C6H13NaO3S
powder. Freely soluble in water and in methanol, and
Sodium hexanitrocobaltate (III) Na3Co(NO2)6
slightly soluble in ethanol (99.5).
[K 8347, Special class]
Identification—(1) Determine the infrared absorption
spectrum of sodium glycocholate for thin-layer chromatog- Sodium hexanitrocobaltate (III) TS Dissolve 10 g of so-
raphy as directed in the potassium bromide disk method dium hexanitrocobaltate (III) in water to make 50 mL,
under Infrared Spectrophotometry <2.25>: it exhibits absorp- and filter if necessary. Prepare before use.
tion at the wave numbers of about 2940 cm－1, 1599 cm－1,
Sodium hyaluronate, purified (C14H20NNaO11)n [Same
1398 cm－1, 1309 cm－1, 1078 cm－1, 1040 cm－1, 982 cm－1 and
as the namesake monograph]
915 cm－1.
(2) Sodium glycocholate for thin-layer chromatography Sodium hyaluronate for assay (C14H20NNaO11)n [Same
responds to the Qualitative Tests <1.09> (1) for sodium salt. as the monograph Purified Sodium Hyaluronate. It contains
Optical rotation <2.49> [a]20D : ＋25 – ＋359(60 mg, meth- not less than 99.0z of sodium hyaluronate [(C14H20NNaO11)
anol, 20 mL, 100 mm). n], calculated on the dried basis.]
Purity Related substances—Dissolve 5 mg of sodium
Sodium hydrogen carbonate NaHCO3 [K 8622, Spe-
glycocholate for thin-layer chromatography in 1 mL of
cial class]
methanol, and use this solution as the sample solution. Pipet
0.2 mL of the sample solution, add methanol to make ex- Sodium hydrogen carbonate for pH determination
actly 10 mL, and use this solution as the standard solution. NaHCO3 [K 8622, for pH determination]
Perform the test with these solutions as directed under Thin-
Sodium hydrogen carbonate TS Dissolve 5.0 g of sodium
layer Chromatography <2.03>. Proceed with 5 mL each of the
hydrogen carbonate in water to make 100 mL.
sample solution and standard solution as directed in the
Identification under Bear Bile: the spots other than the prin- 10z Sodium hydrogen carbonate TS Dissolve 10 g of so-
cipal spot with an R f value of about 0.2 obtained from the dium hydrogen carbonate in water to make 100 mL, and
sample solution are not more intense than the spot obtained sterilize in a tight container in an autoclave at 1219C for 15
from the standard solution. minutes or by filtration through a membrane filter with a
pore size not exceeding 0.22 mm.
Sodium 1-heptane sulfonate C7H15NaO3S White, crys-
tals or crystalline powder. 7z Sodium hydrogen carbonate injection See 7z so-
Purity Clarity and color of solution—Dissolve 1.0 g of dium bicarbonate injection.
sodium 1-heptane sulfonate in 10 mL of water: the solution
Sodium hydrogen sulfite [K 8059, Special class]
is clear and colorless.
Loss on drying <2.41>: not more than 3.0z (1 g, 1059C, Sodium hydrogen sulfite TS Dissolve 10 g of sodium
3 hours). hydrogen sulfite in water to make 30 mL. Prepare before
Content: not less than 98.0z. Assay—Dissolve about use.
0.4 g of sodium 1-heptane sulfonate, previously dried and
Sodium hydrogen tartrate monohydrate
weighed accurately, in 50 mL of water, transfer to a chro-
NaHC4H4O6.H2O [K 8538, (＋)-Sodium hydrogentartrate
matographic column, prepared by packing a chromato-
monohydrate, Special class]
graphic tube 9 mm in inside diameter and 160 mm in height
with 10 mL of strongly acidic ion exchange resin for column Sodium hydrogen tartrate TS Dissolve 1 g of sodium
chromatography (425 to 600 mm in particle diameter, H bitartrate in water to make 10 mL (0.5 mol/L). Prepare be-
type), and flow at a flow rate of about 4 mL per minute. fore use.
Wash the column at the same flow rate with 150 mL of
Sodium hydroxide NaOH [K 8576, Special class]
water, combine the washings with the effluent solution, and
titrate <2.50> with 0.1 mol/L sodium hydroxide VS (indica- Sodium hydroxide-dioxane TS Dissolve 0.80 g of sodium
tor: 10 drops of bromothymol blue TS) until the color of the hydroxide in a mixture of 1,4-dioxane and water (3:1) to
solution changes from yellow to blue. make 100 mL.
Each mL of 0.1 mol/L sodium hydroxide VS Sodium hydroxide-methanol TS Dissolve by thorough
＝ 20.23 mg of C7H15NaO3S shaking 4 g of sodium hydroxide in methanol to make 100
mL. To the supernatant liquid obtained by centrifugation
Sodium 1-hexane sulfonate C6H13NaO3S White, crys-
add methanol to make 500 mL. Prepare before use.
tals or crystalline powder.
Loss on drying <2.41>: not more than 3.0z (1 g, 1059C, Sodium hydroxide TS Dissolve 4.3 g of sodium hydrox-
2 hours). ide in water to make 100 mL (1 mol/L). Preserve in poly-
Content: not less than 98.0z. Assay—Weigh accurately ethylene bottles.
about 0.4 g of sodium 1-hexane sulfonate, previously dried,

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
330 General Tests / 9.41 Reagents, Test Solutions JP XVII
Sodium hydroxide TS, dilute Dissolve 4.3 g of sodium Each mL of 0.1 mol/L sodium thiosulfate VS
hydroxide in freshly boiled and cooled water to make 1000 ＝ 3.722 mg of NaClO.
mL. Prepare before use (0.1 mol/L).
Sodium L-lactate solution for assay C3H5NaO3 [Same
0.01 mol/L Sodium hydroxide TS Dilute 10 mL of so- as the monograph, Sodium L-Lactate Solution]
dium hydroxide TS with water to make 1000 mL. Prepare
Sodium lauryl sulfate [Same as the namesake mono-
before use.
graph]
0.05 mol/L Sodium hydroxide TS To 10 mL of 0.5
Sodium lauryl sulfate TS Dissolve 100 g of sodium lauryl
mol/L sodium hydroxide TS add water to make 100 mL.
sulfate in 900 mL of water, add 10 mL of 1 mol/L hydro-
0.2 mol/L Sodium hydroxide TS Dissolve 8.0 g of so- chloric acid TS, and add water to make 1000 mL.
dium hydroxide in freshly boiled and cooled water to make
0.2z Sodium lauryl sulfate TS Dissolve 0.1 g of sodium
1000 mL. Prepare before use.
lauryl sulfate in 0.1 mol/L sodium phosphate buffer (pH
0.5 mol/L Sodium hydroxide TS Dissolve 22 g of so- 7.0) to make 50 mL.
dium hydroxide in water to make 1000 mL. Preserve in poly-
Sodium metabisulfite See sodium disulfite.
ethylene bottles.
Sodium metabisulfite TS See sodium disulfite TS.
2 mol/L Sodium hydroxide TS Dissolve 86 g of sodium
hydroxide in water to make 1000 mL. Preserve in polyethy- Sodium, metallic See sodium
lene bottles.
Sodium 1-methyl-1H-tetrazole-5-thiolate See sodium 1-
4 mol/L Sodium hydroxide TS Dissolve 168 g of sodium methyl-1H-tetrazole-5-thiolate dihydrate.
hydroxide in water to make 1000 mL. Preserve in polyethy-
Sodium 1-methyl-1H-tetrazole-5-thiolate dihydrate
lene bottles.
C2H3N4NaS.2H2O White, crystals or crystalline powder.
5 mol/L Sodium hydroxide TS Dissolve 210 g of sodium Melting point <2.60>: 90 – 949C
hydroxide in water to make 1000 mL. Preserve in a polyethy- Purity Related substances—Dissolve 10 mg of sodium 1-
lene bottle. methyl-1H-tetrazole-5-thiolate dihydrate in 10 mL of water,
and use the sample solution as the sample solution. Perform
6 mol/L Sodium hydroxide TS Dissolve 252 g of sodium
the test with this solution as directed under Thin-layer Chro-
hydroxide in water to make 1000 mL. Preserve in a polyethy-
matography <2.03>. Spot 5 mL of the sample solution on a
lene bottle.
plate of silica gel with fluorescent indicator for thin-layer
8 mol/L Sodium hydroxide TS Dissolve 336 g of sodium chromatography. Develop the plate with a mixture of ethyl
hydroxide in water to make 1000 mL. Preserve in polyethy- acetate, acetone, water and acetic acid (100) (10:2:1:1) to a
lene bottles. distance of about 10 cm, and air-dry the plate. Examine
under ultraviolet light (main wavelength: 254 nm): any spot
Sodium hypobromite TS To 8 mL of bromine TS add 25
other than the principal spot does not appear.
mL of water and 25 mL of sodium carbonate TS. Prepare
before use. Sodium molybdate See sodium molybdate (VI) dihy-
drate.
Sodium hypochlorite-sodium hydroxide TS To a volume
of sodium hypochlorite TS for ammonium limit test, equiva- Sodium molybdate (VI) dihydrate Na2MoO4.2H2O
lent to 1.05 g of sodium hypochlorite (NaClO: 74.44), add [K 8906, disodium molybdate (VI) dihydrate, Special class]
15 g of sodium hydroxide and water to make 1000 mL. Pre-
Sodium 2-naphthalenesulfonate C10H7NaO3S Pale
pare before use.
brown, crystals or powder.
Sodium hypochlorite TS Prepare the solution by passing Content: not less than 98.0z.
chlorine into sodium hydroxide TS while cooling with ice, so
Sodium b-naphthoquinone sulfonate C10H5NaO5S
as to contain 5z of sodium hypochlorite (NaClO: 74.44).
Yellow to orange-yellow, crystals or crystalline powder.
Prepare before use.
Soluble in water, and practically insoluble in ethanol (95).
10z Sodium hypochlorite TS Prepare by introducing Loss on drying <2.41>: Not more than 2.0z (1 g, in vacu-
chlorine into an aqueous solution of sodium hydroxide while um, 509C).
ice-cooling so that the content of sodium hypochlorite Residue on ignition <2.44>: 26.5 – 28.0z (1 g, after dry-
(NaClO: 74.44) is 10z. Prepare before use. ing).
Sodium hypochlorite TS for ammonium limit test Sodium naphthoquinone sulfonate TS Dissolve 0.25 g of
Clear, colorless or pale green-yellow solution prepared by sodium b-naphthoquinone sulfonate in methanol to make
passing chlorine into sodium hydroxide or sodium carbonate 100 mL.
decahydrate solution, having the odor of chlorine.
Sodium nitrate NaNO3 [K 8562, Special class]
Content: not less than 4.2 g/d L as sodium hypochlorite
(NaClO: 74.44). Assay—Pipet 10 mL of sodium hypochlo- Sodium nitrite NaNO2 [K 8019, Special class]
rite TS for ammonium limit test, and add water to make
Sodium nitrite TS Dissolve 10 g of sodium nitrite in
exactly 100 mL. Transfer exactly 10 mL of this solution to a
water to make 100 mL. Prepare before use.
glass-stoppered flask, add 90 mL of water, then add 2 g of
potassium iodide and 6 mL of diluted acetic acid (1 in 2), Sodium nitroprusside See sodium pentacyanonitrosylfer-
stopper tightly, shake well, and allow to stand for 5 minutes rate (III) dihydrate.
in a dark place. Titrate <2.50> the liberated iodine with 0.1
Sodium nitroprusside TS See sodium pentacyano-
mol/L sodium thiosulfate VS (indicator: 3 mL of starch TS).
nitrosylferrate (III) TS.
Perform a blank determination in the same manner, and
make any necessary correction. Sodium 1-nonanesulfonate CH3(CH2)8SO3Na White

crystalline powder. Freely soluble in water. VS (indicator: 10 drops of bromothymol blue TS) until the
Loss on drying <2.41>: Not more than 1.0z (1 g, 1059C, yellow color of the solution changes to blue.
3 hours).
Each mL of 0.1 mol/L sodium hydroxide VS
Residue on ignition <2.44>: 30 – 32z (0.5 g).
＝ 17.42 mg of C5H11NaO3S
Sodium 1-octane sulfonate CH3(CH2)7SO3Na White,
Sodium perchlorate See sodium perchlorate monohy-
crystals or powder.
drate.
Residue on ignition <2.44>: 32.2 – 33.0z (1.0 g).
Sodium perchlorate monohydrate NaClO4.H2O
Sodium oxalate (standard reagent) C2Na2O4 In addi-
[K 8227, Special class]
tion to JIS K 8005 standard reagent for volumetric analysis,
certified reference material which can be used for volumetric Sodium periodate NaIO4 [K 8256, Special class]
analysis may be used.
Sodium periodate TS Dissolve 60.0 g of sodium perio-
Sodium pentacyanoammine ferroate (II) n-hydrate date in 120 mL of 0.05 mol/L sulfuric acid TS, and add
Na3[Fe(CN)5NH3].x H2O A light yellow to light green-yel- water to make 1000 mL. Keep in a light-resistant vessel.
low crystalline powder.
Sodium peroxide Na2O2 [K 8231, Special class]
Identification—(1) Dissolve 0.2 g of sodium pentac-
yanoammine ferroate (II) x-hydrate in 5 mL of water, add 2 Sodium p-phenol sulfonate See sodium p-phenol sul-
mL of sodium hydroxide solution (1 in 10), and heat: ammo- fonate dihydrate.
nia gas is evolved and a brown precipitate is produced.
Sodium p-phenol sulfonate dihydrate C6H5O4NaS.2H2O
(2) Dissolve 0.25 g of sodium pentacyanoammine ferro-
White to light yellow, crystals or crystalline powder, having
ate (II) n-hydrate in 20 mL of water. To 1 mL of this solu-
a specific odor.
tion add 0.2 mL of iron (II) sulfate TS: a green-blue color
Identification (1) To 10 mL of a solution of sodium p-
develops, which changes to a dark blue color on the addition
phenol sulfonate (1 in 10) add 1 drop of iron (III) chloride
of 2 drops of diluted sodium hypochlorite TS (2 in 5) and 0.2
TS: a purple color develops.
mL of acetic acid (100).
(2) Determine the absorption spectrum of a solution of
Sodium pentacyanonitrosylferrate (III) dihydrate sodium p-phenol sulfonate (1 in 5000) as directed under
Na2[Fe(CN)5(NO)].2H2O [K 8722, Special class] Ultraviolet-visible Spectrophotometry <2.24>: it exhibits
maxima between 269 nm and 273 nm and between 276 nm
Sodium pentacyanonitrosylferrate (III)-potassium hexa-
and 280 nm.
cyanoferrate (III) TS Mix an equal volume of a solution of
Purity Clarity and color of solution—Dissolve 1.0 g of
sodium pentacyanonitrosylferrate (III) dihydrate (1 in 10), a
sodium p-phenol sulfonate in 25 mL of water: the solution is
solution of potassium hexacyanoferrate (III) (1 in 10) and a
clear and colorless.
solution of sodium hydroxide (1 in 10), and allow to stand
Content: not less than 90.0z. Assay—Dissolve about
for 30 minutes. Use after the color of the solution is changed
0.5 g of sodium p-phenol sulfonate, accurately weighed, in
from a dark red to yellow. Prepare before use.
50 mL of water. Transfer the solution to a chromatographic
Sodium pentacyanonitrosylferrate (III)-potassium hexa- column, prepared by pouring strongly acidic ion exchange
cyanoferrate (III) TS, dilute To 5 mL of a solution of resin (H type) for column chromatography (150 to 300 mm in
pentacyanonitrosylferrate (III) dihydrate (3 in 50) add 5 mL particle diameter) into a chromatographic tube about 1 cm in
of a solution of potassium hexacyanoferrate (III) (13 in 200) inside diameter and about 30 cm in height, and allow to
and 2.5 mL of a solution of sodium hydroxide (1 in 10), add flow. Wash the chromatographic column with water until
water to make 25 mL, mix, and use after changing the color the washing is no longer acidic, combine the washings with
of the solution from a dark red to yellow. Prepare before the above effluent solution, and titrate <2.50> with 0.1
use. mol/L sodium hydroxide VS (indicator: 5 drops of
bromocresol green-methyl red TS). Separately, dissolve 0.5 g
Sodium pentacyanonitrosylferrate (III) TS Dissolve 1 g
of sodium p-phenol sulfonate, weighed accurately, in 50 mL
of sodium pentacyanonitrosylferrate (III) dihydrate in water
of water and titrate with 0.1 mol/L sodium hydroxide VS,
to make 20 mL. Prepare before use.
and make any necessary correction.
Sodium 1-pentane sulfonate C5H11NaO3S White, crys-
Each mL of 0.1 mol/L sodium hydroxide VS
tals or crystalline powder. Freely soluble in water, and prac-
＝ 23.22 mg of C6H5O4NaS.2H2O
tically insoluble in acetonitrile.
Purity Clarity and color of solution—Dissolve 1.0 g of Sodium phosphate See trisodium phosphate dodecahy-
sodium 1-pentane sulfonate in 10 mL of water: the solution drate.
is colorless and clear.
Sodium phosphate TS Dissolve 5.68 g of disodium
Water <2.48>: not more than 3.0z (0.2 g).
hydrogen phosphate and 6.24 g of sodium dihydrogen phos-
Content: not less than 99.0z, calculated on the anhydrous
phate dihydrate in water to make 1000 mL.
basis. Assay—Dissolve about 0.3 g of sodium 1-pentane
sulfonate, accurately weighed, in 50 mL of water. Transfer 0.1 mol/L Sodium phosphate buffer solution (pH 7.0)
this solution to a chromatographic column, prepared by Dissolve 17.9 g of disodium hydrogen phosphate dodecahy-
pouring 10 mL of strongly acidic ion-exchange resin (H type) drate in water to make 500 mL. Add to this solution to a 500
(425 – 600 mm in particle diameter) into a chromatographic mL solution prepared by dissolving 7.8 g of sodium dihydro-
tube, 9 mm in inside diameter and 160 mm in height, and gen phosphate dihydrate in water until the pH becomes 7.0.
elute at the rate of about 4 mL per minute. Wash the chro-
Sodium pyruvate CH3COCOONa A white to pale yel-
matographic column with 50 mL of water at the rate of
low crystalline powder. Freely soluble in water, and slightly
about 4 mL per minute, and wash again with 100 mL of
soluble in ethanol (99.5) and in acetone.
water in the same manner. Combine the washings with the
Identification (1) Determine the infrared absorption
eluate, and titrate <2.50> with 0.1 mol/L sodium hydroxide

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
332 General Tests / 9.41 Reagents, Test Solutions JP XVII
spectrum of sodium pyruvate as directed in the potassium Sodium sulfide TS Dissolve 5 g of sodium sulfide ennea-
bromide disk method under Infrared Spectrophotometry hydrate in a mixture of 10 mL of water and 30 mL of glyce-
<2.25>: it exhibits absorption at the wave numbers of about rin. Or dissolve 5 g of sodium hydroxide in a mixture of 30
1710 cm－1, 1630 cm－1, 1410 cm－1, 1360 cm－1, 1190 cm－1, mL of water and 90 mL of glycerin, saturate a half volume
1020 cm－1, 980 cm－1, 830 cm－1,750 cm－1, 630 cm－1 and 430 of this solution with hydrogen sulfide, while cooling, and
cm－1. mix with the remaining half. Preserve in well-filled, light-
(2) A solution of sodium pyruvate (1 in 20) responds to resistant bottles. Use within 3 months.
the Qualitative Tests <1.09> (1) for sodium salt.
Sodium sulfite See sodium sulfite heptahydrate.
Content: Not less than 97.0z. Assay—Weigh accurately
about 0.4 g of sodium pyruvate, and dissolve in water to Sodium sulfite, anhydrous Na2SO3 [K 8061, Sodium
make exactly 200 mL. Pipet 20 mL of this solution into an sulfite, Special class]
iodine bottle, cool to 109C or lower, add exactly 40 mL of
Sodium sulfite heptahydrate Na2SO3.7H2O [K 8060,
0.05 mol/L iodine VS, then add 20 mL of a solution of so-
Special class]
dium hydroxide (17 in 100), and allow to stand at a dark
place for 2 hours. Then add 15 mL of diluted sulfuric acid (1 1 mol/L Sodium sulfite TS Dissolve 1.26 g of anhydrous
in 6), and titrate <2.50> with 0.1 mol/L sodium thiosulfate sodium sulfite in water to make 10 mL.
VS (indicator: starch TS). Perform a blank determination in
Sodium sulfite-sodium dihydrogen phosphate TS Mix
the same manner, and make any necessary correction.
1.5 mL of a solution dissolved 1.26 g of anhydrous sodium
Each mL of 0.05 mol/L iodine VS sulfite in 100 mL of water and 98.5 mL of a solution dis-
＝ 1.834 mg of C3H3NaO3 solved 1.56 g of sodium dihydrogen phosphate dihydrate in
100 mL of water. Prepare before use.
100 mmol/L Sodium pyruvate TS Dissolve 1.1 g of so-
dium pyruvate in water to make 100 mL, and sterilize by Sodium tartrate See sodium tartrate dihydrate.
filtration through a membrane filter with a pore size not
Sodium tartrate dihydrate C4H4Na2O6.2H2O
exceeding 0.22 mm.
[K 8540, sodium (＋)-tartrate dihydrate, Special class]
Sodium salicylate HOC6H4COONa [K 8397, Special
Sodium tauroursodeoxycholate for thin-layer chromatog-
class]
raphy C26H44NNaO6S White to pale brown, crystalline
Sodium salicylate-sodium hydroxide TS Dissolve 1 g of powder or powder. Freely soluble in methanol, soluble in
sodium salicylate in 0.01 mol/L sodium hydroxide VS to water, and sparingly soluble in ethanol (99.5).
make 100 mL. Identification—(1) Determine the infrared absorption
spectrum of sodium tauroursodeoxycholate for thin-layer
Sodium selenite Na2SeO3 A white crystalline powder.
chromamatography as directed in the potassium bromide
Identification—(1) Dissolve 1 g of sodium selenite in 100
disk method under Infrared Spectrophotometry <2.25>: it ex-
mL of water, and use this solution as the sample solution.
hibits absorption at the wave numbers of about 2930 cm－1,
To 10 mL of the sample solution add 2 mL of tin (II) chlo-
1645 cm－1, 1556 cm－1, 1453 cm－1, 1215 cm－1 and 1049
ride TS: a red precipitate is produced.
cm－1.
(2) The sample solution obtained in (1) responds to the
(2) Sodium tauroursodeoxycholate for thin-layer chro-
Qualitative Tests <1.09> (1) for sodium salt.
matography responds to the Qualitative Tests <1.09> (1) for
Preserve in a light-resistant tight container.
sodium salt.
Sodium p-styrenesulfonate C8H7NaO3S White, crystals Optical rotation <2.49> [a]20
D : ＋40 – ＋509(40 mg, meth-
or crystalline powder. Freely soluble in water, slightly solu- anol, 20 mL, 100 mm).
ble in ethanol (99.5), and practically insoluble in diethyl Purity Related substances—Dissolve 10 mg of sodium
ether. tauroursodeoxycholate for thin-layer chromatography in 1
Recrystalize from diluted ethanol (1 in 2), and dry in mL of methanol, and use this solution as the sample solu-
vacuum. tion. Pipet 0.2 mL of the sample solution, add methanol to
Identification—Determine the infrared absorption spec- make exactly 10 mL, and use this solution as the standard
trum of sodium p-styrenesulfonate according to the potas- solution. Perform the test with these solutions as directed
sium bromide disk method under Infrared Spectrophotome- under Thin-layer Chromatography <2.03>. Perform the test
try <2.25>: it exhibits absorption at the wave numbers of with 5 mL each of the sample solution and standard solution
about 1236 cm－1, 1192 cm－1, 1136 cm－1, 1052 cm－1, 844 as directed in the Identification under Bear Bile: the spots
cm－1 and 688 cm－1. other than the principal spot with an R f value of about 0.2
Purity—Perform the test with 10 mL of a solution of so- obtained from the sample solution are not more intense than
dium p-styrenesulfonate (1 in 1000) as directed in the Assay the spot obtained from the standard solution.
under Panipenem: Any obstructive peaks for determination
Sodium tetraborate-calcium chloride buffer solution (pH
of panipenem are not observed.
8.0) Dissolve 0.572 g of sodium tetraborate decahydrate
Sodium sulfate See sodium sulfate decahydrate. and 2.94 g of calcium chloride dihydrate in 800 mL of
freshly boiled and cooled water, adjust the pH to 8.0 with 1
Sodium sulfate, anhydrous Na2SO4 [K 8987, Special
mol/L hydrochloric acid TS, and add water to make 1000
class]
mL.
Sodium sulfate decahydrate Na2SO4.10H2O [K 8986,
Sodium tetraborate decahydrate Na2B4O7.10H2O
Special class]
[K 8866, Special class]
Sodium sulfide See sodium sulfide enneahydrate.
Sodium tetraborate decahydrate for pH determination
Sodium sulfide enneahydrate Na2S.9H2O [K 8949, [K 8866, for pH standard solution]
Special class]
Sodium tetraborate-sulfuric acid TS To 9.5 g of sodium

tetraborate decahydrate add 1000 mL of sulfuric acid, and with plate seal, and then shake gently. Centrifuge for 2
dissolve by shaking for a night. minutes if some solution sticks to the top of the microplate
Purity: To 1 mL of water add gently 5 mL of sodium or elsewhere. Dissolve 0.5 g of bovine serum albumin in 100
tetraborate-sulfuric acid TS, previously cooled in ice water, mL of 0.01 mol/L phosphate buffer-sodium chloride TS (pH
stir while cooling, then heat in a water bath for 10 minutes, 7.4) to make the wash solution. After leaving the microplates
and cool in ice water. Add exactly 0.2 mL of carbazole TS, for 16 to 24 hours at a constant temperature of about 259 C,
stir thoroughly, then heat in a water bath for 15 minutes, remove the solution in each well by aspiration, add 0.25 mL
and cool in ice water to room temperature: a green color of the wash solution, shake gently, and then remove this so-
does not appear. lution by aspiration. Repeat this procedure 2 more times for
each well using 0.25 mL of the wash solution. Add 0.25 mL
Sodium tetraphenylborate (C6H5)4BNa [K 9521, Spe-
of the block buffer solution to each well, gently shake, and
cial class]
let stand for 16 to 24 hours at a constant temperature of
Sodium thioglycolate HSCH2COONa A white powder, about 259C to make solid plates. When using, remove the
having a characteristic odor. solution from the wells by aspiration, add 0.25 mL of the
Identification (1) To a solution (1 in 10) add 0.1 mL of wash solution to each well, shake gently, and then remove
ammonia solution (28) and 1 drop of iron (III) chloride TS: this solution by aspiration. Repeat this procedure 2 more
a dark red-purple color appears. times using 0.25 mL of the wash solution.
(2) Perform the test as directed under Flame Coloration
Soluble starch See starch, soluble.
Test <1.04> (1): a yellow color appears.
Purity Clarity and color of solution—Dissolve 1 g in 10 Soluble starch TS Triturate 1 g of soluble starch in 10
mL of water: the solution is clear and colorless. mL of cooled water, pour gradually into 90 mL of boiled
water while costantly stirring, boil gently for 3 minutes, and
Sodium thiosulfate See sodium thiosulfate pentahydrate.
cool. Prepare before use.
Sodium thiosulfate pentahydrate Na2S2O3.5H2O
Sorbitan sesquioleate [Same as the namesake mono-
[K 8637, Special class]
graph]
Sodium thiosulfate TS Dissolve 26 g of sodium thiosul-
D-Sorbitol C6H14O6 [Same as the namesake mono-
fate pentahydrate and 0.2 g of anhydrous sodium carbonate
graph]
in freshly boiled and cooled water to make 1000 mL (0.1
mol/L). D-Sorbitol for gas chromatography Prepared for gas
chromatography.
Sodium toluenesulfonchloramide trihydrate
C7H7ClNNaO2S.3H2O [K 8318, Sodium p-toluenesulfon- Soybean-casein digest medium See Sterility Test <4.06>.
chloramide trihydrate, Special class]
Soybean oil [Same as the namesake monograph]
Sodium toluenesulfonchloramide TS Dissolve 1 g of so-
Soybean peptone See peptone, soybean.
dium toluenesulfonchloramide trihydrate in water to make
100 mL. Prepare before use. Stachyose for thin-layer chromatography C24H42O21 A
white powder. Very soluble in water, and practically insolu-
Sodium tridecanesulfonate C13H27SO3Na White, crys-
ble in ethanol (99.5). It is deliquescence with the atmospheric
tals or powder.
moisture.
Purity Absorbance—Dissolve 1.43 g of sodium tridecan-
Optical rotation <2.49> [a]20D : ＋144 – ＋1549(50 mg calcu-
sulfonate in 1000 mL of water, and perform the test with this
lated on the anhydrous basis, diluted ammonia solution (28)
solution as directed under Ultraviolet-visible Spectropho-
(1 in 1000), 5 mL, 100 mm).
tometry <2.24>: the absorbances at 230 nm and 254 nm are
Purity Related substances—Dissolve 2 mg of stachyose
not more than 0.05 and 0.01, respectively.
for thin-layer chromatography in 1 mL of a mixture of water
Sodium 3-trimethylsilylpropanesulfonate for nuclear mag- and methanol (1:1), and use this solution as the sample solu-
netic resonance spectroscopy (CH3)3SiCH2CH2CH2SO3Na tion. Perform the test with the sample solution as directed
Prepared for nuclear magnetic resonance spectroscopy. under Thin-layer chromatography <2.03>. Spot 2 mL of the
sample solution on a plate of silica gel for thin-layer chroma-
Sodium 3-trimethylsilylpropionate-d4 for nuclear magnetic
tography. Develop the plate with a mixture of 2-propanol,
resonance spectroscopy (CH3)3SiCD2CD2COONa Pre-
water and methanol (3:2:2) to a distance of about 7 cm, and
pared for nuclear magnetic resonance spectroscopy.
air-dry the plate. Spray evenly 1,3-naphthalenediol TS to the
Sodium 2,4,6-trinitrobenzenesulfonate dihydrate plate, and heat at 1059 C for 10 minutes: a spot other than
C6H2N3NaO9S.2H2O White or pale yellowish, crystals or the principle spot with an Rf value of about 0.5 is not ob-
powder. served.
Sodium tungstate See sodium tungstate (VI) dihydrate. Stacking gel for celmoleukin In 0.5 mol/L Tris buffer
solution (pH 6.8), prepare stacking the gel using ammonium
Sodium tungstate (VI) dihydrate Na2WO4.2H2O
persulfate and N, N, N?, N?-tetramethylenediamine so the
[K 8612, Special class]
acrylamide concentration is 5.2z and the sodium lauryl sul-
Sodium valproate for assay C8H15NaO2 [Same as the fate concentration is 0.1z.
monograph Sodium Valproate. When dried, it contains not
Stannous chloride See tin (II) chloride dihydrate.
less than 99.0z of sodium valproate (C8H15NaO2).]
Stannous chloride-sulfuric acid TS See tin (II) chloride-
Solid plates Dilute anti-E. coli protein antibody stock so-
sulfuric acid TS.
lution by adding 0.2 mol/L Tris hydrochloride buffer (pH
7.4) to a concentration of about 0.02 mg/mL. Add exactly Stannous chloride TS See tin (II) chloride TS.
0.1 mL of this solution to each well in the microplates, cover
Stannous chloride TS, acidic See tin (II) chloride TS,

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
334 General Tests / 9.41 Reagents, Test Solutions JP XVII
acidic. (30).
Starch [K 8658, Special class] Strongly acidic ion exchange resin Contains strong acid
ion exchange residues. Particle diameter is about 100 mm.
Starch-sodium chloride TS Saturate starch TS with so-
dium chloride. Use within 5 to 6 days. Strongly basic ion exchange resin Contains strong basic
ion exchange residues. Particle diameter is about 100 mm.
Starch, soluble A potato starch, dried after treating with
acid, neutralizing and washing with water. A white powder. Strontium TS Dissolve 76.5 g of strontium chloride in
Practically insoluble in ethanol (99.5). Soluble by heating water to make exactly 500 mL. Pipet 20 mL of this solution,
after addition of water. and add water to make exactly 1000 mL (1000 ppm).
pH <2.54>: To 2.0 g of soluble starch add 90 mL of freshly
Strontium chloride See strontium chloride hexahydrate.
boiled and cooled water, and heat to dissolve. After cooling,
add freshly boiled and cooled water to make 100 mL: pH of Strontium chloride hexahydrate SrCl2.6H2O [K 8132,
this solution, measured at 259C, is 4.0 – 7.5. Special class]
Purity: Iron—Place 1.0 g of soluble starch in a crucible,
Strychnine nitrate for assay C21H22N2O2.HNO3 To 1 g
moisten with a little amount of sulfuric acid, and heat gradu-
of strychnine nitrate add 14 mL of water and about 10 mg of
ally at a temperature as lower as possible to carbonize com-
active carbon, heat in a water bath for 10 minutes, filter
pletely. After allowing to cool, moisten the residue with a lit-
while hot, cool the filtrate quickly to form crystals, and filter
tle amount of sulfuric acid, heat gradually until white fumes
the crystals. Add 8 mL of water to the crystals, dissolve by
are on longer evolved, then ignite at 600 ± 509C until the
heating in a water bath, filter while hot, cool quickly, and
residue is completely incinerated. After cooling, dissolve the
filter the crystals formed. Repeat this procedure with 8 mL
residue by adding 1 mL of 7.5 mol/L hydrochloric acid TS
of water, and dry the crystals in a desiccator (in vacuum,
and a suitable amount of water, and evaporate to dryness on
silica gel) for 24 hours. Colorless or white, crystals or crys-
a water bath. Dissolve the residue in 4 mL of 7.5 mol/L hy-
talline powder. Sparingly soluble in water and in glycerin,
drochloric acid TS, and add water to make 40 mL. To 10 mL
slightly soluble in ethanol (95), and practically insoluble in
of this solution add water to make 15 mL, and use this solu-
diethyl ether.
tion as the test solution. Separately, to 1.0 mL of Standard
Purity Related substances—Dissolve 35 mg of strychnine
Iron Solution add 7.5 mol/L hydrochloric acid TS to make
nitrate for assay in 100 mL of the mobile phase and use this
15 mL, and use this solution as the control solution. To the
solution as the sample solution. Pipet 2 mL of the sample so-
test solution and the control solution add 1 mL of a solution
lution, add the mobile phase to make exactly 100 mL, and
of hydroxylammonium chloride (1 in 10), mix, and allow
use this solution as the standard solution (1). Perform the
them to stand for 5 minutes, and add 1 mL of a solution of
test with exactly 20 mL each of the sample solution and
1,10-phenanthrolinium chloride monohydrate (7 in 2500)
standard solution (1) as directed under Liquid Chromatogra-
and 5 mL of a solution of ammonium acetate (1 in 4), and
phy <2.01> according to the following conditions. Determine
add water to make 25 mL. After allowing to stand at 20 –
each peak area of these solutions by the automatic integra-
309 C for 15 minutes, compare the color of both solution
tion method: the total area of the peaks other than strych-
against a white background: the solution obtained from the
nine from the sample solution is not larger than the peak
test solution is not more colored than the solution obtained
area of strychnine from the standard solution (1).
from the control solution (not more than 40 ppm).
Operating conditions
Loss on drying <2.41>—Not more than 20z (1 g, 1059C,
Proceed the operating conditions in the Assay under Nux
2 hours).
Vomica except detection sensitivity and time span of meas-
Sensitivity—Mix thoroughly 2.0 g of soluble starch with
urement.
10 mL of water, then add 90 mL of hot water, and boil for 2
Detection sensitivity: Pipet 1 mL of the standard solution
minutes while stirring to dissolve. After allowing to cool to
(1), add the mobile phase to make exactly 40 mL, and use
room temperature, to 2.5 mL of this solution add 97.5 mL
this solution as the standard solution (2). Adjust the detec-
of water and an amount of 0.005 mol/L iodine VS: a blue to
tion sensitivity so that the peak area of strychnine obtained
blue-purple color appears, and the color disappears on the
from 20 mL of the standard solution (2) can be measured by
addition of 0.01 mol/L sodium thiosulfate VS.
the automatic integration method and the peak height of
Starch TS Triturate 1 g of starch with 10 mL of cold strychnine from 20 mL of the standard solution (1) is about
water, and pour the mixture slowly, with constant stirring, 20z of the full scale.
into 200 mL of boiling water. Boil the mixture until a thin, Time span of measurement: About 3 times as long as the
translucent fluid is obtained. Allow to settle, and use the retention time of strychnine, beginning after the solvent
supernatant liquid. Prepare before use. peak.
Loss on drying <2.41>: not more than 0.5z (0.2 g, 1059C,
Stearic acid for gas chromatography C18H36O2
3 hours).
[K 8585, Special class]
Content: not less than 99.0z calculated on the dried
Stearyl alcohol [Same as the namesake monograph] basis. Assay—Dissolve about 0.5 g of strychnine nitrate
for assay, accurately weighed, in 40 mL of a mixture of
Sterile purified water [Same as the monograph Sterile
acetic anhydride and acetic acid (100) (4:1), heat if neces-
Purified Water in Containers. It is not necessary to confirm
sary, cool, and titrate <2.50> with 0.1 mol/L perchloric acid
if they meet all of the requirement, provided that they are
VS (potentiometric titration). Perform a blank determina-
confirmed to be suitable for the purpose of the relevant test.]
tion, and make any necessary correction.
Strong ammonia water See ammonia solution (28).
Each mL of 0.1 mol/L perchloric acid VS
Strong cupric acetate TS See copper (II) acetate TS, ＝ 39.74 mg of C21H22N2O2.HNO3
strong.
Styrene C8H8 Colorless, clear liquid.
Strong hydrogen peroxide water See hydrogen peroxide Specific gravity <2.56> d: 0.902 – 0.910

Purity—Perform the test with 1 mL of styrene as directed Water <2.48>: Not more than 10.0z (10 mg, coulometric
under Gas Chromatography <2.02> according to the follow- titration).
ing conditions. Determine each peak area by the automatic
Subculture medium for nartograstim test Dissolve an
integration method and calculate the amount of styrene by
amount of Nartograstim (Genetical Recombination),
the area percentage method: it shows the purity of not less
equivalent to 0.20 mg in 20 mL of phosphate-buffered so-
than 99z.
dium chloride TS. To 0.1 mL of this solution add 100 mL of
Operating conditions
potency measuring medium for nartograstim test.
Detector: Thermal conductivity detector.
Column: A glass column, about 3 mm in inside diameter Substrate TS for epoetin alfa Dissolve 30 mg of 4-chlo-
and about 2 m in length, packed with siliceous earth (180 to ro-1-naphthol in 10 ml of methanol, and use as Solution A.
250 mm in particle diameter) coated with polyethylene glycol Mix 30 mL of hydrogen peroxide (30) and 50 mL of 0.02
20 M at the ratio of 10z. mol/L tris buffer solution (pH 7.5) and use as Solution B.
Column temperature: A constant temperature of about Mix Solutions A and B before use.
1009C.
Substrate TS for interferon alfa confirmation Dissolve 9
Temperature of sample vaporization chamber: A constant
mg of 3,3?-diaminobenzidine tetrahydrochloride in phos-
temperature of about 1509 C.
phate-buffered sodium chloride TS to make 30 mL. Add 5
Carrier gas: Helium.
mL of hydrogen peroxide (30) to this solution. Prepare be-
Flow rate: Adjust so that the retention time of styrene is
fore use.
about 10 minutes.
Time span of measurement: About twice as long as the Substrate buffer for celmoleukin Dissolve 32.4 g of
retention time of styrene. tripotassium citrate monohydrate in water to make 1000 mL,
and add 1 mol/L citric acid TS for buffer solution to adjust
Styrene-maleic acid alternating copolymer partial butyl
the pH to 5.5. To 100 mL of this solution add and dissolve
ester A copolymer of styrene and maleic anhydride, poly-
0.44 g of o-phenylenediamine and then 60 mL of hydrogen
merized using cumene as solvent and added 1-butanol or
peroxide (30). Prepare at the time of use.
water to the maleic anhydride groups. Average molecular
mass: about 1600. A white or pale yellowish white powder. Substrate TS for lysozyme hydrochloride To a suitable
Identification—Dissolve 5 mg of the substance to be exa- amount of dried cells of Micrococcus luteus add a suitable
mined in sodium hydrogen carbonate solution (1 in 15) to amount of phosphate buffer solution (pH 6.2) gently shake
make 10 mL. Determine the absorption spectrum of this so- to make a suspension, and add the substrate cells or the same
lution as directed under Ultraviolet-visible Spectrophotome- buffer solution so that the absorbance of the suspension at
try <2.24>: it exhibits a maximum between 256 nm and 260 640 nm is about 0.65. Prepare before use.
nm, and a shoulder between 251 nm and 256 nm.
Substrate TS for peroxidase determination Dissolve
Absorbance <2.24> E 11zcm (258 nm): 6.3 – 7.3 [5 mg calcu-
0.195 mL of hydrogen peroxidase (30), 8.38 g of disodium
lated on the anhydrous basis, sodium hydrogen carbonate
hydrogen phosphate dodecahydrate and 1.41 g of citric acid
solution (1 in 15), 10 mL].
monohydrate in water to make 300 mL. To 15 mL of this so-
Purity—Proceed as directed in the Purity (3) under
lution add 13 mg of o-phenylenediamine dihydrochloride be-
Zinostatin Stimalamer, with the exception of without using
fore use.
of (iii) Standard solution, and changing (iv) Sample solution,
(v) Procedure and (vii) Determination as follows: Substrate TS for kallidinogenase assay (1) Dissolve an
(iv) Sample solution Dissolve 3.0 mg of the substance appropriate amount of H-D-valyl-L-leucyl-L-arginine-4-
to be examined in the buffer solution for sample to make 20 nitroanilide dihydrochloride in 0.1 mol/L tris buffer solu-
mL. tion (pH 8.0) to prepare a solution containing 1 mg of H-D-
(v) Procedure Mount the gel in the electrophoresis ap- valyl-L-leucyl-L-arginine-4-nitroanilide dihydrochloride in 5
paratus. Add a mixture of 200 mL of Solution F and 2 mL mL.
of bromophenol blue solution (1 in 100,000) to the top
Substrate TS for kallidinogenase assay (2) Dissolve 17.7
reservoir (cathode) and 300 mL of Solution F to the lower
mg of N-a-benzoyl-L-arginine ethyl ester hydrochloride in
reservoir (anode). Introduce carefully exactly 100 mL of the
0.1 mol/L tris buffer solution (pH 8.0) to make 100 mL.
sample solution onto the surface of the gel, and allow elec-
trophoresis at room temperature to take place with a current Substrate TS for kallidinogenase assay (3) Suspend 0.6 g
of 2 mA per tube as a bromophenol blue band is passing in of milk casein purified by the Hammerstein's method in 80
the stacking gel and then increase the current to 4 mA per mL of 0.05 mol/L sodium hydrogen phosphate TS, and dis-
tube as the bromophenol blue band is passing in the resolv- solve by warming at 659C for 20 minutes. After cooling,
ing gel, and stop the current when the band reached at 5 cm adjust to pH 8.0 with 1 mol/L hydrochloric acid TS or so-
from the upper end of the gel. dium hydroxide TS, and add water to make exactly 100 mL.
(vii) Determination Determine the peak area, AT, of Prepare before use.
styrene-maleic acid alternating copolymer partial butyl ester
Substrate TS for kallidinogenase assay (4) Dissolve 25
and the total area, A, of the peaks other than styrene-maleic
mg of H-D-valyl-L-leucyl-L-arginine-4-nitroanilide dihydro-
acid alternating copolymer partial butyl ester, based on the
chloride in 28.8 mL of water.
absorbance at 600 nm of the gel determined by using a
densitometer. Calculate the amount of styrene-maleic acid N-Succinimidyl 4-(N-maleidomethyl)cyclohexane-1-car-
alternating copolymer partial butyl ester by the following boxylate See 4-(N-Maleidomethyl cyclohexane-1-carboxylic
formula: not less than 98.0z. acid N-succinimidyl ester.
Amount (z) of styrene-maleic acid alternating Succinic acid C4H6O4 Colorless or white crystalline
copolymer partial butyl ester powder. Very soluble in hot water, soluble in water and in
＝ AT/(AT ＋ A) × 100 ethanol (99.5), and sparingly soluble in diethyl ether.
Melting point <2.60>: About 1859C.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
336 General Tests / 9.41 Reagents, Test Solutions JP XVII
Residue on ignition <2.44>: not more than 0.02z (1 g). Internal standard solution—A solution of ethyl parahy-
Content: not less than 99.5z. Assay—Weigh accurately droxybenzoate in the mobile phase (7 in 1000).
about 1 g of succinic acid, dissolve in 50 mL of water, add Operating conditions
5 drops of phenolphthalein TS, and titrate <2.50> with 1 Detector: Ultraviolet absorption photometer (wave-
mol/L sodium hydroxide VS. Perform a blank titration in length: 220 nm).
the same manner, and make any necessary correction. Column: A stainless steel column 3.9 mm in inside diame-
ter and 30 cm in length, packed with octadecylsilanized silica
Each mL of 1 mol/L sodium hydroxide VS
gel for liquid chromatography (10 mm in particle diameter).
＝ 59.05 mg of C4H6O4
Column temperature: A constant temperature of about
Succinic acid, anhydrous C4H4O3 White or pale yellow- 359C.
ish white, crystals or flakes. It is odorless. Soluble in water, Mobile phase: To 750 mL of 0.005 mol/L tetrabutylam-
freely soluble in hot water, and sparingly soluble in ethanol monium hydroxide TS add 250 mL of acetonitrile for liquid
(95). chromatography.
Purity (1) Chloride <1.03>: not more than 0.005z. Flow rate: Adjust so that the retention time of sulbactam
(2) Iron <1.10>: not more than 0.001z. is about 6 minutes.
Residue on ignition <2.44>: not more than 0.1z (1 g). System suitability
Content: not less than 98.0z. Assay—Dissolve about System performance: When the procedure is run with 10
1 g of anhydrous succinic acid, accurately weighed, in 50 mL mL of the standard solution according to the above operating
of water by warming, cool, and titrate <2.50> with 1 mol/L conditions, sulbactam and the internal standard are eluted in
sodium hydroxide VS (indicator: 2 drops of phenolphthalein this order with the resolution between these peaks being not
TS). less than 1.5.
System repeatability: When the test is repeated 6 times
Each mL of 1 mol/L sodium hydroxide VS
with 10 mL of the standard solution according to the above
＝ 50.04 mg of C4H4O3.
operating conditions, the relative standard deviation of the
Sucrose C12H22O11 [K 8383, Special class] (for reduc- peak areas of sulbactam is not more than 2.0z.
tion liquid for molecular mass determination)
Sulfamic acid (standard reagent) See amido sulfuric acid
Sucrose C12H22O11 [Same as the namesake monograph] (standard reagent).
Sucrose for optical rotation C12H22O11 [K 8383, Su- Sulfanilamide H2NC6H4SO2NH2 [K 9066, Special
crose, Special class] class]
Sudan III C22H16N4O Red-brown powder. It dissolves Sulfanilamide for titration of diazotization
in acetic acid (100) and in chloroform, and insoluble in H2NC6H4SO2NH2 [K 9066, For titration of diazotization]
water, in ethanol (95), in acetone and in ether.
Sulfanilic acid H2NC6H4SO3H [K 8586, Special class]
Melting point <2.60>: 170 – 1909 C
Sulfathiazole C9H9N3O3S2 White crystalline powder.
Sudan III TS Dissolve 10 mg of sudan III in 5 mL of
Melting point <2.60>: 200 – 2049C
ethanol (95), filter, and add 5 mL of glycerin to the filtrate.
Prepare before use. Sulfite oxidase One unit indicates an amount of the en-
zyme which consumes 1 mmol of oxygen in 1 minute at 259C
Sulbactam sodium for sulbactam penicillamine
and pH 8.0 using sulfur dioxide and oxygen as the substrate.
C8H10NNaO5S White to yellowish white crystalline pow-
der. Freely soluble in water, and slightly soluble in ethanol Sulfite oxidase TS Suspend sulfite oxidase in ammonium
(95). sulfate TS so that each mL contains 2.5 units of the activity.
Identification—Determine the infrared absorption spec- Storage—Between 0 and 89C.
trum of sulbactam sodium for sulbactam penicillamine ac-
Sulfosalicylic acid See 5-sulfosalicylic acid dihydrate.
cording to the potassium bromide disk method under In-
frared Spectrophotometry <2.25>: it exhibits the absorption 5-Sulfosalicylic acid dihydrate C7H6O6S.2H2O
at the wave numbers of about 1780 cm－1, 1600 cm－1, 1410 [K 8589, Special class]
cm－1, 1400 cm－1, 1320 cm－1, 1300 cm－1, 1200 cm－1 and
Sulfosalicylic acid TS Dissolve 5 g of 5-sulfosalicylic acid
1130 cm－1.
dihydrate in water to make 100 mL.
Water <2.48>: not more than 1.0z (0.5 g).
Content: not less than 875 mg (potency) per mg, calculated Sulfur S [K 8088, Special class]
on the anhydrous basis. Assay—Weigh accurately an
Sulfur dioxide SO2 Prepare by adding sulfuric acid
amount of sulbactam sodium for sulbactam penicillamine
dropwise to a concentrated solution of sodium bisulfite.
and Sulbactam RS, equivalent to about 0.10 g (potency), dis-
Colorless gas, having a characteristic odor.
solve each in a suitable volume of the mobile phase, add ex-
actly 10 mL of the internal standard solution and the mobile Sulfuric acid H2SO4 [K 8951, Special class]
phase to make 100 mL, and use these solutions as the sample
Sulfuric acid, dilute Cautiously add 5.7 mL of sulfuric
solution and the standard solution, respectively. Perform the
acid to 10 mL of water, cool, and dilute with water to make
test with 10 mL each of these solutions as directed under Liq-
100 mL (10z).
uid Chromatography <2.01> according to the following con-
ditions, and calculate the ratios, QT and QS, of the peak area Sulfuric acid-ethanol TS With stirring, add slowly 3 mL
of sulbactam to that of the internal standard. of sulfuric acid to 1000 mL of ethanol (99.5), and cool.
Amount [ mg (potency)] of sulbactam (C8H11NO5S) Sulfuric acid for readily carbonizable substances To sul-
＝ MS × QT/QS × 1000 furic acid, the content of which has previously been deter-
mined by the following method, add water cautiously, and
MS: amount [mg (potency)] of Sulbactam RS taken
adjust the final concentration to 94.5z to 95.5z of sulfuric

acid (H2SO4). When the concentration is changed owing to Sulpiride for assay C15H23N3O4S [Same as the mono-
absorption of water during storage, prepare freshly. graph Sulpiride. When dried, it contains not less than 99.0z
Assay—Weigh accurately about 2 g of sulfuric acid in a of sulpiride (C15H23N3O4S).]
glass-stoppered flask rapidly, add 30 mL of water, cool, and
Sulpyrine See sulpyrine hydrate.
titrate <2.50> the solution with 1 mol/L sodium hydroxide
VS (indicator: 2 to 3 drops of bromothymol blue TS). Sulpyrine for assay See sulpyrine hydrate for assay.
Each mL of 1 mol/L sodium hydroxide VS Sulpyrine hydrate C13H16N3NaO4S.H2O [Same as the
＝ 49.04 mg of H2SO4 namesake monograph]
Sulfuric acid, fuming H2SO4.nSO3 [K 8741, Special Sulpyrine hydrate for assay C13H16N3NaO4S.H2O
class] [Same as the monograph Sulpyrine Hydrate. Calculated on
the dried basis, it contains not less than 99.0z of sulpyrine
Sulfuric acid-hexane-methanol TS To 230 mL of a mix-
(C13H16N3NaO4S).]
ture of methanol and hexane (3:1) add cautiously 2 mL of
sulfuric acid. Suxamethonium chloride for thin-layer chromatography
See suxamethonium chloride hydrate for thin-layer chroma-
Sulfuric acid-methanol TS Prepare carefully by adding
tography.
60 mL of sulfuric acid to 40 mL of methanol.
Suxamethonium chloride hydrate for thin-layer chroma-
Sulfuric acid-methanol TS, 0.05 mol/L Add gradually 3
tography C14H30Cl2N2O4.2H2O [Same as the monograph
mL of sulfuric acid to 1000 mL of methanol, while stirring,
Suxamethonium Chloride Hydrate]
and allow to cool.
Sweet hydrangea leaf dihydroisocoumarin for thin-layer
Sulfuric acid-monobasic sodium phosphate TS See sul-
chromatography White to pale yellow-brown crystalline
furic acid-sodium dihydrogenphosphate TS.
powder of mainly two components, generally obtained from
Sulfuric acid, purified Place sulfuric acid in a beaker, activated charcoal treated fraction of acetone or methanol
heat until white fumes are evolved, then heat for 3 minutes extracts of crumping upped leaves or tip branches of
cautiously and gently. Use after cooling. Hydrangea macrophylla Seringe var. thunbergii Makino
(Saxifragaceae).
Sulfuric acid-sodium dihydrogenphosphate TS Add 6.8
Identification Dissolve 2 mg of the substance to be ex-
mL of sulfuric acid to 500 mL of water, then dissolve 50 g of
amined in 1 mL of methanol, and perform the test with 5 mL
sodium dihydrogenphosphate dihydrate in this solution, and
of this solution as directed in the Identification under Sweet
add water to make 1000 mL.
Hydrangea Leaf: two consecutive spots are observed at Rf
Sulfuric acid-sodium hydroxide TS With stirring add values of about 0.3.
slowly 120 mL of sulfuric acid to 1000 mL of water, and cool
Swertia herb [Same as the namesake monograph]
(solution A). Dissolve 88.0 g of sodium hydroxide in 1000
mL of freshly boiled and cooled water (solution B). Mix Swertiamarin for thin-layer chromatography C16H22O10
equal volumes of solution A and solution B. A white to light yellow powder. Freely soluble in water and
in ethanol (95).
Sulfuric acid TS Cautiously add 1 volume of sulfuric
Identification Determine the infrared absorption spec-
acid to 2 volumes of water, and while warming on a water
trum of swertiamarin to be examined as directed in the po-
bath add dropwise potassium permanganate TS until a pale
tassium bromide disk method under Infrared Spectropho-
red color of the solution remains.
tometry <2.25>: it exhibits absorption at the wave numbers of
0.05 mol/L Sulfuric acid TS Dilute 100 mL of 0.5 mol/L about 3380 cm－1, 1693 cm－1, 1618 cm－1 and 1068 cm－1.
sulfuric acid TS with water to make 1000 mL. Purity Dissolve 2 mg of swertiamarin to be examined in
1 mL of ethanol (95), and use this solution as the sample so-
0.25 mol/L Sulfuric acid TS With stirring, add slowly 15
lution. Pipet 0.5 mL of the sample solution, add ethanol (95)
mL of sulfuric acid to 1000 mL of water, then cool.
to make exactly 25 mL, and use this solution as the standard
0.5 mol/L Sulfuric acid TS With stirring, add slowly 30 solution. Perform the test with these solutions as directed
mL of sulfuric acid to 1000 mL of water, then cool. under Thin-layer Chromatography <2.03>. Spot 20 mL each
of the sample solution and standard solution on a plate of
1 mol/L Sulfuric acid TS Add 60 mL of sulfuric acid in
silica gel with fluorescent indicator for thin-layer chromatog-
1000 mL of water slowly with stirring, then allow to cool.
raphy. Develop the plate with a mixture of ethyl acetate, 1-
2 mol/L Sulfuric acid TS To 1000 mL of water add propanol and water (6:4:3) to a distance of about 10 cm and
gradually 120 mL of sulfuric acid with stirring, and cool. air-dry the plate. Examine under ultraviolet light (main
wavelength: 254 nm): any spot other than the principal spot
5 mol/L Sulfuric acid TS Add 300 mL of sulfuric acid in
with an Rf value of about 0.5 obtained from the sample so-
1000 mL of water slowly with stirring, then allow to cool.
lution is not more intense than the spot obtained from the
Sulfurous acid See sulfurous acid solution. standard solution.
Sulfurous acid solution A clear and colorless liquid con- Syntheric zeolite for drying A mixture of
taining more than 5z of SO2, having a pungent odor. 6(Na2O).6(Al2O3).12(SiO2) and 6(K2O).6(Al2O3).12(SiO2)
Specific gravity: about 1.03 g/mL. prepared for drying. Usually, use the spherically molded
Identification—To 1 mL of iodine TS add 20 mL of water, form, 2 mm in diameter, prepared by adding a binder. White
and add 1 mL of sulfurous acid solution: the color of the so- to grayish white, or color transition by adsorbing water.
lution disappears, and this solution forms a white precipitate Average fine pore diameter is about 0.3 nm, and the surface
upon addition of 1 mL of barium chloride TS. area is 500 to 700 m2 per g.
Preserve at a cold place. Loss on ignition <2.43>: not more than 2.0z [2 g, 550 –
6009C, 4 hours, allow to stand in a desiccator (phosphorus

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
338 General Tests / 9.41 Reagents, Test Solutions JP XVII
(V) oxide).] Identification—Determine the absorption spectrum of a
solution of terphenyl in methanol (1 in 250,000) as directed
System suitability test solution for filgrastim Filgrastim
under Ultraviolet-visible Spectrophotometry <2.24>: it exhib-
(Genetical Recombination) containing about 2z charge
its a maximum between 276 nm and 280 nm.
isomer.
p-Terphenyl See terphenyl.
Talc [Same as the namesake monograph]
Test bacteria inoculation medium for teceleukin Dissolve
Taltirelin hydrate for assay C17H23N7O5.4H2O [Same
6.0 g of peptone, 3.0 g of yeast extract, 1.5 g of meat extract,
as the monograph Taltirelin Hydrate. It contains not less
1.0 g of glucose, and 13.0 to 20.0 g of agar in water to make
than 99.0z of taltirelin (C17H23N7O5), calculated on the an-
1000 mL and sterilize. The pH is 6.5 to 6.6.
hydrous basis.]
Test bacteria inoculation medium slant for teceleukin
Tamsulosin hydrochloride C20H28N2O5S.HCl [Same as
Sterilized slant culture obtained by adding approximately 9
the namesake monograph]
mL of bacteria inoculation medium for teceleukin to a test
Tamsulosin hydrochloride for assay C20H28N2O5S.HCl tube with an inside diameter of 16 mm.
[Same as the monograph Tamsulosin Hydrochloride. When
Testosterone C19H28O2 White, crystals or crystalline
dried, it contains not less than 99.0z of tamsulosin hydro-
powder.
chloride (C20H28N2O5S.HCl).]
Identification—Determine the infrared absorption spec-
Tannic acid [Same as the namesake monograph] trum of testosterone as directed in the potassium bromide
disk method under Infrared Spectrophotometry <2.25>: it
Tannic acid TS Dissolve 1 g of tannic acid in 1 mL of
exhibits the absorption at the wave numbers of about 3530
ethanol (95), and add water to make 10 mL. Prepare before
cm－1, 3380 cm－1, 1612 cm－1, 1233 cm－1, 1067 cm－1 and
use.
1056 cm－1.
Tartaric acid See L-tartaric acid.
Testosterone propionate C22H32O3 [Same as the name-
L-Tartaric acid C 4 H 6 O6 [K 8532, L(＋)-Tartaric acid, sake monograph]
Special class].
Tetrabromophenolphthalein ethyl ester potassium salt
Tartrate buffer solution (pH 3.0) Dissolve 1.5 g of L-tar- C22H13Br4KO4 [K 9042, Special class]
taric acid and 2.3 g of sodium tartarate dihydrate in water to
Tetrabromophenolphthalein ethyl ester TS Dissolve
make 1000 mL.
0.1 g of tetrabromophenolphthalein ethyl ester potassium
Taurine H2NCH2CH2SO3H White, crystals or crystal- salt in acetic acid (100) to make 100 mL. Prepare before use.
line powder.
Tetra-n-butylammonium bromide [CH3(CH2)3]4NBr
Contents: not less than 95.0z. Assay—Weigh accurately
White, crystals or crystalline powder, having a slight, char-
about 0.2 g, dissolve in 50 mL of water, add 5 mL of formal-
acteristic odor.
dehyde solution, and titrate <2.50> with 0.1 mol/L sodium
Melting point <2.60>: 101 – 1059C
hydroxide VS (indicator: 3 drops of phenolphthalein TS).
Purity Clarity and color of solution—Dissolve 1.0 g of
Perform a blank determination in the same manner, and
tetra-n-butylammonium bromide in 20 mL of water: the so-
make any necessary correction.
lution is clear and colorless.
Each mL of 0.1 mol/L sodium hydroxide VS Content: not less than 98.0z. Assay—Dissolve about
＝ 12.52 mg of C2H7NO3S 0.5 g of tetra-n-butylammonium bromide, accurately
weighed, in 50 mL of water, add 5 mL of dilute nitric acid,
Telmisartan for assay C33H30N4O2 [Same as the mono-
and titrate <2.50> with 0.1 mol/L silver nitrate VS while
graph Telmisartan]
strongly shaking (potentiometric titration). Perform a blank
Temocapril hydrochloride for assay C23H28N2O5S2.HCl determination and make any necessary correction.
[Same as the monograph Temocapril Hydrochloride. It
Each mL of 0.1 mol/L silver nitrate VS
contains not less than 99.5z of temocapril hydrochloride
＝ 32.24 mg of C16H36NBr
(C23H28N2O5S2.HCl: 513.07), calculated on the anhydrous
basis.] Tetra-n-butylammonium chloride C16H36ClN White
crystals, and it is deliquescent.
Terbinafine hydrochloride for assay C21H25N.HCl
Water <2.48>: not more than 6.0z (0.1 g).
[Same as the monograph Terbinafine Hydrochloride]
Content: not less than 95.0z, calculated on the anhydrous
Terephthalic acid C6H4(COOH)2 White, crystals or basis. Assay—Weigh accurately about 0.25 g of tetra-n-
crystalline powder. Slightly soluble in ethanol (95), and prac- butylammonium chloride, dissolve in 50 mL of water, and
tically insoluble in water and in diethyl ether. titrate <2.50> with 0.1 mol/L silver nitrate VS (potentiomet-
Residue on ignition <2.44>: not more than 0.3z (1 g). ric titration).
Content: not less than 95.0z. Assay—Weigh accurately
Each mL of 0.1 mol/L silver nitrate VS
about 2 g of terephthalic acid, dissolve in exactly 50 mL of 1
＝ 27.79 mg of C16H36ClN
mol/L sodium hydroxide VS, and titrate <2.50> with 1
mol/L hydrochloric acid VS (indicator: 3 drops of phenol- Tetrabutylammonium hydrogensulfate C16H37NO4S
phthalein TS). Perform a blank determination in the same White crystalline powder.
manner. Content: not less than 98.0z. Assay—Weigh accurately
about 0.7 g of tetrabutylammonium hydrogensulfate, dis-
Each mL of 1 mol/L sodium hydroxide VS
solve in 100 mL of freshly boiled and cooled water, and
＝ 83.07 mg of C8H6O4
titrate <2.50> with 0.1 mol/L sodium hydroxide VS (indica-
Terphenyl C18H14 White crystalline powder. Melting tor: 3 drops of bromocresol green-methyl red TS).
point: 208 – 2139C

Each mL of 0.1 mol/L sodium hydroxide VS Amount [ mg (potency)] of tetracycline (C22H24N2O8)

＝ 33.95 mg of C16H37NO4S ＝ MS × (AT/AS) × 1000
40z Tetrabutylammonium hydroxide TS A solution MS: Amount [mg (potency)] of Tetracycline Hydrochlo-
containing 40 g/dL of tetrabutylammonium hydroxide ride RS taken
[(C4H9)4NOH: 259.47].
Tetracycline Hydrochloride C22H24N2O8.HCl Yellow,
Content: 36 – 44 g/dL. Assay—Pipet 10 mL of 40z
crystals or crystalline powder.
tetrabutylammonium hydroxide TS, and titrate <2.50> with 1
Purity Related substances—Dissolve 20 mg of tetracy-
mol/L hydrochloric acid VS (indicator: 3 drops of methyl
cline hydrochloride in 0.01 mol/L hydrochloric acid TS to
red TS).
make 25 mL, and use this solution as the sample solution.
Each mL of 1 mol/L hydrochloric acid VS Proceed the test with 20 mL of the sample solution as di-
＝ 259.5 mg of C16H37NO rected in the Purity (2) under Oxytetracycline Hydrochlo-
ride, determine each peak area by the automatic integration
0.005 mol/L Tetrabutylammonium hydroxide TS To 10
method, and calculate the amounts of them by the area per-
mL of tetrabutylammonium hydroxide TS add 700 mL of
centage method: the total amount of the peaks other than
water, adjust to pH 4.0 with diluted phosphoric acid (1 in
tetracycline is not more than 10z.
10), and add water to make 1000 mL.
Tetradecyl trimethylammonium bromide
Tetrabutylammonium hydroxide-methanol TS Methanol
CH3(CH2)13N(CH3)3Br A white powder.
solution containing 25 g/dL of tetrabutylammonium hy-
Purity Clarity and color of solution—Dissolve 1.0 g of
droxide [(C4H9)4NOH: 259.47]. Colorless to pale yellow
tetradecyl trimethylammonium bromide in 20 mL of water:
solution, having an ammonium-like odor.
the solution is clear and colorless.
Content: 22.5 – 27.5 g/dL. Assay—Pipet 15 mL of
Content: not less than 98.0z. Assay—Weigh accurately
tetrabutylammonium hydroxide-methanol TS and titrate
about 0.5 g of tetradecyl trimethylammonium bromide, dis-
<2.50> with 1 mol/L hydrochloric acid VS (indicator: 3 drops
solve in 100 mL of water, add 5 mL of a mixture of water
of methyl red TS).
and nitric acid (2:1), and titrate <2.50> with 0.1 mol/L silver
Each mL of 1 mol/L hydrochloric acid VS nitrate VS (potentiometric titration). Perform a blank test in
＝ 259.5 mg of C16H37NO the same manner, and make any necessary correction.
10z Tetrabutylammonium hydroxide-methanol TS A Each mL of 0.1 mol/L silver nitrate VS
methanol solution containing 10 g/dL of tetrabutylammoni- ＝ 33.64 mg of C17H38NBr
um hydroxide [(C4H9)4NOH: 259.47].
Tetraethylammonium hydroxide TS A solution contain-
Content: 9.0 – 11.0 g/dL. Assay—Pipet 2 mL of 10z
ing 10z of tetraethylammonium hydroxide [(C2H5)4NOH:
tetrabutylammonium hydroxide-methanol TS, transfer to a
147.26]. A clear, colorless liquid, having a strong ammonia
glass-stoppered flask containing 20 mL of water, and titrate
odor. It is a strong basic and easily absorbs carbon dioxide
<2.50> with 0.1 mol/L hydrochloric acid VS (indicator: 3
from the air.
drops of methyl red TS).
Content: 10.0 – 11.0z Assay—Weigh accurately about 3 g
Each mL of 0.1 mol/L hydrochloric acid VS of tetraethylammonium hydroxide in a glass-stoppered flask
＝ 25.95 mg of C16H37NO containing 15 mL of water, and titrate <2.50> with 0.1 mol/L
hydrochloric acid VS (indicator: 3 drops of methyl red TS).
Tetrabutylammonium hydroxide TS A solution con-
Perform a blank determination in the same manner, and
taining 13 g/dL of tetrabutylammonium hydroxide
make any necessary correction.
[(C4H9)4NOH: 259.47].
Content: 11.7 – 14.3 g/dL. Assay—Pipet a quantity, Each mL of 0.1 mol/L hydrochloric acid VS
equivalent to about 0.3 g of tetrabutylammonium hydroxide ＝ 14.73 mg of C8H21NO
[(C4H9)4NOH], transfer to a glass-stoppered flask containing
Tetra-n-heptylammonium bromide [CH3(CH2)6]4NBr
15 mL of water, accurately weighed, and titrate <2.50> with
White, crystals or crystalline powder, having a slight, char-
0.1 mol/L hydrochloric acid VS (indicator: 3 drops of
acteristic odor.
methyl red TS).
Melting point <2.60>: 87 – 899C
Each mL of 0.1 mol/L hydrochloric acid VS Content: not less than 98.0z. Assay—Dissolve about
＝ 25.95 mg of C16H37NO 0.5 g of tetra-n-heptylammonium bromide, accurately
weighed, in 50 mL of diluted acetonitrile (3 in 5),and 5 mL
Tetrabutylammonium phosphate (C4H9)4NH2PO4
of dilute nitric acid, and titrate <2.50> with 0.1 mol/L silver
White powder. It is soluble in water.
nitrate VS while strongly shaking (potentiometric titration).
Content: not less than 97.0z. Assay—Weigh accurately
Perform a blank determination and make any necessary cor-
1.5 g of tetrabutylammonium phosphate, dissolve in 80 mL
rection.
of water, and titrate <2.50> with 0.5 mol/L sodium hydrox-
ide VS (potentiometric titration). Perform a blank determi- Each mL of 0.1 mol/L silver nitrate VS
nation, and make any necessary correction. ＝ 49.07 mg C28H60NBr
Each mL of 0.5 mol/L sodium hydroxide VS Tetrahydrofuran CH2(CH2)2CH2O [K 9705, Special
＝ 169.7 mg of (C4H9)4NH2PO4 class]
Tetracycline C22H24N2O8 Yellow to dark yellow, crys- Tetrahydrofuran for gas chromatography Use tetra-
tals or crystalline powder. Sparingly soluble in ethanol, and hydrofuran prepared by distilling with iron (II) sulfate hep-
very slightly soluble in water. tahydrate.
Content: it contains not less than 870 mg (potency) per mg. Storage—Preserve in containers, in which the air has been
Assay—Proceed as directed in the Assay under Tetracycline displaced by nitrogen, in a dark, cold place.
Hydrochloride. However, use the following formula.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
340 General Tests / 9.41 Reagents, Test Solutions JP XVII
Tetrahydrofuran for liquid chromatography C4H8O (1 in 10).
Clear and colorless liquid.
3,3?,5,5?-Tetramethylbenzidine dihydrochlorate dihydrate
Refractive index <2.45> n 20
D : 1.406 – 1.409
C16H22Cl2N2.2H2O White to slightly reddish-white crystal-
Density <2.56> 0.884 – 0.889 g/mL (209 C)
line powder.
Purity Ultraviolet absorbing substances—Determine the
absorption spectrum of tetrahydrofuran for liquid chroma- N, N, N?, N?-Tetramethylethylenediamine
tography as directed under Ultraviolet-visible Spectropho- (CH3)2NCH2CH2N(CH3)2 Pale yellow clear liquid.
tometry <2.24>, using water as the blank: the absorbences at Specific gravity <2.56> d 20
4 : 0.774 – 0.799
240 nm, 254 nm, 280 nm, 290 nm, and between 300 nm and Content: not less than 99.0z.
400 nm are not more than 0.35, 0.20, 0.05, 0.02 and 0.01, re-
Tetramethylsilane for nuclear magnetic resonance spec-
spectively.
troscopy (CH3)4Si Prepared for nuclear magnetic reso-
Peroxide—Perform the test according to the method
nance spectroscopy.
described in JIS K 9705: not more than 0.01z.
Tetra-n-pentylammonium bromide [CH3(CH2)4]4NBr
Tetrahydroxyquinone C6H4O6 Dark blue crystals. Its
White, crystals or crystalline powder. It is hygroscopic.
color changes to yellow on exposure to light. Soluble in
Melting point <2.60>: 100 – 1019C
ethanol (95) and sparingly soluble in water.
Tetraphenylboron sodium See sodium tetraphenyl-
Tetrahydroxyquinone indicator Mix 1 g of tetrahydroxy-
borate.
quinone with 100 g of sucrose homogeneously.
Tetra-n-propylammonium bromide [CH3CH2CH2]4NBr
Tetrakishydroxypropylethylenediamine for gas chroma-
White, crystals or crystalline powder.
tography Prepared for gas chromatography.
Purity Clarity and color of solution—Dissolve 1.0 g of
Tetramethylammonium hydroxide (CH3)4NOH Ordi- tetra-n-propylammonium bromide in 20 mL of water: the so-
narily, available as an approximately 10z aqueous solution, lution is clear and colorless.
which is clear and colorless, and has a strong ammonia-like Content: not less than 98.0z. Assay—Weigh accurately
odor. Tetramethylammonium hydroxide is a stronger base about 0.4 g of tetra-n-propylammonium bromide, dissolve in
than ammonia, and rapidly absorbs carbon dioxide from the 50 mL of water, add 5 mL of dilute nitric acid, and titrate
air. Use a 10z aqueous solution. <2.50> with 0.1 mol/L silver nitrate VS while shaking
Purity Ammonia and other amines—Weigh accurately a strongly (potentiometric titration).
quantity of the solution, corresponding to about 0.3 g of
Each mL of 0.1 mol/L silver nitrate VS
tetramethylammonium hydroxide [(CH3)4NOH], in a weigh-
＝ 26.63 mg of C12H28NBr
ing bottle already containing 5 mL of water. Add a slight
excess of 1 mol/L hydrochloric acid TS (about 4 mL), and Theophylline C7H8N4O2 White powder. Slightly solu-
evaporate on a water bath to dryness. The mass of the ble in water.
residue (tetramethylammonium chloride), dried at 1059C for Melting point <2.60>: 269 – 2749C
2 hours and multiplied by 0.8317, represents the quantity of Purity Caffeine, theobromine or paraxanthine—To
tetramethylammonium hydroxide [(CH3)4NOH], and cor- 0.20 g of theophylline add 5 mL of potassium hydroxide TS
responds to ±0.2z of that found in the Assay. or 5 mL of ammonia TS: each solution is clear.
Residue on evaporation: not more than 0.02z (5 mL, Loss on drying <2.41>: not more than 0.5z (1 g, 1059C,
1059C, 1 hour). 4 hours).
Content: not less than 98z of the labeled amount. Content: not less than 99.0z. Assay—Weigh accurately
Assay—Accurately weigh a glass-stoppered flask containing about 0.25 g of theophylline, previously dried, dissolve it in
about 15 mL of water. Add a quantity of the solution, 40 mL of N, N-dimethylformamide, and titrate <2.50> with
equivalent to about 0.2 g of tetramethylammonium hydrox- 0.1 mol/L sodium methoxide VS (indicator: 3 drops of
ide [(CH3)4NOH], weigh again, and titrate <2.50> with 0.1 thymol blue-N, N-dimethylformamide TS). Perform a blank
mol/L hydrochloric acid VS (indicator: methyl red TS). determination and make any necessary correction.
Each mL of 0.1 mol/L hydrochloric acid VS Each mL of 0.1 mol/L sodium methoxide VS
＝ 9.115 mg of C4H13NO ＝ 18.02 mg of C7H8N4O2
Tetramethylammonium hydroxide-methanol TS A meth- Theophylline for assay C7H8N4O2 [Same as the mono-
anol solution containing of 10 g/dL of tetramethylammo- graph Theophylline meeting the following additional spe-
nium hydroxide [(CH3)4NOH: 91.15] cifications.]
Content: 9.0 – 11.0 g/dL. Assay—Pipet 2 mL of tetra- Purity Related substances—Dissolve 50 mg of theophyl-
methylammonium hydroxide-methanol TS, transfer to a line for assay in water to make 100 mL, and use this solution
glass-stoppered flask containing 20 mL of water, and titrate as the sample solution. Pipet 1 mL of the sample solution,
<2.50> with 0.1 mol/L hydrochloric acid VS (indicator: add water to make exactly 200 mL, and use this solution as
bromocresol green-methyl red TS). the standard solution. Perform the test with exactly 20 mL of
the sample solution and standard solution as directed under
Each mL of 0.1 mol/L hydrochloric acid VS
Liquid Chromatography <2.01>. Determine each peak area
＝ 9.115 mg of C4H13NO
from both solutions by the automatic integration method:
Tetramethylammonium hydroxide TS Pipet 15 mL of the total area of peaks other than theophylline obtained
tetramethylammonium hydroxide, and add ethanol (99.5) to from the sample solution is not larger than the peak area of
make exactly 100 mL. theophylline obtained from the standard solution.
Operating conditions
Tetramethylammonium hydroxide TS (pH 5.5) To 10
Detector: An ultraviolet absorption photometer (wave-
mL of tetramethylammonium hydroxide add 990 mL of
length: 270 nm).
water, and adjust the pH to 5.5 with diluted phosphoric acid
Column: A stainless steel column 6 mm in inside diameter

and 15 cm in length, packed with octadecylsilanized silica gel 1169C.

for liquid chromatography (5 mm in particle diameter).
Thioacetamide-alkaline glycerin TS To 0.2 mL of a solu-
Column temperature: A constant temperature of about
tion of thioacetamide (1 in 25) add 1 mL of alkaline glycerin
409 C.
TS, and heat for 20 seconds in a water bath. Prepare before
Mobile phase: A mixture of diluted acetic acid (100) (1 in
use.
100) and methanol (4:1).
Flow rate: Adjust so that the retention time of theophyl- Thioacetamide TS To 0.2 mL of a solution of thioa-
line is about 10 minutes. cetamide (1 in 25) add 1 mL of a mixture of 15 mL of so-
Time span of measurement: About 3 times as long as the dium hydroxide TS, 5 mL of water and 20 mL of 85z glyce-
retention time of theophylline. rin, and heat in a water bath for 20 seconds. Prepare before
System suitability use.
Test for required detectability: Pipet 5 mL of the standard
Thiodiglycol S(CH2CH2OH)2 [ b-Thiodiglycol for
solution, and add water to make exactly 25 mL. Confirm
amino acid autoanalysis] Colorless or pale yellow, clear
that the peak area of theophylline obtained from 20 mL of
liquid.
this solution is equivalent to 15 to 25z of that of theophyl-
Specific gravity <2.56> d 20
20: 1.180 – 1.190
line obtained from 20 mL of the standard solution.
Water <2.48>: not more than 0.7z.
System performance: When the procedure is run with 20
mL of the standard solution under the above operating con- Thioglycolate medium I for sterility test See fluid
ditions, the number of theoretical plates and the symmetry thioglycolate medium.
factor of the peak of theophylline are not less than 3000 and
Thioglycolate medium II for sterility test See alternative
not more than 1.5, respectively.
thioglycolate medium.
System repeatability: When the test is repeated 6 times
with 20 mL of the standard solution under the above operat- Thioglycolic acid See mercapto acetic acid.
ing conditions, the relative standard deviation of the peak
Thionyl chloride SOCl2 A colerless or light yellow,
area of theophylline is not more than 3.0z.
clear liquid, having a pungent odor.
Thermolysin It has the activity of 50 – 100 units per mg Specific gravity <2.56> d 20
20: about 1.65 (Method 3).
protein. Origin: Bacillus thermoproteolyticus rokko. Content: not less than 95.0z. Assay—Weigh accurately
0.1 g of thionyl chloride in a weighing bottle, put the bottle
Thiamine nitrate C12H17N5O4S [Same as the namesake
in a glass-stoppered conical flask containing 50 mL of water
monograph]
cooled to about 59 C, stopper immediately, dissolve the sam-
Thianthol [Same as the monograph Thianthol. Proceed ple thoroughly, and transfer the solution to a 200-mL
as directed in the Identification (3) under Sulfur, Salicylic beaker. Wash the conical flask and the weighing bottle in it
Acid and Thianthol Ointment: any spot other than the prin- with 30 mL of water, and combine the washings and the so-
cipal spot does not appear.] lution in the beaker. Add 1 drop of an aqueous solution of
polyvinyl alcohol (1 in 10), and titrate <2.50> with 0.1 mol/L
3-Thienylethylpenicillin sodium C14H15N2NaO4S2
silver nitrate VS (potentiometric titration). Perform a blank
White to pale yellowish white powder. Very soluble in water,
determination in the same manner, and make any necessary
freely soluble in methanol, and sparingly soluble in ethanol
correction.
(95).
Optical rotation <2.49> [a]20
D : ＋265 – ＋2909(0.5 g calcu- Each mL of 0.1 mol/L silver nitrate VS
lated on the anhydrous bases, water, 50 mL, 100 mm). ＝ 5.949 mg of SOCl2
Water <2.48>: Not more than 10.0z (0.2 g, volumetric
Thiopental for assay C11H18N2O2S Dissolve 10 g of
titration, direct titration).
thiopental sodium in 300 mL of water. To this solution add
Content: not less than 90z calculated on the anhydrous
slowly 50 mL of dilute hydrochloric acid with stirring. Take
basis. Assay—Weigh accurately about 0.1 g of 3-
the produced crystals by filtration, wash with water until the
thienylethylpenicillin sodium, dissolve in 35 mL of water,
filtrate indicates no reaction to chloride, and air-dry. Add
add 0.75 mL of 0.1 mol/L hydrochloric acid TS, and adjust
diluted ethanol (3 in 5), dissolve by heating in a water bath,
to pH 8.5 with 0.1 mol/L sodium hydroxide TS. To this so-
allow to stand, and take the produced crystals by filtration.
lution add 2 mL of a penicillinase solution prepared by dis-
Air-dry the crystals in air, and dry again at 1059C for 4
solving penicillinase, equivalent to 513,000 Levy units, in 25
hours. White, odorless crystals.
mL of water and neutralizing with dilute sodium hydroxide
Melting point <2.60>: 159 – 1629C
TS until a pale red color appears with 1 drop of a solution of
Purity (1) Clarity and color of solution—Dissolve 1.0 g
phenolphthalein in ethanol (95) (1 in 1000) as indicator, and
of thiopental for assay in 10 mL of ethanol (99.5): the solu-
allow to stand at 259 C for 5 minutes. Titrate <2.50> this solu-
tion is clear and light yellow.
tion with 0.1 mol/L sodium hydroxide VS until the solution
(2) Related substances—Dissolve 50 mg of thiopental for
reaches pH 8.5 (potentiometric titration). Use the water
assay in 15 mL of acetonitrile, add water to make 50 mL,
freshly boiled and cooled.
and use this solution as the sample solution. Pipet 1 mL of
Each mL of 0.1 mol/L sodium hydroxide VS the sample solution, add the mobile phase in the Purity (4)
＝ 36.24 mg of C14H15N2NaO4S2 under Thiopental Sodium to make exactly 200 mL, and use
this solution as the standard solution. Proceed as directed in
Thimerosal C9H9HgNaO2S White or yellowish crystal-
Purity (4) under Thiopental Sodium.
line powder. Freely soluble in water.
Loss on drying <2.41>: not more than 0.20z (1 g, 1059C,
Melting point <2.60>: 107 – 1149
C
3 hours).
Thioacetamide C2H5NS A white crystalline powder or Content: not less than 99.0z. Assay—Weigh accurately
colorless crystals, having a characteristic odor. Freely solu- about 0.35 g of thiopental for assay, previously dried, dis-
ble in water and in ethanol (99.5). Melting point: 112 – solve in 5 mL of ethanol (99.5) and 50 mL of chloroform,

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
342 General Tests / 9.41 Reagents, Test Solutions JP XVII
and titrate <2.50> with 0.1 mol/L potassium hydroxide- as directed in the Purity (3) under Aceglutamide Aluminum:
ethanol VS (potentiometric titration). Perform a blank de- any peak does not appear at the retention time of acegluta-
termination, and make any necessary correction. mide.
Each mL of 0.1 mol/L potassium hydroxide-ethanol VS Thymine for liquid chromatography C5H6N2O2 Occurs
＝ 24.23 mg of C11H18N2O2S as a white powder.
Purity—Dissolve 10 mg of the substance to be examined in
Thiopental sodium C11H17N2NaO2S [Same as the
100 mL of methanol, add the mobile phase to make 250 mL,
namesake monograph]
and use this solution as the sample solution. Pipet 5 mL of
Thiosemicarbazide H2NCSNHNH2 White, crystals or this solution, add the mobile phase to make exactly 100 mL,
crystalline powder. and use this solution as the standard solution. Pipet 10 mL
Identification—Determine the infrared absorption spec- each of these solutions and perform the test as directed in the
trum as directed in the potassium bromide disk method as di- Purity (3) under Zidovudine. Determine the area of each
rected under Infrared Spectrophotometry <2.25>: it exhibits peak in the sample and standard solutions by the automatic
absorption at the wave numbers of about 3370 cm－1, 3180 integration method: the total area of peaks other than thy-
cm－1, 1648 cm－1, 1622 cm－1, 1535 cm－1, 1288 cm－1, 1167 mine from the sample solution is not larger than that from
cm－1, 1003 cm－1 and 803 cm－1. the standard solution. However, the time span of meas-
urement is about 10 times the retention time of thymine, be-
Thiourea H2NCSNH2 [K 8635, Special class]
ginning after the solvent peak.
Thiourea TS Dissolve 10 g of thiourea in water to make
Thymol CH3C6H3(OH)CH(CH3)2 [Same as the name-
100 mL.
sake monograph]
L-Threonine C4H9NO3 [Same as the namesake mono-
Thymol blue C27H30O5S [K 8643, Special class]
graph]
Thymol blue-N, N-dimethylformamide TS Dissolve 0.1 g
Threoprocaterol hydrochloride C16H22N2O3.HCl To
of thymol blue in 100 mL of N, N-dimethylformamide.
procaterol hydrochloride add 10 volumes of 3 mol/L hydro-
chloric acid TS, heat, and reflux for 3 hours. After cooling, Thymol blue-1,4-dioxane TS Dissolve 50 mg of thymol
neutralize (pH 8.5) with sodium hydroxide TS, and collect blue in 100 mL of 1,4-dioxane, and filter if necessary. Pre-
the crystals produced. Suspend the crystals in water, dissolve pare before use.
by acidifying the solution at pH 1 to 2 with addition of hy-
Thymol blue TS Dissolve 0.1 g of thymol blue in 100 mL
drochloric acid, neutralize (pH 8.5) by adding sodium hy-
of ethanol (95), and filter if necessary.
droxide TS, and separate the crystals produced. Suspend the
crystals in 2-propanol, and acidify the solution at pH 1 to 2 Thymol blue TS, dilute Dissolve 50 mg of thymol blue in
by adding hydrochloric acid. The crystals are dissolved and 100 mL of ethanol (99.5), and filter if necessary. Prepare be-
reproduced. Collect the crystals, dry at about 609C while fore use.
passing air. White to pale yellowish white, odorless, crystals
Thymol for assay C10H14O [Same as the monograph
or crystalline powder. Melting point: about 2079C (with de-
Thymol. It contains not less than 99.0z of thymol
composition).
(C10H14O).]
Purity—Dissolve 0.10 g of threoprocaterol hydrochloride
in 100 mL of diluted methanol (1 in 2), and use this solution Thymol for spraying test solution C10H14O White,
as the sample solution. Perform the test with 2 mL of the crystals or crystalline powder, having an aromatic odor.
sample solution as directed under Liquid Chromatography Very soluble in methanol and in ethanol (99.5), and practi-
<2.01> according to the operating conditions in the Purity (3) cally insoluble in water.
under Procaterol Hydrochloride Hydrate. Measure each Identification—Determine the infrared absorption spec-
peak area by the automatic integration method, and calcu- trum as directed in the potassium bromide disk method
late the amount of threoprocaterol by the area percentage under Infrared Spectrophotometry <2.25>: it exhibits absorp-
method: it shows the purity of not less than 95.0z. Adjust tion at the wave numbers of about 2960 cm－1, 1420 cm－1,
the detection sensitivity so that the peak height of 1290 cm－1, 1090 cm－1 and 810 cm－1.
threoprocaterol obtained from 2 mL of the solution prepared Melting point <2.60>: 49 – 529C
by diluting 5.0 mL of the sample solution with diluted meth- Purity Otherphenols—Shake vigorously 1.0 g of the
anol (1 in 2) to make 100 mL, is 5 to 10z of the full scale, substance to be examined with 20 mL of warm water for 1
and the time span of measurement is about twice as long as minute, and filter. To 5 mL of the filtrate add 1 drop of a so-
the retention time of threoprocaterol, beginning after the lution of iron (III) chloride hexahydrate (27 in 100): the solu-
solvent peak. tion reveals a green but not a blue to purple color.
Thrombin [Same as the namesake monograph] Thymolphthalein C28H30O4 [K 8642, Special class]
Thymine C5H6N2O2 Thymolphthalein TS Dissolve 0.1 g of thymolphthalein
Identification—Determine the infrared absorption spec- in 100 mL of ethanol (95), and filter if necessary.
trum of thymine, previously dried at 1059C for 3 hours, as
Thymol-sulfuric acid-methanol TS for spraying Dissolve
directed in the potassium bromide disk method under In-
1.5 g of thymol for spraying test solution in 100 mL of meth-
frared Spectrophotometry <2.25>: it exhibits absorption at
anol, and add 5.7 mL of sulfuric acid.
the wave numbers of about 3030 cm－1, 1734 cm－1, 1676
cm－1, 1446 cm－1 and 814 cm－1. Tiaramide hydrochloride for assay C15H18ClN3O3S‚HCl
Purity Related substances—Dissolve 50 mg of thymine [Same as the monograph Tiaramide Hydrochloride. When
in 100 mL of methanol. To 10 mL of this solution add the dried, it contains not less than 99.0z of tiaramide hydro-
mobile phase to make 100 mL, and use this solution as the chloride (C15H18ClN3O3S.HCl).]
sample solution. Proceed with 10 mL of the sample solution
Tiapride hydrochloride for assay C15H24N2O4S.HCl

[Same as the monograph Tiapride Hydrochloride] drochloric acid. Preserve in glass-stoppered bottles in which
a fragment of tin has been placed. Use within 1 month.
Ticlopidine hydrochloride for assay C14H14ClNS.HCl
[Same as the monograph Ticlopidine Hydrochloride. It Tin (II) chloride TS, acidic Dissolve 8 g of Tin (II) chlo-
meets the following additional requirements.] ride dihydrate in 500 mL of hydrochloric acid. Preserve in
Purity Related substances—Dissolve 0.2 g of ticlopidine glass-stoppered bottles. Use within 3 months.
hydrochloride for assay in 100 mL of a mixture of water and
Tipepidine hibenzate for assay C15H17NS2.C14H10O4
methanol (1:1), and use this solution as the sample solution.
[Same as the monograph Tipepidine Hibenzate. When dried,
Pipet 1 mL of the sample solution, add a mixture of water
it contains not less than 99.0z of tipepidine hibenzate
and methanol (1:1) to make exactly 100 mL. Pipet 1 mL of
(C15H17NS2.C14H10O4).]
this solution, add a mixture of water and methanol (1:1) to
make exactly 10 mL, and use this solution as the standard Titanium dioxide See titanium (IV) oxide.
solution. Perform the test with exactly 10 mL each of the
Titanium dioxide TS See titanium (IV) oxide TS.
sample solution and standard solution as directed under Liq-
uid Chromatography <2.01> according to the following con- Titanium (III) chloride (20) TiCl3 [K 8401, Titanium
ditions, and determine each peak area by the automatic in- (III) chloride solution, Special class] Store in light-
tegration method: the area of the peak other than ticlopidine resistant, glass-stoppered containers.
obtained from the sample solution is not larger than the peak
Titanium (III) chloride-sulfuric acid TS Mix carefully 20
area of ticlopidine obtained from the standard solution, and
mL of titanium (III) chloride TS and 13 mL of sulfuric acid,
the total area of the peaks other than ticlopidine from the
add carefully hydrogen peroxide (30) in small portions until
sample solution is not larger than 2 times the peak area of
a yellow color develops, and heat until white fumes evolve.
ticlopidine from the standard solution.
After cooling, add water, heat again in the same manner,
Operating conditions
repeat this procedure until the solution is colorless, and add
Detector: An ultraviolet absorption photometer (wave-
water to make 100 mL.
length: 220 nm).
Column: A stainless steel column 4.6 mm in inside diame- Titanium (III) chloride TS To titanium (III) chloride (20)
ter and 15 cm in length, packed with octadecylsilanized silica add dilute hydrochloric acid to obtain a solution containing
gel for liquid chromatography (5 mm in particle diameter). 15 g/dL of titanium (III) chloride (TiCl3). Prepare before
Column temperature: A constant temperature of about use.
409 C. Content: 14.0 – 16.0 g/dL. Assay—To exactly 2 mL of
Mobile phase: A mixture of 0.05 mol/L phosphate buffer titanium (III) chloride TS add 200 mL of water and 5 mL of
solution (pH 3.5) and methanol (1:1). a hydrochloric acid solution (2 in 3), and titrate <2.50> with
Flow rate: Adjust so that the retention time of ticlopidine 0.1 mol/L ferric ammonium sulfate VS under carbon diox-
is about 8 minutes. ide until a slight red color develops in the solution (indicator:
Time span of measurement: About 7 times as long as the 5 mL of ammonium thiocyanate TS).
retention time of ticlopidine, beginning after the solvent
Each mL of 0.1 mol/L ferric ammonium sulfate VS
peak.
＝ 15.42 mg of TiCl3
System suitability
Test for required detectability: Pipet 2 mL of the standard Titanium (IV) oxide TiO2 [K 8703, Special class]
solution, and add a mixture of water and methanol (1:1) to
Titanium (IV) oxide TS To 100 mL of sulfuric acid add
make exactly 10 mL. Confirm that the peak area of ticlopi-
0.1 g of titanium (IV) oxide, and dissolve by gradually heat-
dine obtained with 10 mL of this solution is equivalent to 14
ing on a flame with occasional gentle shaking.
to 26z of that obtained with 10 mL of the standard solution.
System performance: When the procedure is run with 10 Titanium trichloride See titanium (III) chloride (20).
mL of the standard solution under the above operating con-
Titanium trichloride-sulfuric acid TS See titanium (III)
ditions, the number of theoretical plates and the symmetry
chloride-sulfuric acid TS.
factor of the peak of ticlopidine are not less than 5000 and
not more than 1.5, respectively. Titanium trichloride TS See titanium (III) chloride TS.
System repeatability: When the test is repeated 6 times
Titanium yellow C28H19N5Na2O6S4 A dark yellow to
with 10 mL of the standard solution under the above operat-
dark yellow-brown, powder or masses.
ing conditions, the relative standard deviation of the peak
Identification—Determine the infrared absorption spec-
area of ticlopidine is not more than 2.0z.
trum of titanium yellow, previously dried at 1059 C for 4
Tin Sn [K 8580, Special class] hours, as directed in the potassium bromide disk method as
directed under Infrared Spectrophotometry <2.25>: it exhib-
Tin (II) chloride dihydrate SnCl2.2H2O [K 8136, Spe-
its absorption at the wave numbers of about 1603 cm－1,
cial class]
1467 cm－1, 1394 cm－1, 1306 cm－1, 1040 cm－1, 988 cm－1,
Tin (II) chloride-hydrochloric acid TS To 20 g of tin add 820 cm－1 and 644 cm－1.
85 mL of hydrochloric acid, heat until hydrogen gas no lon- Preserve in a light-resistant tight container.
ger are evolved, and allow to cool. Mix 1 volume of this so-
Tocopherol C29H50O2 [Same as the namesake mono-
lution and 10 volume of dilute hydrochloric acid. Prepare
graph]
before use.
Tocopherol acetate C31H52O3 [Same as the namesake
Tin (II) chloride-sulfuric acid TS Dissolve 10 g of tin (II)
monograph]
chloride dihydrate in diluted sulfuric acid (3 in 200) to make
100 mL. Tocopherol calcium succinate C66H106CaO10 [Same as
the namesake monograph]
Tin (II) chloride TS Dissolve 1.5 g of Tin (II) chloride di-
hydrate in 10 mL of water containing a small amount of hy- Tocopherol succinate C33H54O5 Wet 0.5 g of toco-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
344 General Tests / 9.41 Reagents, Test Solutions JP XVII
pherol calcium succinate with 5 mL of acetic acid (100), add (2) A solution in ethanol (95) (1 in 200) shows a blue
10 mL of toluene, and warm at 709 C for 30 minutes with oc- color.
casional shaking. After cooling, add 30 mL of water, shake (3) A solution shows a maximum absorption at around
thoroughly, and allow to stand. Remove the water layer, 630 nm.
wash the toluene layer with several 30-mL portions of water
Tranilast for assay C18H17NO5 [Same as the mono-
until the washings become neutral, and allow to stand.
graph Tranilast. When dried, it contains not less than 99.5z
Shake the toluene extract with 3 g of anhydrous sodium sul-
of tranilast (C18H17NO5).]
fate, decant the toluene layer, distil the toluene under
reduced pressure, and obtain a light yellow, viscous liquid. Triamcinolone acetonide C24H31FO6 [Same as the
When preserved at room temperature for a long time, it namesake monograph]
becomes a pale yellowish solid.
Trichloroacetic acid CCl3COOH [K 8667, Special
Absorbance <2.24> E 11zcm (286 nm): 38.0 – 42.0 (10 mg,
class]
chloroform, 100 mL).
Trichloroacetic acid-gelatin-tris buffer solution To 1
Tolbutamide C12H18N2O3S [Same as the namesake
volume of a solution of trichloroacetic acid (1 in 5) add 6
monograph]
volume of gelatin-tris buffer solution (pH 8.0) and 5 volume
Toluene C6H5CH3 [K 8680, Special class] of water.
o-Toluene sulfonamide C7H9NO2S Colorless crystals Trichloroacetic acid TS Dissolve 1.80 g of trichloroacetic
or white crystalline powder. Soluble in ethanol (95), and acid, 2.99 g of sodium acetate trihydrate and 1.98 g of acetic
sparingly soluble in water. acid (31) in water to make 100 mL.
Melting point <2.60>: 157 – 1609 C
Trichloroacetic acid TS for serrapeptase Dissolve 1.80 g
Purity p-Toluene sulfonamide—Use a solution of o-
of trichloroacetic acid and 1.80 g of anhydrous sodium ace-
toluene sulfonamide in ethyl acetate (1 in 5000) as the sample
tate in 5.5 mL of 6 mol/L acetic acid TS and water to make
solution. Perform the test with 10 mL of the sample solution
100 mL.
as directed under Gas Chromatography <2.02> according to
the operating conditions in the Purity (5) under Saccharin Trichloroethylene C2HCl3 [K 8666, Special class]
Sodium Hydrate: any peak other than the peak of o-toluene
Trichlorofluoromethane CCl3F A colorless liquid or
sulfonamide does not appear. Adjust the flow rate so that
gas.
the retention time of o-toluene sulfonamide is about 10
Specific gravity <2.56> d 17.2
4 : 1.494
minutes, and adjust the detection sensitivity so that the peak
Boiling point <2.57>: 23.79C
height of o-toluene sulfonamide obtained from 10 mL of the
sample solution is about 50z of the full scale. Time span of 1,1,2-Trichloro-1,2,2-trifluoroethane CFCl2CF2Cl
measurement is about twice as long as the retention time of Colorless volatile liquid. Miscible with acetone and with
o-toluene sulfonamide, beginning after the solvent peak. diethyl ether, and not with water.
Water <2.48>: not more than 0.5z (4 g, use 25 mL of Purity Related substances—Perform the test with 0.1 mL
methanol for water determination and 5 mL of pyridine for of 1,1,2-trichloro-1,2,2-trifluroethane as directed under Gas
water determination). Chromatography <2.02> according to the operating condi-
Content: not less than 98.5z, calculated on the anhy- tions in the Purity (5) under Halothane: any peak other than
drous basis. Assay—Weigh accurately about 25 mg of the peak of 1,1,2-trichloro-1,2,2-trifluoroethane does not
o-toluene sulfonamide, and perform the test as directed appear.
under Nitrogen Determination <1.08>.
Tricine C6H13NO5 White crystalline powder. Melting
Each mL of 0.005 mol/L sulfuric acid VS point: 182 to 1849C (with decomposition).
＝ 1.712 mg of C7H9NO2S
Trientine hydrochloride for assay C6H18N4.2HCl
p-Toluene sulfonamide CH3C6H4SO2NH2 White, crys- [Same as the monograph Trientine Hydrochloride or puri-
tals or crystalline powder. Melting point: about 1379C fied Trientine Hydrochloride according to the method of
Purity Related substances—Dissolve 30 mg of p-toluene purification shown below. It contains not less than 98.0z of
sulfonamide in acetone to make exactly 200 mL. Proceed trientine hydrochloride (C6H18N4.2HCl), calculated on the
with 10 mL of this solution as directed in the Purity (3) under dried basis, and meets the following requirements.]
Tolazamide: any spot other than the principal spot at the R f Purity Related substances—Dissolve 0.10 g of trientine
value of about 0.6 does not appear. hydrochloride for assay in 10 mL of methanol, and use this
solution as the sample solution. Pipet 5 mL of the sample so-
p-Toluene sulfonic acid See p-toluenesulfonic acid
lution, and add methanol to make exactly 50 mL. Pipet 3
monohydrate.
mL of this solution, add methanol to make exactly 100 mL,
p-Toluenesulfonic acid monohydrate and use this solution as the standard solution. Perform the
CH3C6H4SO3H.H2O [K 8681, Special class] test with these solutions as directed under Thin-layer Chro-
matography <2.03>. Spot 3 mL each of the sample solution
o-Toluic acid C8H8O2 White, crystals or crystalline
and standard solution on two plates of silica gel for thin-
powder.
layer chromatography. Develop one of the plate with a mix-
Melting point <2.60>: 102 – 1059
C
ture of 2-propanol and ammonia solution (28) (3:2) to a
Content: not less than 98.0z.
distance of about 6 cm, and air-dry the plate. Spray evenly
Toluidine blue See toluidine blue O ninhydrin-butanol TS on the plate, and heat at 1309C for 5
minutes: the spot other than the principal spot and the spot
Toluidine blue O C15H16ClN3S Dark green powder,
nearby the original point obtained with the sample solution
soluble in water, and slightly soluble in ethanol (95).
is not more intense than the spot obtained with the standard
Identification—
solution. Develop the other plate with a mixture of ammonia
(1) A solution (1 in 100) shows a blue to purple color.

solution (28), diethyl ether, acetonitrile and ethanol (99.5) 2,4,6-trinitrobenzenesulfonic acid See 2,4,6-trinitroben-
(10:4:3:3), and perform the test in the same manner as zenesulfonic acid dihydrate.
above: the spot nearby the original point with the sample so-
2,4,6-Trinitrobenzenesulfonic acid dihydrate
lution is not more intense than the spot with the standard so-
C6H2(NO2)3SO3H.2H2O Pale yellow to light yellow pow-
lution.
der.
Method of purification: Dissolve Trientine Hydrochloride
Water <2.48>: 11 – 15z (0.1 g, volumetric titration, direct
in water while warming, and recrystallize by addition of
titration).
ethanol (99.5). Or dissolve Trientine Hydrochloride in water
Content: not less than 98z, calculated on the anhydrous
while warming, allow to stand after addition of activated
basis. Assay—Weigh accurately about 0.3 g of 2,4,6-
charcoal in a cool and dark place for one night, and filter.
trinitrobenzenesulfonic acid, dissolve in 50 mL of a mixture
To the filtrate add ethanol (99.5), allow to stand in a cool
of water and ethanol (99.5) (1:1), and titrate <2.50> with 0.1
and dark place, and recrystallize. Dry the crystals under
mol/L sodium hydroxide VS (potentiometric titration). Per-
reduced pressure not exceeding 0.67 kPa at 409 C until
form a blank determination and make any necessary correc-
ethanol odor disappears.
tion.
Triethanolamine See 2,2?,2!-nitrilotriethanol.
Each mL of 0.1 mol/L sodium hydroxide VS
Triethylamine (C2H5)3N Clear colorless liquid, having ＝ 29.32 mg of C6H2(NO2)3SO3H
a strong amines odor. Miscible with methanol, with ethanol
2,4,6-Trinitrophenol HOC6H2(NO2)3 Light yellow to
(95) and with diethyl ether.
yellow, moist crystals. It is added 15 to 25z of water for the
Specific gravity <2.56> d 20
4 : 0.722 – 0.730
sake of safety, because it might explode by heating, mechan-
Boiling point <2.57>: 89 – 909C
ical shocking and friction when it is dried.
Triethylamine buffer solution (pH 3.2) To 4 mL of Identification—To 0.1 g add 10 mL of water, dissolve by
triethylamine add 2000 mL of water, and adjust the pH to warming, and add 12 mL of a mixture of 1z copper (II)
3.2 with phosphoric acid. sulfate solution and ammonia TS (5:1): green precipitates
appear.
Triethylamine for epoetin beta (C2H5)3N A clear and
Content: not less than 99.5z. Assay—Weigh accurately
colorless liquid.
about 0.25 g, previously dried in a desiccator (silica gel) for
Specific gravity <2.56> d 20
4 : 0.724 – 0.730
24 hours, dissolve in 50 mL of water by warming, and titrate
Water <2.48>: not more than 0.2z.
<2.50> with 0.1 mL sodium hydroxide VS (indicator: 3 drops
1z Triethylamine-phosphate buffer solution (pH 3.0) of phenolphthalein TS). Perform a blank determination in
Dissolve 10 g of triethylamine in 950 mL of water, adjust the the same manner, and make any necessary correction.
pH to 3.0 with phosphoric acid, and make exactly 1000 mL.
Each mL of 0.1 mol/L sodium hydroxide VS
Triethylamine-phosphate buffer solution (pH 5.0) To ＝ 22.91 mg of HOC6H2(NO2)3
1.0 mL of triethylamine add 900 mL of water, adjust the pH
2,4,6-Trinitrophenol-ethanol TS Dissolve 1.8 g of 2,4,6-
to 5.0 with diluted phosphoric acid (1 in 10), and add water
trinitrophenol in 50 mL of diluted ethanol (99.5) (9 in 10)
to make 1000 mL.
and 30 mL of water, and add water to make 100 mL.
Trifluoroacetic acid CF3COOH Colorless, clear liquid,
2,4,6-Trinitrophenol TS Dissolve 1 g of 2,4,6-trinitro-
having a pungent odor. Miscible well with water.
phenol in 100 mL of hot water, cool, and filter if necessary.
Specific gravity <2.56> d 20
20: 1.535
Boiling point <2.57>: 72 – 739C 2,4,6-Trinitrophenol TS, alkaline Mix 20 mL of 2,4,6-
trinitrophenol TS with 10 mL of a solution of sodium hy-
Trifluoroacetic acid for epoetin beta CF3COOH
droxide (1 in 20), and add water to make 100 mL. Use within
A clear and colorless liquid.
2 days.
Purity: When determine the absorbance of 50 volz solu-
tion of trifluoroacetic acid for epoetin beta as directed under Triphenylantimony Sb(C6H5)3 White to pale yellow-
Ultraviolet-visible Spectrophotometry <2.24>: not more than brown, crystals or crystalline powder or masses.
0.10 at 270 nm, not more than 0.02 at 280 nm, and not more Content: not less than 95.0z. Assay—Weigh accurately
than 0.01 between 300 nm and 400 nm. about 0.3 g of triphenylantimony, dissolve in 100 mL of
ethanol (95), add 1 g of sodium hydrogen carbonate, and
Trifluoroacetic acid for nuclear magnetic resonance spec-
titrate <2.50> with 0.05 mol/L iodine VS. Perform a blank
troscopy CF3COOH Prepared for nuclear magnetic reso-
determination in the same manner.
nance spectroscopy.
Each mL of 0.05 mol/L iodine VS
Trifluoroacetic acid TS To 1 mL of trifluoroacetic acid
＝ 17.65 mg of Sb(C6H5)3
add water to make 1000 mL.
Triphenylchloromethane (C6H5)3CCl White to grayish
Trifluoroacetic anhydride for gas chromatography
or yellowish white, crystals or crystalline powder.
(CF3CO)2O Colorless, clear liquid, having a pungent odor.
Melting point <2.60>: 107 – 1159C
Boiling point <2.57>: 40 – 459C
Triphenylmethane C19H16 A white to pale yellowish,
Trimetazidine hydrochloride for assay C14H22N2O3.2HCl
crystalline powder.
[Same as the monograph Trimetazidine Hydrochloride. It
Melting point <2.60>: 93 – 959C
contains not less than 99.0z of trimetazidine hydrochloride
(C14H22N2O3.2HCl), calculated on the anhydrous basis.] Triphenylmethanol for thin-layer chromatography
C19H15OH Occurs as a white powder.
Trimethylsilyl imidazole C6H12N2Si Clear, colorless to
Purity—Dissolve 0.1 g of triphenylmethanol for thin-layer
pale yellow liquid.
chromatography in 100 mL of methanol and perform the
Refractive index <2.45> n 20
D : 1.4744 – 1.4764
test as directed in the Purity (2) under Zidovudine: spots

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
346 General Tests / 9.41 Reagents, Test Solutions JP XVII
other than the principal spot with an R f value of about 0.73 amount of water, adjust to pH 7.3 with hydrochloric acid or
are not observed. 6 mol/L hydrochloric acid TS, and add water to make 200
mL.
Triphenyltetrazolium chloride See 2,3,5-triphenyl-2H-
tetrazolium chloride. 0.02 mol/L Tris buffer solution (pH 7.4) Dissolve 2.4 g
of 2-amino-2-hydroxymethyl-1,3-propanediol in 800 mL of
Triphenyltetrazolium chloride TS See 2,3,5-triphenyl-
water, adjust to pH 7.4 with 1 mol/L hydrochloric acid TS,
2H-tetrazolium chloride TS.
and add water to make 1000 mL.
2,3,5-Triphenyl-2H-tetrazolium chloride C19H15ClN4
0.02 mol/L Tris buffer solution (pH 7.5) Dissolve 2.4 g
[K 8214, Special class]
of 2-amino-2-hydroxymethyl-1,3-propanediol and 29.2 g of
2,3,5-Triphenyl-2H-tetrazolium chloride TS Dissolve sodium chloride in a suitable amount of water, adjust to pH
0.25 g of 2,3,5-triphenyl-2H-tetrazolium chloride in ethanol 7.5 with hydrochloric acid, and add water to make 1000 mL.
(99.5) to make 100 mL. Prepare before use.
1 mol/L Tris buffer solution (pH 7.5) Dissolve 12.11 g
2,3,5-Triphenyl-2H-tetrazolium chloride-methanol TS for of 2-amino-2-hydroxymethyl-1,3-propanediol in 90 mL of
spraying Solution A: A solution of 2,3,5-triphenyl-2H- water, adjust to pH 7.5 with hydrochloric acid, and add
tetrazolium chloride in methanol (1 in 25). Solution B: A so- water to make 100 mL.
lution of sodium hydroxide in methanol (1 in 125). Mix an
0.1 mol/L Tris buffer solution (pH 8.0) Dissolve 2.42 g
equal volume of the solution A and solution B just before
of 2-amino-2-hydroxymethyl-1,3-propanediol in 100 mL of
use.
water, adjust the pH to 8.0 with 0.2 mol/L hydrochloric acid
Tripotassium citrate monohydrate C6H5K3O7.H2O TS, and add water to make 200 mL.
White, crystals or crystalline powder. Very soluble in water,
1 mol/L Tris buffer solution (pH 8.0) Dissolve 121 g of
and practically insoluble in ethanol (95).
2-amino-2-hydroxymethyl-1,3-propanediol in 800 mL of
Content: 99.0z or more Assay—Accurately weigh about
water, adjust to pH 8.0 with hydrochloric acid, add water to
0.2 g of tripotassium citrate monohydrate, add 50 mL of
make 1000 mL, and sterilize in an autoclave.
acetic acid for nonaqueous titration, dissolve by warming on
a water bath, cool, and then titrate <2.50> with 0.1 mol/L of 0.5 mol/L Tris buffer solution (pH 8.1) Dissolve 12.1 g
perchloric acid VS (potentiometric titration). Correct by of 2-amino-2-hydroxymethyl-1,3-propanediol in 160 mL of
conducting a blank test using the same method. water, adjust to pH 8.1 with 1 mol/L hydrochloric acid TS,
and add water to make 200 mL.
Each mL of 0.1 mol/L perchloric acid VS
＝ 32.44 mg of C6H5K3O7.H2O Tris buffer solution (pH 8.2) Dissolve 24.2 g of 2-amino-
2-hydroxymethyl-1,3-propanediol and 0.5 g of polysorbate
Tris-acetic acid buffer solution (pH 6.5) Dissolve 13.57 g
20 in 800 mL of water, adjust to pH 8.2 with 1 mol/L hydro-
of 2-amino-2-hydroxymethyl-1,3-propanediol and 6.73 g of
chloric acid TS, and add water to make 1000 mL.
acetic acid (100) in water to make 1000 mL.
Tris buffer solution (pH 8.3) Dissolve 3.03 g of 2-amino-
Tris-acetic acid buffer solution (pH 8.0) Dissolve 1.2 g
2-hydroxymethyl-1,3-propanediol, 1.0 g of sodium lauryl
of 2-amino-2-hydroxymethyl-1,3-propanediol in 800 mL of
sulfate, and 14.4 g of glycine in 900 mL of water, adjust to
water, adjust to pH 8.0 with acetic acid (100), and add water
pH 8.3 with 5 mol/L hydrochloric acid TS, and add water to
to make 1000 mL.
make 1000 mL.
Tris buffer solution for bacterial endotoxins test Dis-
Tris buffer solution (pH 8.4) Dissolve 6.1 g of 2-amino-
solve 18.2 g of 2-amino-2-hydroxymethyl-1,3-propanediol in
2-hydroxymethyl-1,3-propanediol and 10.2 g of sodium
800 mL of water for bacterial endotoxins test, add 100 mL
chloride in 800 mL of water, adjust to pH 8.4 with 1 mol/L
of 0.1 mol/L hydrochloric acid TS and water for bacterial
hydrochloride TS, and add water to make 1000 mL.
endotoxins test to make 1000 mL, and sterilize by heating in
an autoclave at 1219C for 90 minutes. 0.05 mol/L Tris buffer solution (pH 8.6) Dissolve 6.1 g
of 2-amino-2-hydroxymethyl-1,3-propanediol in 950 mL of
Tris buffer solution (pH 6.8) Dissolve 30.3 g of 2-amino-
water, add 2 mol/L hydrochloric acid TS to adjust the pH to
2-hydroxymethyl-1,3-propanediol and 2.0 g of sodium lauryl
8.6, and then add water to make 1000 mL.
sulfate in 800 mL of water, adjust to pH 6.8 with 5 mol/L
hydrochloric acid TS, and add water to make 1000 mL. Tris buffer solution (pH 8.8) Dissolve 22.7 g of 2-amino-
2-hydroxymethyl-1,3-propanediol and 1.5 g of sodium lauryl
0.5 mol/L Tris buffer solution (pH 6.8) Dissolve 6 g of
sulfate in 140 mL of water, adjust to pH 8.8 with 5 mol/L
2-amino-2-hydroxymethyl-1,3-propanediol in 50 mL of
hydrochloric acid TS, and add water to make 200 mL.
water, add 2 mol/L hydrochloric acid TS to adjust the pH to
6.8, and then add water to make 100 mL. Filter if necessary. 1.5 mol/L Tris buffer solution (pH 8.8) Dissolve 18.2 g
of 2-amino-2-hydroxymethyl-1,3-propanediol in 75 mL of
0.05 mol/L Tris buffer solution (pH 7.0) Dissolve 6.06 g
water, add 5 mol/L hydrochloric acid TS to adjust the pH to
of 2-amino-2-hydroxymethyl-1,3-propanediol in about 750
8.8, and then add water to make 100 mL. Filter if necessary.
mL of water, adjust to pH 7.0 with 1 mol/L hydrochloric
acid TS, and add water to make 1000 mL. Tris buffer solution (pH 9.5) Dissolve 36.3 g of 2-amino-
2-hydroxymethyl-1,3-propanediol in 1000 mL of water,
Tris buffer solution (pH 7.0) Dissolve 24.3 g of 2-amino-
and adjust the pH to 9.5 by adding 1 mol/L hydrochloric
2-hydroxymethyl-1,3-propanediol in 1000 mL of water,
acid TS.
and adjust the pH to 7.0 with 0.1 mol/L hydrochloric acid
TS. 0.01 mol/L tris buffer solution – sodium chloride TS (pH
7.4) Dissolve 1.2 g of 2-amino-2-hydroxymethyl-1,3-
0.1 mol/L Tris buffer solution (pH 7.3) Dissolve 2.42 g
propanediol, 29.2 g of sodium chloride and 0.5 g of polysor-
of 2-amino-2-hydroxymethyl-1,3-propanediol in a suitable
bate 20 in 800 mL of water, adjust to pH 7.4 with 1 mol/L

hydrochloric acid TS, and add water to make 1000 mL. mined as directed under Ultraviolet-visible spectrophotome-
try <2.24> using water as the blank. If necessary adjust the
Tris(4-t-butylphenyl)phosphate [(CH3)3CC6H4O]3PO
absorbance by addition of the diluting solution or substrate
White, crystals or crystalline powder.
stock solution.
Melting point <2.60>: 100 – 1049 C
(iv) Procedure—Transfer exactly 3 mL of the substrate
Tris-calcium chloride buffer solution (pH 6.5) Dissolve solution, previously warmed to 25 ± 0.19C, into a 1-cm
6.1 g of 2-amino-2-hydroxymethyl-1,3-propanediol and 15 quartz cell, add exactly 0.2 mL of the sample solution, im-
mg of calcium chloride dihydrate in 800 mL of water, adjust mediately start the timer, and determine the change of the
to pH 6.5 with dilute hydrochloric acid, and add water to absorbance at 253 nm at 25 ± 0.19C for 5 minutes, using the
make 1000 mL. control prepared by adding exactly 0.2 mL of 0.001 mol/L
hydrochloric acid TS to exact 3 mL of the substrate solution.
Tris-glycine buffer solution (pH 6.8) Dissolve 1.22 g of
Obtain the variation per minute of the absorbance, A, from
2-amino-2-hydroxymethyl-1,3-propanediol, 0.76 g of gly-
the part where the changing rate of the absorbance is con-
cine, 8.8 g of sodium chloride and 0.1 g of polysorbate 80 in
stant for at least 3 minutes.
800 mL of water, adjust to pH 6.8 with 1 mol/L hydrochlo-
(v) Calculation—Calculate trypsin unit per mg using the
ric acid TS, and add water to make 1000 mL.
following equation. Where, one trypsin unit is the quantity
0.2 mol/L Tris-hydrochloride buffer solution (pH 7.4) of enzyme that gives the variation of the absorbance 0.003
Dissolve 6.61 g of 2-amino-2-hydroxymethyl-1,3- per minute.
propanediol hydrochloride and 0.97 g of 2-amino-2-hydrox-
Trypsin unit per mg ＝ A/0.003 × 1/M
ymethyl-1,3-propanediol in water to make 250 mL.
M: Amount (mg) of the substance to be assayed in 0.2 mL
0.05 mol/L Tris-hydrochloride buffer solution (pH 7.5)
of the sample solution
Dissolve 6.35 g of 2-amino-2-hydroxymethyl-1,3-
propanediol hydrochloride and 1.18 g of 2-amino-2-hydrox- Storage—At a cold place.
ymethyl-1,3-propanediol in water to make 1000 mL.
Trypsin for epoetin alfa liquid chromatography Bovine
Tris-sodium chloride buffer solution (pH 8.0) Dissolve pancreas origin. It has not less than 180 units per mg, as 1
2.42 g of 2-amino-2-hydroxymethyl-1,3-propanediol and unit is equivalent to the amount of enzyme necessary to hy-
1.64 g of sodium chloride in 900 mL of water, adjust to pH drolysis 1 mmol of p-toluenesulfonyl-L-arginine methyl ester
8.0 with dilute hydrochloric acid, and add water to make per minute at 259C, pH 8.2.
1000 mL.
Trypsin for liquid chromatography An enzyme obtained
Trishydroxymethylaminomethane See 2-amino-2-hydro- from the bovine pancreas. This one part digests 250 parts of
xymethyl-1,3-propanediol. casein in the following reaction system.
Casein solution—To 0.1 g of milk casein add 30 mL of
Trisodium citrate dihydrate See sodium citrate hydrate.
water, disperse the casein well, add 1.0 mL of diluted sodium
0.1 mol/L Trisodium citrate TS Dissolve 29.4 g of triso- hydroxide TS (1 in 10) to dissolve, and add water to make 50
dium citrate dihydrate in water to make 1000 mL. mL. Prepare before use.
Sample solution—Dissolve 10 mg of trypsin for liquid
Trisodium ferrous pentacyanoamine TS See iron (II)
chromatography in 500 mL of water.
trisodium pentacyanoamine TS.
Procedure—To 5 mL of the casein solution add 2 mL of
Trisodium phosphate dodecahydrate Na3PO4.12H2O the sample solution and 3 mL of water, mix, then allow to
[K 9012, Special class] stand at 409C for 1 hour, and add 3 drops of a mixture of
ethanol (95), water and acetic acid (100) (10:9:1): no precipi-
Trypsin Obtained from bovine or hog pancreas, and pre-
tate appears.
pared for biochemistry or to meet the following require-
ments. White to light yellowish, crystals or powder. Trypsin inhibitor Produced by purifying soybean. Each
Loss on drying: Not more than 5.0z (609C, in vacuum, mg of trypsin inhibitor inhibits 10,000 to 30,000 BAEE Units
4 hours). of trypsin. One BAEE Unit means a trypsin activity to indi-
Content: Not less than 220 trypsin units per mg. Assay cate an absorbance difference of 0.001 at 253 nm per minute
(i) Sample solution—Weigh accurately about 20 mg of the when 3.2 mL of the solution is reacted at 259C and pH 7.6,
substance to be assayed, and dissolve in 0.001 mol/L hydro- using N-a-benzoyl-L-arginine ethyl ester as substrate.
chloric acid TS so that each mL contains about 3000 trypsin
Trypsin inhibitor TS Dissolve 5 mg of trypsin inhibitor
units. To a suitable amount of this solution add 0.001 mol/L
in 0.05 mol/L phosphate buffer solution (pH 7.0) to make
hydrochloric acid TS so that each mL contains about 40
10 mL.
trypsin units, and use this solution as the sample solution.
(ii) Diluting solution—Dissolve 4.54 g of potassium di- Trypsin TS Dissolve 0.5 g of trypsin and 0.2 g of disodi-
hydrogen phosphate in water to make exactly 500 mL (Solu- um dihydrogen ethylenediamine tetraacetate dihydrate in
tion I). Dissolve 4.73 g of anhydrous disodium hydrogen phosphate buffer solution for cytotoxicity test to make 1000
phosphate in water to make exactly 500 mL (Solution II). To mL, and sterilize by filtration through a membrane filter
80 mL of Solution II add a suitable amount of Solution I to with a pore size not exceeding 0.22 mm.
adjust to pH 7.6.
Trypsin TS for epoetin alfa Dissolve 0.5 mg of trypsin
(iii) Substrate solution—Dissolve 85.7 mg of N-a-ben-
for epoetin alfa liquid chromatography in 2.5 mL of water.
zoyl-L-ethylarginine hydrochloride in water to make exactly
100 mL, and use this solution as the substrate stock solution. Trypsin TS for test of elcatonin Dissolve 5 mg of trypsin
Pipet 10 mL of the substrate stock solution add diluting so- for liquid chromatography in 20 mL of a solution of ammo-
lution to make exactly 100 mL, and use this solution as the nium hydrogen carbonate (1 in 100). Prepare before use.
substrate solution. The substrate solution gives an absor-
Trypsin TS for test of ulinastatin Dissolve crystalline
bance of between 0.575 and 0.585 at 253 nm when deter-

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
348 General Tests / 9.41 Reagents, Test Solutions JP XVII
trypsin for ulinastatin assay in ice-cooled 1 mmol/L hydro- Uracil C4H4N2O2 Needle crystals. Freely soluble in hot
chloric acid TS containing 1 mmol/L calcium chloride dihy- water, and slightly soluble in cold water.
drate so that each mL of the solution contains 180 mg of Melting point <2.60>: 3359C
trypsin. Prepare before use, and preserve in an ice-cooled
Urea H2NCONH2 [K 8731, Special class]
water bath.
Urea-EDTA TS Dissolve 48.0 g of urea and 0.2 g of dis-
L-Tryptophan C11H12N2O2 [Same as the namesake
odium ethylenediamine tetraacetate dihydrate in 0.5 mol/L
monograph]
tris buffer solution (pH 8.1) to make 100 mL.
Tulobuterol for assay C12H18ClNO [Same as the mono-
Urethane See ethyl carbamate.
graph Tulobuterol. It contains not less than 99.0z of
tulobuterol (C12H18ClNO), calculated on the anhydrous Ursodeoxycholic acid C24H40O4 [Same as the namesake
basis.] monograph]
Turpentine oil [Same as the namesake monograph] Ursodeoxycholic acid for assay C24H40O4 [Same as the
monograph Ursodeoxycholic Acid. However, when dried, it
L-Tyrosine C9H11NO3 White, crystals or crystalline pow-
contains not less than 99.0z of ursodeoxycholic acid
der. Odorless and tasteless. Freely soluble in formic acid,
(C24H40O4) meeting the following additional specifications.]
very slightly soluble in water, and practically insoluble in
Purity Related substances—Dissolve 0.15 g of ursodeox-
ethanol (95) and in diethyl ether. It dissolves in dilute hydro-
ycholic acid for assay in 5 mL of methanol for liquid chro-
chloric acid and in dilute nitric acid.
matography, and use this solution as the sample solution.
Optical rotation <2.49> [a]20
D : －10.5 – －12.59(after dry-
Pipet 2 mL of the sample solution and add methanol for liq-
ing, 2.5 g, 1 mol/L hydrochloric acid TS, 50 mL, 100 mm).
uid chromatography to make exactly 50 mL. Pipet 2.5 mL of
Loss on drying <2.41>: not more than 0.30z (1 g, 1059C,
this solution, add methanol for liquid chromatography to
3 hours).
make exactly 20 mL, and use this solution as the standard
Content: not less than 99.0z. Assay—Weigh accurately
solution. Perform the test with exactly 5 mL each of the sam-
about 0.3 g of L-tyrosine, previously dried, dissolve in 6 mL
ple solution and standard solution as directed under Liquid
of formic acid, add 50 mL of acetic acid (100), and titrate
Chromatography <2.01> according to the following condi-
<2.50> with 0.1 mol/L perchloric acid VS (potentiometric
tions. Determine each peak area of both solutions by the
titration). Perform a blank determination, and make any
automatic integration method: the area of the peak, having
necessary correction.
the relative retention time of about 2.5 to ursodeoxycholic
Each mL of 0.1 mol/L perchloric acid VS acid, obtained from the sample solution is not larger than
＝ 18.12 mg of C9H11NO3 the peak area of ursodeoxycholic acid obtained from the
standard solution, and the area of the peak, having the rela-
Ubenimex for assay C16H24N2O4 [Same as the mono-
tive retention time of about 5.5, from the sample solution is
graph Ubenimex. When dried, it contains not less than
not larger than 1/5 times the peak area of ursodeoxycholic
99.0z of ubenimex (C16H24N2O4).]
acid from the standard solution. Furthermore, the total area
Ubiquinone-9 Yellow to orange crystalline powder. of the peaks other than ursodeoxycholic acid and the peaks
Odorless and no taste. mentioned above from the sample solution is not larger than
Absorbance <2.24> E 11zcm (275 nm): 163 – 190 (ethanol 1/5 times the peak area of ursodeoxycholic acid from the
(99.5)) standard solution.
Melting point <2.60>: about 449C Operating conditions
Detector: An ultraviolet absorption photometer (wave-
Umbelliferone for thin-layer chromatography C9H6O3
length: 210 nm).
White or light brown powder. Sparingly soluble in methanol
Column: A stainless steel column 3 mm in inside diameter
and in ethanol (99.5), and practically insoluble in water.
and 7.5 cm in length, packed with octylsilanized silica gel for
Melting point: about 2329 C.
liquid chromatography (5 mm in particle diameter).
Identification—(1) Determine the absorption spectrum
Column temperature: A constant temperature of about
of a solution of umbelliferone for thin-layer chromatogra-
409C.
phy in methanol (1 in 300,000) as directed under Ultraviolet-
Mobile phase: A mixture of methanol for liquid chroma-
visible Spectrophotometry <2.24>: it exhibits maxima be-
tography, diluted phosphoric acid (1 in 1000) and aceto-
tween 214 nm and 218 nm, and between 322 nm and 326 nm.
nitrile for liquid chromatography (96:69:35).
(2) Determine the infrared absorption spectrum of um-
Flow rate: Adjust so that the retention time of ursodeoxy-
belliferone for thin-layer chromatography as directed in the
cholic acid is about 2.3 minutes.
potassium bromide disk method under Infrared Spectropho-
Time span of measurement: About 7 times as long as the
tometry <2.25>: it exhibits absorption at the wave numbers
retention time of ursodeoxycholic acid, beginning after the
of about 3160 cm－1, 1681 cm－1, 1604 cm－1, 1323 cm－1, 990
solvent peak.
cm－1 and 903 cm－1.
System suitability
Purity Related substances－Dissolve 1.0 mg of umbel-
Test for required detectability: Pipet 2 mL of the standard
liferone for thin-layer chromatography in 10 mL of metha-
solution, and add methanol for liquid chromatography to
nol, and use this solution as the sample solution. Pipet 1 mL
make exactly 20 mL. Confirm that the peak area of ur-
of the sample solution, add methanol to make exactly 50
sodeoxycholic acid obtained from 5 mL of this solution is
mL, and use this solution as the standard solution. Perform
equivalent to 8 to 12z of that of ursodeoxycholic acid ob-
the test with 5 mL each of the sample solution and standard
tained from 5 mL of the standard solution.
solution as directed in the Identification under Artemisia
System performance: To 30 mg of chenodeoxycholic acid
Leaf: the spot other than the principal spot having an R f
for thin-layer chromatography and 30 mg of lithocholic acid
value of about 0.5 obtained with the sample solution is not
for thin-layer chromatography, add 1 mL of the sample so-
more intense than the spot obtained with the standard solu-
lution, dissolve in methanol for liquid chromatography to
tion.

make 50 mL. When the procedure is run with 5 mL of this so- Constituent amino acids—Perform the test as directed in
lution under the above operating conditions, ursodeoxychol- the Constituent amino acids under Oxytocin, and calculate
ic acid, chenodeoxycholic acid, and lithocholic acid are the respective molar ratios with respect to glycine: 0.9 – 1.1
eluted in this order with the resolution between these peaks for aspartic acid, 0.9 – 1.1 for glutamic acid, 0.9 – 1.1 for
being not less than 7, respectively. proline, 0.8 – 1.1 for tyrosine, 0.9 – 1.1 for phenylalanine,
System repeatability: When the test is repeated 6 times 0.9 – 1.1 for arginine and 0.8 – 1.1 for cystine, and not more
with 5 mL of the standard solution under the above condi- than 0.03 for other amino acids.
tions, the relative standard deviation of the peak area of ur-
Verapamil hydrochloride for assay C27H38N2O4.HCl
sodeoxycholic acid is not more than 2.0z.
[Same as the monograph Verapamil Hydrochloride. When
n-Valerianic acid CH3(CH2)3COOH Clear, colorless to dried, it contains not less than 99.0z of verapamil hydro-
pale yellow liquid, having a characteristic odor. Miscible chloride (C27H38N2O4.HCl).]
with ethanol (95) and with diethyl ether, and soluble in
Verbascoside for thin-layer chromatography C29H36O15
water.
A white to very pale yellow, odorless, crystalline powder or
Specific gravity <2.56> d 20
4 : 0.936 – 0.942
powder. Soluble in methanol, sparingly soluble in ethanol
Distilling range <2.57>: 186 – 1889C, not less than 98
(99.5), and slightly soluble in water.
volz.
Identification Determine the infrared absorption spec-
L-Valine C5H11NO2 [Same as the namesake mono- trum of verbascoside for thin-layer chromatography as di-
graph] rected in the potassium bromide disk method under Infrared
Spectrophotometry <2.25>: it exhibits absorption at the wave
L-Valine for assay C5H11NO2 [Same as the monograph
numbers of about 1604 cm－1, 1446 cm－1, 1272 cm－1 and 815
L-Valine. When dried, it contains not less than 99.0z of
cm－1.
L-valine (C5H11NO2).]
Purity Related substances—Dissolve 10 mg of verbasco-
H-D-Valyl-L-leucyl-L-arginine-4-nitroanilide dihydrochlo- side for thin-layer chromatography in 2 mL of methanol,
ride C23H38N8O5.2HCl White to pale yellow, powder or and use this solution as the sample solution. Pipet 1 mL of
masses. Sparingly soluble in water. the sample solution, add methanol to make exactly 50 mL,
Absorbance <2.24> E 11zcm (316 nm): 214 – 236 (10 mg, and use this solution as the standard solution. Perform the
water, 500 mL). test with 20 mL each of the sample solution and standard
solution as directed in the Identification under Cistanche
Vanadium pentoxide See vanadium (V) oxide.
Herb: the spot other than the principal spot that appears at
Vanadium pentoxide TS See vanadium (V) oxide TS. an Rf value of about 0.35 obtained from the sample solution
is not more intense than the spot obtained from the standard
Vanadium pentoxide TS, dilute See vanadium (V) oxide
solution.
TS, dilute.
Vinblastine sulfate C46H58N4O9.H2SO4 [Same as the
Vanadium (V) oxide V2O5 Orangish yellow to yellow-
namesake monograph]
brown powder.
Identification—Dissolve 0.3 g in 10 mL of ammonia TS Vincristine sulfate C46H56N4O10.H2SO4 [Same as the
and 15 mL of water. To 2 mL of this solution add 20 mL of namesake monograph]
water, mix, and add gently 1 mL of copper (II) sulfate TS:
Vinyl acetate C4H6O2 Clear, colorless liquid.
yellow precipitates appear.
Specific gravity <2.56>: 0.932 – 0.936
Vanadium (V) oxide TS Add vanadium (V) oxide to Water <2.48>: not more than 0.2z
phosphoric acid, saturate with vanadium (V) oxide by shak-
Vinyl chloride C2H3Cl Colorless gas.
ing vigorously for 2 hours, and filter through a glass filter.
Boiling point <2.57>: －149C
Vanadium (V) oxide TS, dilute Dilute 10 mL of vanadi- Melting point <2.60>: －1609C
um (V) oxide TS with water to make 100 mL. Prepare before
2-Vinylpyridine C7H7N A clear, colorless or dark
use.
brown liquid.
Vanillin C6H3CHO(OCH3)(OH) A white to light yel- Refractive index <2.45> n 20
D : 1.546 – 1.552
low crystalline powder, having a characteristic odor. Sepcific gravity <2.56> d 20
20: 0.975 – 0.982
Melting point <2.60>: 80.5 – 83.59C
4-Vinylpyridine C7H7N A pale yellow to blackish
Preserve in a light-resistant tight container.
brown liquid.
Vanillin-hydrochloric acid TS Dissolve 5 mg of vanillin Refractive index <2.45> n20D : 1.5500 – 1.5530
in 0.5 mL of ethanol (95), and to this solution add 0.5 mL of Specific gravity <2.56> d 20
20: 0.9850 – 0.9880
water and 3 mL of hydrochloric acid. Prepare before use.
1-Vinyl-2-pyrrolidone C6H9NO Clear liquid.
Vanillin-sulfuric acid-ethanol TS Dissolve 3 g of vanillin Purity—Perform the test with 0.5 mL of 1-vinyl-2-pyrroli-
in ethanol (99.5) to make 100 mL, and add 0.5 mL of sulfu- done as directed under Gas Chromatography <2.02> accord-
ric acid. ing to the following conditions. Determine each peak area of
the solutions by the automatic integration method, and cal-
Vanillin-sulfuric acid-ethanol TS for spraying Dissolve
culate the amount of 1-vinyl-2-pyrrolidone by the area per-
3 g of vanillin in 30 mL of ethanol (99.5), and add 100 mL of
centage method: it is not less than 99.0z.
dilute sulfuric acid.
Operating conditions
Vanillin-sulfuric acid TS Add cautiously 75 mL of sulfu- Detector: A hydrogen flame-ionization detector.
ric acid to 25 mL of ice-cold ethanol (95). After cooling, add Column: A hollow, capillary glass column about 0.53 mm
1 g of vanillin to dissolve. Prepare before use. in inside diameter and about 30 m in length, having an about
1.0 mm layer of polyethylene glycol 20 M for gas chromatog-
Vasopressin C46H65N15O12S2 A white powder.

The JP Drugs are to be tested according to the provisions given in the pertinent monographs, General Notices, General Rules for Crude Drugs,
General Rules for Preparations, and General Tests for their conformity to the Japanese Pharmacopoeia. (See the General Notices 5.)
350 General Tests / 9.41 Reagents, Test Solutions JP XVII
raphy on the inner side. gent, and is suitable for bacterial endotoxins test.
Column temperature: Maintain the temperature at 809C
Water for ICP analysis See Inductively Coupled Plasma-
for 1 minute, then raise at the rate of 109 C per minute to
Atomic Emission Spectrometry and Inductively Coupled
1909C, and hold constant to the temperature for 20 minutes.
Plasma-Mass Spectrometry <2.63>.
Temperature of sample vaporization chamber: A constant
temperature of about 1909 C. Water for injection [Use the water prescribed by the
Carrier gas: Helium. monographs of Water for Injection or Sterile Water for
Flow rate: Adjust so that the retention time of 1-vinyl-2- Injection in Containers. It is not necessary to check the con-
pyrrolidone is about 15 minutes. formity to all the specification items of the monograph, if it
Detection sensitivity: Adjust the detection sensitivity so is confirmed that the water to be used is suitable for the pur-
that the peak height of 1-vinyl-2-pyrrolidone from 0.5 mL of pose of relevant test.]
1-vinyl-2-pyrrolidone is about 70z of the full scale.
Water, nuclease-free Water in which nuclease is not in-
Time span of measurement: About twice as long as the
cluded.
retention time of 1-vinyl-2-pyrrolidone, beginning after the
solvent peak. Water, sterile purified [Use the water prescribed by the
Water <2.48>—Take 50 mL of methanol for water deter- monograph of Sterile Purified Water in Containers. It is not
mination and 10 mL of butyrolactone in a dry titration flask, necessary to check the conformity to all the specification
and titrate with Karl Fischer TS for water determination items of the monograph, if it is confirmed that the water to
until end point. Weigh accurately about 2.5 g of 1-vinyl-2- be used is suitable for the purpose of relevant test.]
pyrrolidone, transfer immediately to a titration flask, and
Wijs' TS Transfer 7.9 g of iodine trichloride and 8.9 g of
perform the test: water is not more than 0.1z.
iodine to separate flasks, dissolve each with acetic acid (100),
V8 protease A protease obtained from Staphylococus mix both solutions, and add acetic acid (100) to make 1000
aureus strain. When an amount of the enzyme hydrolyzes 1 mL. Preserve in light-resistant, glass containers.
mmol of N-t-butoxycarbonyl-L-glutamic acid-a-phenyl ester
Wogonin for thin-layer chromatography C16H12O5 Yel-
in 1 minute at pH 7.8 and 379C is defined as 1 unit, it con-
low, crystals or crystalline powder. Slightly soluble in metha-
tains 500 – 1000 units per mg.
nol and in ethanol (99.5), and practically insoluble in water.
V8 protease for insulin glargine A protease obtained Melting point: 204 – 2089C
from Staphylococcus aureus strain. When an amount of the Identification—Determine the absorption spectrum of a
enzyme which hydrolyzes 1 mmol of carbobenzoxy- solution in methanol (1 in 200,000) as directed under Ultravi-
phenylalanyl-leucyl-glutamyl-4-nitroanilide in 1 minute at olet-visible Spectrophotometry <2.24>: it exhibits maxima be-
pH 7.8 and 259C is defined as 1 unit, it contains not less tween 207 nm and 211 nm, and between 273 nm and 277 nm.
than 20 units per mg. Purity Related substances—Dissolve 1 mg in 1 mL of
methanol, and perform the test with 10 mL of this solution as
V8 protease TS Dissolve V8 protease in water to make a
directed in the Identification (3) under Saireito Extract: no
solution of 1 mg/mL. Keep at a cold place and use within 6
spot other than the principal spot (R f value is about 0.4) ap-
days after preparation.
pears.
Voglibose for assay C10H21NO7 [Same as the mono-
Xanthene C13H10O White to light yellow, crystals or
graph Voglibose]
crystalline powder, having a slight, characteristic odor.
Warfarin potassium for assay C19H15KO4 [Same as the Melting point <2.60>: 98 – 1029 C
monograph Warfarin Potassium. When dried, it contains Water <2.48>: not more than 0.5z (0.15 g).
not less than 99.0z of warfarin potassium (C19H15KO4).]
Xanthene-9-carboxylic acid C14H10O3 Dissolve 0.25 g
Washing fluid for nartograstim test Dissolve 1 mL of of propantheline bromide in 5 mL of water and 10 mL of
polysorbate 20 in phosphate-buffered sodium chloride TS to sodium hydroxide TS, heat the mixture to boiling, then
make 1000 mL. continue to heat for 2 minutes. Cool to 609C, add 5 mL of
dilute sulfuric acid, cool, filter the precipitate, and wash
25z Water containing benzoyl peroxide See Benzoyl
thoroughly with water. Recrystallize the residue from dilute
peroxide, 25z water containing.
ethanol, and dry for 3 hours in a desiccator (in vacuum,
Water for ammonium limit test To 1500 mL of water silica gel).
add cautiously 4.5 mL of sulfuric acid, distil using a hard Melting point <2.60>: 217 – 2229C
glass distiller, discard the sufficient volume of first distillate,
Xanthone C13H8O2 Light yellow powder. Freely solu-
and use the remaining distillate (ammonium-free water) as
ble in chloroform, and slightly soluble in hot water and in
the water for ammonium limit test.
diethyl ether.
Purity—Mix 40 mL of water for ammonium limit test with
Melting point <2.60>: 174 – 1769C
6.0 mL of phenol-sodium pentacyanonitrosylferrate (III) TS.
Purity Related substances—Dissolve 50 mg of xanthone
Add 4.0 mL of sodium hypochlorite-sodium hydroxide TS,
in chloroform to make exactly 10 mL. Perform the test with
mix, and allow to stand for 60 minutes. Perform the test
5 mL of this solution as directed in the Purity under Propan-
with this solution as directed under Ultraviolet-visible Spec-
theline Bromide: any spot other than the principal spot at the
trophotometry <2.24>, using water as the blank: the absor-
R f value of about 0.7 does not appear.
bance at 640 nm is not more than 0.010.
Xanthydrol C13H10O2 White to pale yellow powder.
Water for bacterial endotoxins test Use the water
Dissolves in ethanol (95), in acetic acid (100), in chloroform,
prescribed by the monographs of Water for Injection or
and in diethyl ether, and is practically insoluble in water.
Sterile Water for Injection in Containers, or the water pro-
Melting point <2.60>: 121 – 1249C
duced by other procedures that shows no reaction with the
Residue on ignition <2.44>: not more than 2.0z (0.5 g).
lysate reagent employed, at the detection limit of the rea-

Xylene C6H4(CH3)2 [K 8271, First class] make 1000 mL.

o-Xylene C6H4(CH3)2 Colorless, clear liquid. 0.04 mol/L Zinc chloride TS Dissolve 5.452 g of zinc
Refractive index <2.45> n 20
D : 1.501 – 1.506 chloride in water to make 1000 mL.
Specific gravity <2.56> d 20
4 : 0.875 – 0.885
Zinc dibutyldithiocarbamate [(C4H9)2NCSS]2Zn
Distilling range <2.57>: 143 – 1469C, not less than 95
A white powder. Melting point: 106 – 1109C.
volz.
Content: Not less than 95.0z. Assay—Weigh accurately
Xylene cyanol FF C25H27N2NaO6S2 [K 8272, Special about 1.0 g of zinc dibutyldithiocarbamate, add 10 mL of
class] water and 5 mL of hydrochloric acid, and evaporate to dry-
ness on a hot plate. To the residue add 15 mL of diluted hy-
Xylenol orange C31H30N2Na2O13S [K 9563, Special
drochloric acid (1 in 3), dissolve by warming, then add 50
class]
mL of water and 40 mL of ammonia-ammonium chloride
Xylenol orange TS Dissolve 0.1 g of xylenol orange in buffer solution (pH 10.7) and titrate <2.50> with 0.1 mol/L
water to make 100 mL. disodium dihydrogen ethylenediamine tetraacetate VS until
the color of the solution changes from red to blue (indicator:
Xylitol C5H12O5 [Same as the namesake monograph]
0.1 mL of eriochrome black T TS).
Xylose See D-xylose.
Each mL of 0.1 mol/L disodium dihydrogen
D-Xylose C5H10O5 [Same as the monograph D-Xylose ethylenediamine tetraacetate VS
of the Japanese Standards of Food Additives] ＝ 47.41 mg of [(C4H9)2NCSS]2Zn
Yeast extract A peptone-like substance which represents Zinc diethyldithiocarbamate [(C2H5)2NCSS]2Zn
all the soluble product of yeast cells (Saccharomyces) pre- A white to pale yellow powder. Melting point: 177 – 1829 C.
pared under optimum conditions, clarified, and dried by Content: 94.0 – 108.0z. Assay—Weigh accurately
evaporating to a powder. Yeast extract (1 g) represents not about 0.8 g of zinc diethyldithiocarbamate, add 50 mL of
less than 7.5 g of yeast. A reddish yellow to brown powder, water and 15 mL of diluted hydrochloric acid (1 in 3), and
having a characteristic but not putrescent odor. Soluble in boil to dissolve. After cooling, add 40 mL of ammonia-
water, forming a yellow to brown solution, having a slight ammonium chloride buffer solution (pH 10.7) and titrate
acidic reaction. It contains no added carbohydrate. <2.50> with 0.1 mol/L disodium dihydrogen ethylenediamine
Purity (1) Chloride <1.03> (calculated as NaCl): not tetraacetate VS until the color of the solution changes from
more than 5z. red to blue (indicator: 0.1 mL of eriochrome black T TS).
(2) Coagulable protein—On heating a solution of yeast
Each mL of 0.1 mol/L disodium dihydrogen
extract (1 in 20) to boiling, no precipitate is produced.
ethylenediamine tetraacetate VS
Loss on drying <2.41>: not more than 5z (1059C, con-
＝ 36.19 mg of [(C2H5)2NCSS]2Zn
stant mass).
Residue on ignition <2.44>: not more than 15z (0.5 g). Zinc disodium ethylenediamine tetraacetate See zinc dis-
Nitrogen content <1.08>: 7.2 – 9.5z (after drying). odium ethylenediamine tetraacetate tetrahydrate.
Yellow beeswax [Same as the namesake monograph] Zinc disodium ethylenediamine tetraacetate tetrahydrate
C10H12N2Na2O8Zn.4H2O White powder. The pH of a solu-
Zaltoprofen C17H14O3S [Same as the namesake mono-
tion of zinc disodium ethylenediamine tetraacetate (1 in 100)
graph]
is between 6.0 and 9.0.
Zaltoprofen for assay C17H14O3S [Same as the mono- Purity Clarity and color of solution—Dissolve 0.10 g of
graph Zaltoprofen. When dried, it contains not less than zinc disodium ethylenediamine tetraacetate tetrahydrate in
99.5z of zaltoprofen (C17H14O3S)]. 10 mL of freshly boiled and cooled water: the solution is
clear and colorless.
Zinc Zn [K 8012, Special class]
Content: not less than 98.0z. Assay—Dissolve about
Zinc (standard reagent) Zn In addition to JIS K 8005 0.5 g of zinc disodium ethylenediamine tetraacetate tetrahy-
standard reagent for volumetric analysis, certified reference drate, accurately weighed, in water to make exactly 100 mL.
material which can be used for volumetric analysis may be Pipet 10 mL of this solution, adjust the pH to about 2 with
used. 80 mL of water and dilute nitric acid, and titrate <2.50> with
0.01 mol/L bismuth nitrate VS until the color of the solution
Zinc acetate See zinc acetate dihydrate.
changes from yellow to red (indicator: 2 drops of xylenol
0.25 mol/L Zinc acetate buffer solution (pH 6.4) Dis- orange TS).
solve 54.9 g of zinc acetate dihydrate in 150 mL of acetic
Each mL of 0.01 mol/L bismuth nitrate VS
acid (100) and 600 mL of water, add 150 mL of ammonia
＝ 4.716 mg of C10H12N2Na2O8Zn.4H2O
water (28), gently mix, and allow to cool to a room tempera-
ture. Adjust to pH 6.4 with ammonia water (28), and add Zinc dust See zinc powder.
water to make 1000 mL.
Zinc for arsenic analysis Zn [K 8012] Use granules of
Zinc acetate dihydrate Zn(CH3COO)2.2H2O [K 8356, about 800 mm.
Special class]
Zinc iodide-starch TS To 100 mL of boiling water add a
Zinc, arsenic-free See zinc for arsenic analysis. solution of 0.75 g of potassium iodide in 5 mL of water, a
solution of 2 g of zinc chloride in 10 mL of water and a
Zinc chloride ZnCl2 [K 8111, Special class]
smooth suspension of 5 g of starch in 30 mL of water, with
Zinc chloride TS Dissolve 10 g of zinc chloride and 10 g stirring. Continue to boil for 2 minutes, then cool.
of potassium hydrogen phthalate in 900 mL of water, adjust Sensitivity—Dip a glass rod into a mixture of 1 mL of 0.1
the pH to 4.0 with sodium hydroxide TS, and add water to mol/L sodium nitrite VS, 500 mL of water and 10 mL of hy-

Packings for Chromatography Gel-type strong acid cation-exchange resin for liquid chro-
matography (degree of cross-linkage: 8 z) Prepared for
a1-Acid glycoprotein binding silica gel for liquid chroma- liquid chromatography.
tography Silica gel bond a1-acid glycoprotein, prepared for
Gel type strong acid ion-exchange resin for liquid chroma-
liquid chromatography.
tography (degree of cross-linkage: 6 z) Prepared for liq-
Aminopropylsilanized silica gel for liquid chromato- uid chromatography.
graphy Prepared for liquid chromatography.
Gel type strong basic ion-exchange resin for liquid chro-
Butylsilanized silica gel for liquid chromatography Pre- matography Prepared for liquid chromatography.
pared for liquid chromatography.
Glycol etherifized silica gel for liquid chromatography
Carbamoyl group bound silica gel for liquid chromatogra- Glycol group is bound to silica gel for liquid chromatogra-
phy Prepared for liquid chromatography. phy.
Cellulose derivative-bonded silica gel for liquid chroma- Graphite carbon for gas chromatography Prepared for
tography Prepared for liquid chromatography. gas chromatography.
Cellulose for thin-layer chromatography Use a high- Graphite carbon for liquid chromatography Prepared

for liquid chromatography. m2/g) A porous acrylonitrile-divinylbenzene copolymer

prepared for gas chromatography.
Hexasilanized silica gel for liquid chromatography Pre-
pared for liquid chromatography. Porous ethyl vinylbenzene-divinylbenzene copolymer for
gas chromatography Prepared for gas chromatography.
Hydrophilic silica gel for liquid chromatography Dio-
lized porous silica gel prepared for liquid chromatography Porous ethylvinylbenzene-divinylbenzene copolymer for
(5–10 mm in particle diameter). gas chromatography (average pore diameter: 0.0075 mm,
500 – 600 m2/g) A porous ethylvinylbenzene-divinylben-
2-Hydroxypropyl-b-cyclodextrin onto silica gel for liquid
zene copolymer prepared for gas chromatography. The
chromatography Prepared for liquid chromatography.
average pore diameter is 0.0075 mm, and surface area is 500
Hydroxypropylsilanized silca gel for liquid chromatogra- to 600 m2 per g.
phy Prepared for liquid chromatography.
Porous polymer beads for gas chromatography Prepared
Neutral alumina for chromatography Prepared for chro- for gas chromatography.
matography (75 – 180 mm in particle diameter).
Porous polymethacrylate for liquid chromatography
Neutral alumina for column chromatography Prepared Prepared for liquid chromatography.
for column chromatography.
Porous silica gel for liquid chromatography A porous
Octadecylsilanized polyvinyl alcohol gel polymer for liq- silica gel prepared for liquid chromatography.
uid chromatography Prepared for liquid chromatography.
Porous styrene-divinylbenzene copolymer for gas chroma-
Octadecylsilanized porous glass for liquid chromatogra- tography (average pore diameter: 0.0085 mm, 300 – 400 m2/
phy Prepared for liquid chromatography. g) A porous styrene-divinylbenzene copolymer prepared
for gas chromatography. The average pore diameter is
Octadecylsilanized silica gel for liquid chromatography
0.0085 mm, and surface area is 300 to 400 m2/g.
Prepared for liquid chromatography.
Porous styrene-divinylbenzene copolymer for gas chroma-
Octadecylsilanized silica gel for thin-layer chromatogra-
tography (0.3 – 0.4 mm in mean pore size, not exceeding 50
phy Octadecylsilanized silica gel prepared for thin-layer
m2/g) Prepared for gas chromatography.
chromatography.
Porous styrene-divinylbenzene copolymer for liquid chro-
Octadecylsilanized silica gel with fluorescent indicator for
matography A porous styrene-divinylbenzen copolymer
thin-layer chromatography Octadecylsilanized silica gel for
prepared for liquid chromatography.
thin-layer chromatography containing fluorescent indicator.
Quaternary alkylaminated styrene-divinylbenzene copoly-
Octadecylsilanized silicone polymer coated silica gel for
mer for liquid chromatography Prepared for liquid chro-
liquid chromatography Prepared for liquid chromatogra-
matography.
phy.
Quaternary ammonium group bound to Hydrophilic vinyl
Octylsilanized silica gel for liquid chromatography Pre-
polymer gel for liquid chromatography Prepared for liquid
pared for liquid chromatography.
chromatography.
Ovomucoid-chemically bonded amino silica gel for liquid
Silica gel coated with cellulose tris(4-methylbenzoate) for
chromatography Prepared for liquid chromatography.
liquid chromatography Prepared for liquid chromatogra-
Palmitamide propylsilanized silica gel for liquid chroma- phy.
tography Prepared for liquid chromatography.
Silica gel for gas chromatography A silica gel prepared
Pentaethylenehexaaminated polyvinyl alcohol polymer for gas chromatography.
bead for liquid chromatography Prepared for liquid chro-
Silica gel for liquid chromatography A silica gel pre-
matography.
pared for liquid chromatography.
Perfluorohexylpropylsilanized silica gel for liquid chroma-
Silica gel for liquid chromatography with attached car-
tography Prepared for liquid chromatography.
bamoyl groups Prepared for liquid chromatography.
Phenylated silica gel for liquid chromatography Pre-
Silica gel for thin-layer chromatography A silica gel pre-
pared for liquid chromatography.
pared for thin-layer chromatography.
Phenylhexylsilanized silica gel for liquid chromatography
Silica gel for thin-layer chlomatography (particle size 5 – 7
Prepared for liquid chromatography.
mm, with fluorescent indication) Prepared for high-perfor-
Phenylsilanized silica gel for liquid chromatography Pre- mance thin-layer chromatography.
pared for liquid chromatography.
Silica gel with complex fluorescent indicator for thin-layer
Polyamide for column chromatography Prepared for chromatography A silica gel for thin-layer chromatogra-
column chromatography. phy containing suitable complex fluorescent indicators.
Polyamide for thin-layer chromatography Prepared for Silica gel with fluorescent indiator for thin-layer chroma-
thin-layer chromatography. tography A silica gel for thin-layer chromatography con-
taining a suitable fluorescent indicator.
Polyamide with fluorescent indicator for thin-layer chro-
matography Add a fluorescent indicator to polyamide for Siliceous earth for chromatography A siliceous earth
thin-layer chromatography. prepared for chromatography.
Porous acrylonitrile-divinylbenzene copolymer for gas Siliceous earth for gas chromatography A siliceous earth
chromatography (pore diameter: 0.06 – 0.08 mm, 100 – 200 prepared for gas chromatography.

9.43 Filter Papers, Filters for Potassium iodide-starch paper Impregnate filter paper
with freshly prepared potassium iodide-starch TS, and dry in
filtration, Test Papers, a clean room.
Crucibles, etc. Storage—Store in a glass-stoppered bottle, protected from
light and moisture.
Filter paper [P 3801, Filter paper (for chemical analy-
Red litmus paper See litmus paper, red.
sis), Filter paper for qualitative analysis]
No.1: For bulky gelatinous precipitate Turmeric paper Macerate 20 g of powdered dried rhi-
No.2: For moderate-sized precipitate zome of Curcuma longa Linn áe with four 100 mL-portions of
No.3: For fine precipitate cold water, decant the supernatant liquid each time, and dis-
No.4: Hardened filter paper for fine precipitate card it. Dry the residue at a temperature not over 1009 C.
Macerate the dried residue with 100 mL of ethanol (95) for
Filter paper for quantitative analysis [P 3801, Filter

several days, and filter. Immerse filter paper in this ethanol

decoction, and allow the ethanol (95) to evaporate spontane- Transmission rate
Type of filter for calibration (z) Product name
ously in clean air.
Sensitivity—Dip a strip of turmeric paper, about 1.5 cm Optical filter for calibration 1 JCRM 101
length, in a solution of 1 mg of boric acid in a mixture of within the visible 10 JCRM 110
1 mL of hydrochloric acid and 4 mL of water, after 1 minute wavelength range 20 JCRM 120
30 JCRM 130
remove the paper from the liquid, and allow it to dry spon-
40 JCRM 140
taneouly: the yellow color changes to brown. When the strip
50 JCRM 150
is moistened with ammonia TS, the color of the strip changes
to greenish black. Optical filter for calibration 10 JCRM 210 A
within the ultraviolet
Zinc iodide-starch paper Impregnate the filter paper for wavelength range 50 JCRM 250 A
volumetric analysis with freshly prepared zinc iodide-starch
TS, and dry in the clean room. Preserve in a glass-stoppered Optical filter for calibration 10 JCRM 310
within the near-ultraviolet 30 JCRM 330
bottle, protected from light and moisture.
wavelength range 50 JCRM 350

9.44 Standard Particles, etc.

a-Alumina for specific surface area determination
9.62 Measuring Instruments,
a-Al2O3 Prepared for specific surface area determination. Appliances
Calibration ball for particle density measurement Measuring Instruments are the instruments or machines
Calibration ball with a known volume prepared for meas- used for measuring mass or volume in the JP tests, and Ap-
urement of particle density. The volume of the calibration pliances are the instruments specified in order to make test
ball must be accurately determined to the nearest 0.001 cm3. conditions as consistent as possible in those tests.

Indium for thermal analysis In Prepared for thermal Balances and weights (1) Chemical balances—Use
analysis. balances readable to the extent of 0.1 mg.
Content: not less than 99.99z. (2) Semimicrobalances—Use balances readable to the ex-
tent of 10 mg.
Standard particles for calibrating light-shielded automatic (3) Microbalances—Use balances readable to the extent
fine particle counter Use plastic spherical particles of of 1 mg.
known size and number. (4) Ultramicrobalances—Use balances readable to the
Tin for thermal analysis Sn [K 8580 (Tin). Content: extent of 0.1 mg.
not less than 99.99z] (5) Weights—Use calibrated weights.
Carbon dioxide measuring detector tube [Gas detector
tube measurement system K 0804] Packed with meas-
Measuring Instruments and urement packing for carbon dioxide.

Appliances, Thermometers, etc. Carbon monoxide measuring detector tube [Gas detector
tube measurement system K 0804] Packed with meas-
urement packing for carbon monoxide.
9.61 Optical Filters for Cassia flask Use glass-stoppered flasks, shown in Fig.
Wavelength and Transmission 9.62-1, made of hard glass and having graduation lines of
volume on the neck.
Rate Calibration
Use optical filters for wavelength calibration and those for
transmission rate calibration shown in Table 9.61-1 and
Table 9.61-2, respectively. The optical filters for transmis-
sion rate calibration are also used for the calibration of ab-
sorbances.

Table 9.61-1 Optical Filters for Wavelength Calibration

Range of
Type of filter wavelength
calibration Product name
(nm)
Neodymium optical filter for
400 – 750 JCRM 001
wavelength calibration
Holmium optical filter for
250 – 600 JCRM 002
wavelength calibration

Table 9.61-2 Optical Filters for Transmission Rate

Calibration Fig. 9.62-1 Fig. 9.62-2

9.63 Thermometers
Thermometers Ordinarily, use calibrated thermometers
with an immersion line (rod) or calibrated total immersion
mercury-filled thermometers according to the Japanese In-
dustrial Standards. Use the thermometers with the immer-
sion line (rod), shown in Table 9.63-1, for the tests in Con-
gealing Point, Melting Point (Method 1), Boiling Point and
Distilling Range.

Fig. 9.62-3

Gas mixer Use the apparatus, shown in Fig. 9.62-3,

made of hard glass.
Nessler tube Use colorless, glass-stoppered cylinders 1.0
to 1.5 mm in thickness, shown in Fig. 9.62-2, made of hard
glass. The difference of the height of the graduation line of
50 mL from the bottom among cylinders does not exceed 2
mm.
Sieves Sieves conform to the specifications in Table
9.62-1. Use the sieve number of nominal size as the designa-
tion.
Volumetric measures Use volumetric flasks, transfer
pipets, piston pipets, burets and measuring cylinders con-
forming to the Japanese Industrial Standard.

Table 9.62-1 Specification of Sieves

Nominal Nominal Permissible variation

Sieve Diameter of wire (mm)
size opening of opening (mm)
number
( mm) (mm) Average Maximum Recommended Maximum Minimum

3.5 5600 5.60 ±0.18 0.47 1.60 1.90 1.30

4 4750 4.75 ±0.15 0.41 1.60 1.90 1.30
4.7 4000 4.00 ±0.13 0.37 1.40 1.70 1.20

5.5 3350 3.35 ±0.11 0.32 1.25 1.50 1.06

6.5 2800 2.80 ±0.09 0.29 1.12 1.30 0.95
7.5 2360 2.36 ±0.08 0.25 1.00 1.15 0.85
8.6 2000 2.00 ±0.07 0.23 0.90 1.04 0.77

10 1700 1.70 ±0.06 0.20 0.80 0.92 0.68

12 1400 1.40 ±0.05 0.18 0.71 0.82 0.60
14 1180 1.18 ±0.04 0.16 0.63 0.72 0.54
16 1000 1.00 ±0.03 0.14 0.56 0.64 0.48

18 850 0.850 ±0.029 0.127 0.500 0.580 0.430

22 710 0.710 ±0.025 0.112 0.450 0.520 0.380
26 600 0.600 ±0.021 0.101 0.400 0.460 0.340
30 500 0.500 ±0.018 0.089 0.315 0.360 0.270

36 425 0.425 ±0.016 0.081 0.280 0.320 0.240

42 355 0.355 ±0.013 0.072 0.224 0.260 0.190
50 300 0.300 ±0.012 0.065 0.200 0.230 0.170
60 250 0.250 ±0.0099 0.058 0.160 0.190 0.130

70 212 0.212 ±0.0087 0.052 0.140 0.170 0.120

83 180 0.180 ±0.0076 0.047 0.125 0.150 0.106
100 150 0.150 ±0.0066 0.043 0.100 0.115 0.085
119 125 0.125 ±0.0058 0.038 0.090 0.104 0.077

140 106 0.106 ±0.0052 0.035 0.071 0.082 0.060

166 90 0.090 ±0.0046 0.032 0.063 0.072 0.054
200 75 0.075 ±0.0041 0.029 0.050 0.058 0.043
235 63 0.063 ±0.0037 0.026 0.045 0.052 0.038

282 53 0.053 ±0.0034 0.024 0.036 0.041 0.031

330 45 0.045 ±0.0031 0.022 0.032 0.037 0.027
391 38 0.038 ±0.0029 0.020 0.030 0.035 0.024

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GENERAL TESTS, PROCESSES

Fig. 1.02-2 Vacuum distilling apparatus for ammonium

1.05 Mineral Oil Test

amount of Standard Iron Solution specified in indivisual

1.11 Arsenic Limit Test

2. Melting point Table 1.13-1

and 809C under reduced pressure (about 2.67 kPa) for 30

In assay, ``System performance'' should be defined by the

Fig. 2.21-1 FT-NMR spectrometer Fig. 2.21-2 CW-NMR spectrometer

sity in a fully relaxed spectrum of a compound is directly F ＝ kI0qecl

2.22 Fluorometry 2.23 Atomic Absorption

4. Note length which shows minimal transmission rate, the differ-

atmosphere can be utilized. 3. Identification

2.42 Congealing Point

Concentration Acetic acid Heptane

rapidly to the original position when the titration system has

the needle of the millivoltmeter is at the original position 

Conductivity Measurement is a method for the measuring Concentration Conductivity k Resistivity r

Fig‚ 2.52-1 Thermogram Fig‚ 2.52-2 Thermal diagrams according to purity

Table 2.53-1 Specifications of the Ubbelohde-type

0.005 0.46 3.0 1– 5

Fig. 2.53-2a Coaxial double cylinder-type rotational

liquid. Consequently, torque, T, is generated by the forced

Table 2.56-1 Density of water

trinsic vibration period T in proportion to the mass of the

Fig. 2.56-2 Oscillator-type density meter

ously adjusted to a specified temperature t?9C. Wash the

However, use a 200-mL distilling flask A with a neck 18 to

2.58 X-Ray Powder Diffraction

Fig. 2.58-3 Geometric arrangement of the Bragg-Brentano

2.60 Melting Point Determination

calibration ball of known volume for particle density meas-

Fig. 3.03-1 Schematic diagram of a gas pycnometer

Table 3.04-1. Size of standard sieve series in range of interest

ISO Nominal Aperture

11.20 mm 11.20 mm 11.20 mm 11200

the endotoxin concentration of the sample solution as less Table 4.01-4

Water 1000 mL a, b, c, d, e: Average transmittance or absorbance values

trate, exactly measure the subsequent 2 mL of the filtrate,

organisms used for inoculation are not more than 5 passages

Growth promotion Suitability of counting method in

Table 4.05-I-2 Common neutralizing agents for interfering substances

Interfering substance Potential neutralizing method

Glutaraldehyde, Mercurials Sodium hydrogen sulfite (Sodium bisulfite)

Phenolics, Alcohol, Aldehydes, Sorbate Dilution

Quaternary Ammonium Compounds (QACs), Lecithin

QAC, Iodine, Parabens Polysorbate

Mercurials, Halogens, Aldehydes Thiosulfate

EDTA (edetate) Mg or Ca ions

Observed combinations of numbers of tubes

Table 4.05-II-1 Growth promoting, inhibitory and indicative properties of media

Medium Property Test strains

Test for bile-tolerant gram-negative bacteria

Enterobacteria enrichment broth- Growth promoting E. coli

Test for Escherichia coli

MacConkey broth Growth promoting E. coli

MacConkey agar Growth promoting＋ E. coli

Test for Salmonella

Test for Pseudomonas aeruginosa

Cetrimide agar Growth promoting P. aeruginosa

Test for Staphylococcus aureus

Mannitol salt agar Growth promoting＋ S. aureus

Test for Clostridia

Reinforced medium for Clostridia Growth promoting Cl. sporogenes

Columbia agar Growth promoting Cl. sporogenes

Sabouraud-dextrose broth Growth promoting C. albicans

Sabouraud-dextrose agar Growth promoting＋ C. albicans

3.1.2. Test for absence 3.1.3. Quantitative test

Results for each quantity of product Probable number of bacteria

＋ ＋ ＋ more than 103

3.1.3.2. Interpretation filter the volume of sample corresponding to 1 patch of the

hours. Agar 15.0 g

Table 5.02-I-1 Preparation and use of test micro-organisms

Suitability of counting method

Staphylococcus Soybean-Casein Soybean-Casein Soybean-Casein

Pseudomonas aer- Soybean-Casein Soybean-Casein Soybean-Casein

Bacillus subtilis Soybean-Casein Soybean-Casein Soybean-Casein

Aspergillus Sabouraud Glucose Soybean-Casein Sabouraud Glucose Soybean-Casein Sabouraud Glucose

＋＋＋ more than 103

less than 102 and －－－ less than 10