USP 36
Standard Solution—[NOTE—The concentration can be USP Reference Standard specified in the individual mono-
adjusted depending on the amount of acetate or acetic acid
expected to be present in the test material.] Dissolve an
accurately weighed quantity of USP Glacial Acetic Acid RS in
Diluent to obtain a solution having a known concentration
of about 0.1 mg per mL.
Test Solution—Prepare as directed in the individual
monograph. The amount of material used can be adapted
depending on the amount of acetic acid expected.
Chromatographic System (see Chromatography 〈621〉)—
The liquid chromatograph is equipped with a 210-nm de-
tector and a 4.6-mm × 25-cm column that contains not
greater than 5-µm packing L1. The flow rate is about
1.2 mL per minute. The chromatograph is programmed as
follows.
Time Solution A Solution B
(minutes) (%) (%) Elution
0 95 5 equilibration
0–5 95 5 isocratic
5–10 95→50 5→50 linear gradient
10–20 50 50 isocratic
20–22 50→95 50→5 linear gradient
Chromatograph the Standard Solution, and record the peak
responses as directed for Procedure: the retention time of
acetic acid is between 3 and 4 minutes; and the relative
standard deviation for replicate injections is not more than
5%.
Procedure—Separately inject equal volumes (about
10 µL) of the Standard Solution and the Test Solution into the
chromatograph, record the chromatograms, and measure
the responses for the acetic acid peaks. Calculate the per-
centage of acetic acid in the portion of test material taken
by the formula:
100(CS/M)(rU/rS)
in which CS is the concentration of acetic acid in the Stan-
dard Solution; M is the concentration, in mg per mL, of the
Test Solution, based on the weight of test material taken and
the extent of dilution; and rU and rS are the acetic acid peak
responses obtained from the Test Solution and the Standard
Solution, respectively.
〈511〉 SINGLE-STEROID ASSAY
In the following procedure, the steroid to be assayed is
separated from related foreign steroids and excipients by
thin-layer chromatography and determined following recov-
ery from the chromatogram.
Preparation of the Plate—Prepare a slurry from 30 g of
chromatographic silica gel with a suitable fluorescing sub-
stance by the gradual addition, with mixing, of about 65 mL
of a mixture of water and alcohol (5:2). Transfer the slurry
to a clean, 20- × 20-cm plate, spread to make a uniform
layer 250 µm thick, and allow to dry at room temperature
for 15 minutes. Heat the plate at 105° for 1 hour, and store
in a desiccator.
Solvent A—Mix methylene chloride with methanol
(180:16).
Solvent B—Mix chloroform with acetone (4:1).
Standard Preparation—Dissolve in a mixture of equal
volumes of chloroform and alcohol a suitable quantity of the
Chemical Tests / 〈525〉 Sulfur Dioxide 209
graph, previously dried as directed (see USP Reference Stan-
dards 〈11〉) and accurately weighed, to obtain a solution
having a known concentration of about 2 mg per mL.
Assay Preparation—Prepare as directed in the individual
monograph.
Procedure—Divide the area of the chromatographic
plate into three equal sections, the left and right sections to
be used for the Assay Preparation and the Standard Prepara-
tion, respectively, and the center section for the blank. Ap-
ply 200 µL each of the Assay Preparation and the Standard
Preparation as streaks 2.5 cm from the bottom of the appro-
priate section of the plate. Dry the solution as it is being
applied, with the aid of a stream of air. Using the Solvent
specified in the individual monograph, develop the chro-
matogram in a suitable chamber, previously equilibrated
and lined with absorbent paper, until the solvent front has
moved 15 cm above the initial streaks.
Remove the plate, evaporate the solvent, and locate the
principal band occupied by the Standard Preparation by
viewing under UV light. Mark this band, as well as corre-
sponding bands in the Assay Preparation and blank sections
of the plate. Remove the silica gel from each band sepa-
rately, either by scraping onto glazed weighing papers or by
using a suitable vacuum collecting device, and transfer it to
a glass-stoppered, 50-mL centrifuge tube. To each tube add
25.0 mL of alcohol, and shake for not less than 2 minutes.
Centrifuge the tubes for 5 minutes, pipet 20 mL of the su-
pernatant from each tube into a glass-stoppered, 50-mL
conical flask, add 2.0 mL of a solution prepared by dissolv-
ing 50 mg of blue tetrazolium in 10 mL of methanol, and
mix. Proceed as directed for Procedure under Assay for Ster-
oids 〈351〉, beginning with “Then to each flask.”
〈525〉 SULFUR DIOXIDE
The following methods are provided for the determination
of sulfur dioxide in pharmaceutical excipients.
METHOD I
Procedure
Mix 20 g of the test specimen, accurately weighed, with
200 mL of an appropriate solvent as indicated in each indi-
vidual monograph, and stir until a smooth suspension is ob-
tained. Allow the test specimen mixture to remain undis-
turbed until most of the test specimen has settled, and filter
the aqueous portion through paper (Whatman No. 1 or
equivalent). To 100 mL of the clear filtrate add an additional
solvent as indicated in each individual monograph, add
3 mL of starch TS, and titrate with 0.01 N iodine solution VS
to the first permanent blue or purple color. Each 1.0 mL of
0.01 N iodine solution VS consumed corresponds to
0.003% of sulfur dioxide found.
METHOD II
Procedure
Transfer about 50 to 100 g of the substance to be tested,
accurately weighed, to a 250-mL conical flask, add 100 to
150 mL of water, and mix. Cool to between 5° and 10°.
210 〈525〉 Sulfur Dioxide / Chemical Tests USP 36

While stirring with a magnetic stirrer, add 10 mL of cold

1.5 N sodium hydroxide (at a temperature between 5° and
10°). Stir for an additional 20 seconds, and add 10 mL of
starch indicator solution, prepared as follows: mix 10 g of
soluble starch with 50 mL of cold water, transfer to 1000 mL
of boiling water, stir until completely dissolved, cool, and
add 1 g of salicylic acid preservative. [NOTE—Discard the so-
lution after 1 month.] Add 10 mL of 2.0 N sulfuric acid (at a
temperature between 5° and 10°), and titrate immediately
with 0.005 N iodine VS until a light blue color persists for
1 minute (see Titrimetry 〈541〉). Perform a blank determina-
tion, using 200 mL of water treated similarly to the solution
under test, and make any necessary correction. Each mL of
0.005 N iodine is equivalent to 0.16 mg of SO2.

METHOD III

Procedure
Dissolve 20.0 g of the test specimen in 150 mL of hot
water in a flask having a round bottom and a long neck,
add 5 mL of phosphoric acid and 1 g of sodium bicarbo-
nate, and at once connect the flask to a condenser. [NOTE—
Excessive foaming can be alleviated by the addition of a few
drops of a suitable antifoaming agent.] Distill 50 mL, receiv-
ing the distillate under the surface of 50 mL of 0.1 N iodine.
Acidify the distillate with a few drops of hydrochloric acid,
add 2 mL of barium chloride TS, and heat on a steam bath
until the liquid is nearly colorless. The precipitate of barium
sulfate, if any, when filtered, washed, and ignited, weighs
not more than 3 mg, corresponding to not more than
0.004% of sulfur dioxide, correction being made for any
sulfate that may be present in 50 mL of the 0.1 N iodine.
METHOD IV
In this test, sulfur dioxide is released from the test speci-
men in a boiling acid medium and is removed by a stream
of carbon dioxide. The separated gas is collected in a dilute
hydrogen peroxide solution, in which the sulfur dioxide is
oxidized to sulfuric acid and titrated with standard alkali,
using a pH meter to control the pH value and titration. This
test is performed under conditions such that the require-
ments specified in the system suitability test are met.
Special Reagents
Carbon Dioxide—Use carbon dioxide with a flow regula-
tor that will maintain a flow of 100 ± 10 mL per minute.
Hydrogen Peroxide Solution—Dilute 30% hydrogen
peroxide with water to obtain a 3% solution. Neutralize the
3% hydrogen peroxide solution with 0.01 N sodium hy-
droxide to a pH of 4.1 determined potentiometrically.
Potassium Metabisulfite Solution—Transfer 0.87 g of
potassium metabisulfite (K2S2O5) and 0.2 g of edetate diso-
dium to a 1000-mL volumetric flask. Dilute with water to
volume before mixing. [NOTE—Edetate disodium is used to
protect sulfite ion from oxidation.]
Apparatus
A suitable apparatus for sulfur dioxide determination is
shown in the accompanying diagram (Figure 1).
Figure 1. Apparatus for Method IV.
The apparatus consists of a 500-mL three-neck, round-bot-
tom boiling flask, A; a separatory funnel, B, having a capac-
ity of 100 mL or greater; a gas inlet tube of sufficient length
to permit introduction of the carbon dioxide within 2.5 cm
of the bottom of the boiling flask; a reflux condenser, C,
having a jacket length of 200 mm; and a delivery tube, E,
connecting the upper end of the reflux condenser to the
bottom of a receiving test tube, D. Apply a thin film of
stopcock grease to the sealing surfaces of all joints except
the joint between the separatory funnel and the boiling
flask, and clamp the joints to ensure tightness.
System Suitability Test
Test A—Using the Potassium Metabisulfite Solution as the
standard, proceed as directed for Procedure, except replace
the 25.0 g of test substance with 20 mL of Potassium Meta-
bisulfite Solution. Calculate the content, in µg per mL, of
sulfur dioxide in the Potassium Metabisulfite Solution taken
by the formula:
1000(32.03)VN/VP
in which the factor 1000 converts mg to µg; 32.03 is the
milliequivalent weight of sulfur dioxide; V is the volume, in
mL, of titrant consumed; N is the normality of the titrant;
and VP is the volume, in mL, of the Potassium Metabisulfite
Solution taken for the test.
Test B—In a 100-mL conical flask, add 20 mL of 0.02 N
iodine solution and 5 mL of 2 N hydrochloric acid. Add
1 mL of starch TS, and titrate with the Potassium Metabisul-
fite Solution until the first discoloration is observed. Calculate
the content, in µg per mL, of sulfur dioxide in the Potassium
Metabisulfite Solution by the formula:
1000(32.03)VINI/VP
in which 1000 and 32.03 are defined above; VI is the vol-
ume, in mL, of the iodine solution used in the test; NI is the
normality of the iodine solution; and VP is the volume, in
mL, of the Potassium Metabisulfite Solution consumed.
The difference between the sulfur dioxide contents ob-
tained from Test A and Test B is not more than 5% of their
mean value. Test B shall be performed within 15 minutes
USP 36 Chemical Tests / 〈525〉 Sulfur Dioxide 211

after completion of Test A. [NOTE—This avoids a potential hydroxide while maintaining an atmosphere of nitrogen in
variation of the sulfur dioxide content in the Potassium the bottle.
Metabisulfite Solution when stored at room temperature.] Potassium Metabisulfite Solution—Transfer 0.87 g of
potassium metabisulfite (K2S2O5) and 0.2 g of edetate diso-
Procedure dium to a 1000-mL volumetric flask. Dilute with water to
volume before mixing. [NOTE—Edetate disodium is used to
Add 150 mL of water to the boiling flask (A). Close the protect sulfite ion from oxidation.]
stopcock of the separatory funnel, and begin the flow of
carbon dioxide at a rate of 100 ± 5 mL per minute through Apparatus
the apparatus. Start the condenser coolant flow. Place
10 mL of Hydrogen Peroxide Solution in the receiving test The apparatus (see Figure 2) is designed to effect the se-
tube (D). After 15 minutes, without interrupting the flow of lective transfer of sulfur dioxide from the specimen in boil-
carbon dioxide, remove the separatory funnel (B) from the ing aqueous hydrochloric acid to the Hydrogen Peroxide So-
boiling flask, and transfer 25.0 g of the test specimen to the lution in vessel G. The backpressure is limited to the
boiling flask with the aid of 100 mL of water. Apply stop- unavoidable pressure due to the height of the Hydrogen Per-
cock grease to the outer joint of the separatory funnel, and oxide Solution above the tip of the bubbler, F. Keeping the
replace the separatory funnel in the boiling flask. Close the backpressure as low as possible reduces the likelihood that
stopcock of the separatory funnel, and add 80 mL of 2 N sulfur dioxide will be lost through leaks. Preboil vinyl and
hydrochloric acid to the separatory funnel. Open the stop- silicone tubing. Apply a thin film of stopcock grease to the
cock of the separatory funnel to permit the hydrochloric sealing surfaces of all joints, except the joint between the
acid solution to flow into the boiling flask, guarding against separatory funnel and the flask, and clamp the joints to en-
escape of sulfur dioxide into the separatory funnel by clos- sure tightness. The separatory funnel, B, has a capacity of
ing the stopcock before the last few mL of hydrochloric acid 100 mL or greater. The inlet adapter, A, with a hose con-
drain out. Boil the mixture for 1 hour. Open the stopcock of nector, provides a means of applying headpressure over the
the funnel, stop the flow of carbon dioxide, discontinue solution. [NOTE—A pressure-equalizing dropping funnel is
heating the flask, and turn off the cooling water in the con- not recommended because condensate, which may contain
denser. Remove the receiving test tube, and transfer its con- sulfur dioxide, is deposited in the funnel and the side arm.]
tents to a 200-mL wide-necked, conical flask. Rinse the re-
ceiving test tube with a small portion of water, add the
rinsing to the 200-mL conical flask, and mix. Heat on a
water bath for 15 minutes, and allow to cool. Add 0.1 mL of
bromophenol blue TS, and titrate the contents with 0.1 N
sodium hydroxide VS until the color changes from yellow to
violet-blue, with the color change lasting for at least
20 seconds. Perform a blank determination and make any
necessary correction (see Titrimetry 〈541〉). Calculate the
content, in µg per g, of sulfur dioxide in the test specimen
taken by the formula:
1000(32.03)VN/W
in which the factor 1000 converts mg to µg; 32.03 is the
milliequivalent weight of sulfur dioxide; V is the volume, in
mL, of titrant consumed; N is the normality of the titrant;
and W is the weight, in g, of the test specimen taken.

METHOD V
In this method, similar to Method IV, sulfur dioxide is re-
leased from the test specimen in a boiling acid medium and
is removed by a stream of nitrogen. The separated gas is
collected in a dilute hydrogen peroxide solution, in which
the sulfur dioxide is oxidized to sulfuric acid and titrated
with standard alkali, using methyl red as an indicator. This
test is performed under conditions such that the require-
ments specified in the system suitability test are met.

Special Reagents
Hydrogen Peroxide Solution—Dilute a portion of
30 percent hydrogen peroxide with water to obtain a 3%
solution. Just before use, add 3 drops of methyl red TS, and
neutralize to a yellow endpoint with 0.01 N sodium hydrox-
ide. Do not exceed the endpoint.
Nitrogen—Use high-purity nitrogen with a flow regulator
that will maintain a flow of 200 ± 10 mL per minute. Guard
against the presence of oxygen by passing the nitrogen
through a scrubber, such as alkaline pyrogallol, prepared as
follows: add 4.5 g of pyrogallol to a gas-washing bottle,
purge the bottle with nitrogen for 3 minutes, and add a Figure 2. Apparatus for Method V.
solution containing 85 mL of water and 65 g of potassium
212 〈525〉 Sulfur Dioxide / Chemical Tests
The round-bottom flask, C, is a 1000-mL flask with three
24/40 tapered joints. The gas inlet tube, D, is long enough
to permit introduction of the nitrogen to within 2.5 cm of
the bottom of the flask. The Allihn condenser, E, has a
jacket length of 300 mm. The bubbler, F (see Figure 3), is
fabricated from glass according to the dimensions given in
Figure 3. The Hydrogen Peroxide Solution is contained in the
vessel, G, having an inside diameter of about 2.5 cm and a
depth of about 18 cm. Circulate coolant, such as a mixture
of water and methanol (4:1) maintained at 5°, to chill the
condenser.
Figure 3. Bubbler (F) for apparatus in Method V.
System Suitability Test
Test A—Using the Potassium Metabisulfite Solution as the
standard, proceed as directed for Procedure, except replace
the 50.0 g of test substance with 20 mL of Potassium Meta-
bisulfite Solution. Calculate the content, in µg per mL, of
sulfur dioxide in the Potassium Metabisulfite Solution taken
by the formula:
1000(32.03)VN/VP
in which the factor 1000 converts mg to µg; 32.03 is the
milliequivalent weight of sulfur dioxide; V is the volume, in
mL, of titrant consumed; N is the normality of the titrant;
and VP is the volume, in mL, of Potassium Metabisulfite Solu-
tion taken for the test.
Test B—In a 100-mL conical flask, add 20 mL of 0.02 N
iodine solution and 5 mL of 2 N hydrochloric acid. Add
1 mL of starch TS, and titrate with the Potassium Metabisul-
fite Solution until the first discoloration is observed. Calculate
the content, in µg per mL, of sulfur dioxide in the Potassium
Metabisulfite Solution by the formula:
1000(32.03)VINI/VP
in which 1000 and 32.03 are defined above; VI is the vol-
ume, in mL, of iodine solution used in the test; NI is the
normality of the iodine solution; and VP is the volume, in
mL, of Potassium Metabisulfite Solution consumed.
The difference between the sulfur dioxide contents ob-
tained from Test A and Test B is not more than 5% of their
mean value. Test B shall be performed within 15 minutes
after completion of Test A. [NOTE—This avoids a potential
USP 36
variation of the sulfur dioxide content in the Potassium
Metabisulfite Solution when stored at room temperature.]
Procedure
Position the apparatus in a heating mantle controlled by a
power-regulating device. Add 400 mL of water to the flask.
Close the stopcock of the separatory funnel, and add 90 mL
of 4 N hydrochloric acid to the separatory funnel. Begin the
flow of nitrogen at a rate of 200 ± 10 mL per minute. Start
the condenser coolant flow. Add 30 mL of Hydrogen Peroxide
Solution to the vessel (G). After 15 minutes, remove the sep-
aratory funnel, and transfer a mixture of 50.0 g of the test
specimen, accurately weighed, and 100 mL of alcohol solu-
tion (5 in 100) to the flask. Apply stopcock grease to the
outer joint of the separatory funnel, return the separatory
funnel to the tapered joint flask, and concomitantly resume
the nitrogen flow. Apply headpressure above the hydrochlo-
ric acid solution in the separatory funnel with a rubber bulb
equipped with a valve. Open the stopcock of the separatory
funnel to permit the hydrochloric acid solution to flow into
the flask. Continue to maintain sufficient pressure above the
hydrochloric acid solution to force it into the flask. [NOTE—
The stopcock may be temporarily closed, if necessary, to
increase the pressure.] To guard against escape of sulfur di-
oxide into the separatory funnel, close the stopcock before
the last few mL of hydrochloric acid drain out. Apply power
to the heating mantle sufficient to cause about 85 drops of
reflux per minute. After refluxing for 1.75 hours, remove the
vessel (G), add 3 drops of methyl red TS, and titrate the
contents with 0.01 N sodium hydroxide VS, using a 10-mL
buret with an overflow tube and a hose connection to a
carbon dioxide-absorbing tube, to a yellow endpoint that
persists for at least 20 seconds. Perform a blank determina-
tion, and make any necessary correction (see Titrimetry
〈541〉). Calculate the quantity, in µg, of SO2 in each g of the
test specimen taken by the formula:
1000(32.03)VN/W
in which the factor 1000 converts mg to µg; 32.03 is the
milliequivalent weight of sulfur dioxide; V is the volume, in
mL, of titrant consumed; N is the normality of the titrant;
and W is the weight, in g, of the test specimen taken.
〈531〉 THIAMINE ASSAY
USP Reference Standards 〈11〉—USP Thiamine Hydrochlo-
ride RS.
The following procedure is provided for the determination
of thiamine as an ingredient of Pharmacopeial preparations
containing other active constituents.
Special Solutions and Solvents—
POTASSIUM FERRICYANIDE SOLUTION—Dissolve 1.0 g of potas-
sium ferricyanide in water to make 100 mL. Prepare fresh on
the day of use.
OXIDIZING REAGENT—Mix 4.0 mL of Potassium Ferricyanide
Solution with sufficient 3.5 N sodium hydroxide to make
100 mL. Use this solution within 4 hours.
QUININE SULFATE STOCK SOLUTION—Dissolve 10 mg of quinine
sulfate in 0.1 N sulfuric acid to make 1000 mL. Preserve this
solution, protected from light, in a refrigerator.
QUININE SULFATE STANDARD SOLUTION—Dilute 0.1 N sulfuric
acid with Quinine Sulfate Stock Solution (39:1). This solution
fluoresces to approximately the same degree as the thi-
ochrome obtained from 1 µg of thiamine hydrochloride and