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USP 37
Official Monographs / Azathioprine 1883
the rinse to the aqueous phase from which the respective
supernatant was removed. Insert the stoppers, rotate each
tube for about 10 minutes, and centrifuge. Transfer each supernatant to the respective supernatant previously collected.
Pipet 15 mL of 0.1 N hydrochloric acid into each centrifuge
tube containing the combined supernatants, insert the stoppers, rotate each tube for about 15 minutes, and centrifuge.
Remove and discard the supernatants. Concomitantly determine the absorbances of the solutions in 1-cm cells at the
wavelength of maximum absorbance at about 283 nm, with
a suitable spectrophotometer zeroed with 0.1 N hydrochloric acid, using the prepared reagent blank. Calculate the
quantity, in mg, of C20H22N2 · 2C4H4O4 in the portion of
Tablets taken by the formula:
25C(AU / AS)
in which C is the concentration, in mg per mL, of USP
Azatadine Maleate RS in the Standard preparation; and AU
and AS are the absorbances of the solutions from the Assay
preparation and the Standard preparation, respectively.
.
Azathioprine
C9H7N7O2S
277.26
1H-Purine, 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)thio]-;
6-[(1-Methyl-4-nitroimidazol-5-yl)thio]purine [446-86-6].
DEFINITION
Azathioprine contains NLT 98.0% and NMT 101.5% of
azathioprine (C9H7N7O2S), calculated on the dried basis.
IDENTIFICATION
• A. INFRARED ABSORPTION 〈197K〉
• B. The principal spot from the Sample solution shows the
same RF value as that obtained from Standard solution A
in the test for Limit of Mercaptopurine.
ASSAY
• PROCEDURE
Sample solution: Dissolve 300 mg of Azathioprine in
80 mL of dimethylformamide, and add 5 drops of a
1-in-100 solution of thymol blue in dimethylformamide.
Titrimetric system
Mode: Direct titration
Titrant: 0.1 N tetrabutylammonium hydroxide VS
Endpoint detection: Visual
Analysis: Titrate the Sample solution with Titrant, using
a magnetic stirrer, and taking precautions to prevent
absorption of atmospheric carbon dioxide. Perform a
blank determination. Each mL of 0.1 N tetrabutylammonium hydroxide is equivalent to 27.73 mg of azathioprine (C9H7N7O2S).
Acceptance criteria: 98.0%–101.5% on the dried basis
IMPURITIES
• RESIDUE ON IGNITION 〈281〉: NMT 0.1%
• LIMIT OF MERCAPTOPURINE
Standard solution A: 20 mg/mL of USP Azathioprine
RS in 6 N ammonium hydroxide
Standard solution B: 200 µg/mL of USP Mercaptopurine RS, on the anhydrous basis, in 6 N ammonium hydroxide
Sample solution: 20 mg/mL of Azathioprine in 6 N ammonium hydroxide
Official from December 1, 2014
Copyright (c) 2015 The United States Pharmacopeial Convention. All rights reserved.
USP Monographs
Packaging and storage—Preserve in well-closed containers.
USP Reference standards 〈11〉—
USP Azatadine Maleate RS
Identification—Transfer 15.0 mL of the Standard preparation and 15.0 mL of the Assay preparation, respectively, prepared as directed in the Assay, to separate 50-mL centrifuge
tubes fitted with glass stoppers. To each centrifuge tube add
10.0 mL of 1.0 N sodium hydroxide and 20 mL of solvent
hexane, insert the stoppers, rotate the centrifuge tubes for
about 15 minutes, and centrifuge. Transfer the solvent hexane extracts (upper phase) from each centrifuge tube to
separate 50-mL conical flasks fitted with glass stoppers.
Evaporate the solvent hexane extracts on a steam bath
under a stream of nitrogen to dryness, pipet 1 mL of solvent
hexane into each flask, insert the stoppers, and mix by use
of a vortex mixer (or equivalent) until the residues have dissolved. Use these solutions as the Standard solution and the
test solution, respectively. Apply separately 100 µL each of
the test solution and the Standard solution to a suitable
thin-layer chromatographic plate (see Chromatography
〈621〉) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the
chromatogram in a solvent system consisting of toluene,
isopropyl alcohol, and diethylamine (10:10:1) until the solvent front has moved about three-fourths of the length of
the plate. Remove the plate from the developing chamber,
mark the solvent front, and allow the plate to air-dry. Examine the plate under short-wavelength UV light: the RF
value and intensity of the principal spot in the chromatogram of the test solution correspond to those obtained from
the chromatogram of the Standard solution.
Dissolution 〈711〉—
Medium: 0.01 N hydrochloric acid; 500 mL.
Apparatus 2: 50 rpm.
Time: 30 minutes.
Procedure—Determine the amount of C20H22N2 · 2C4H4O4
dissolved by employing UV absorption at the wavelength of
maximum absorbance at about 283 nm on filtered portions
of the solution under test, diluted with Medium, if necessary,
in comparison with a Standard solution having a known
concentration of USP Azatadine Maleate RS in the same Medium.
Tolerances—Not less than 80% (Q) of the labeled amount
of C20H22N2 · 2C4H4O4 is dissolved in 30 minutes.
Uniformity of dosage units 〈905〉: meet the requirements.
Assay—
Standard preparation—Dissolve an accurately weighed
quantity of USP Azatadine Maleate RS in 0.1 N hydrochloric
acid, and dilute quantitatively, and stepwise if necessary,
with 0.1 N hydrochloric acid to obtain a solution having a
known concentration of about 0.06 mg per mL.
Assay preparation—Weigh and finely powder not fewer
than 20 Tablets. Transfer an accurately weighed portion of
the powder, equivalent to about 1.5 mg of azatadine
maleate, to a 50-mL flask fitted with a glass stopper. Add
25.0 mL of 0.1 N hydrochloric acid, insert the stopper, and
shake the mixture by mechanical means for about 30 minutes. Filter the mixture into a suitable glass-stoppered vessel,
discarding the first 5 mL of the filtrate.
Procedure—Separately transfer 15.0 mL of the Standard
preparation, 15.0 mL of the Assay preparation, and 15.0 mL
of 0.1 N hydrochloric acid to provide the reagent blank to
three 50-mL centrifuge tubes fitted with glass stoppers. To
each centrifuge tube add 10.0 mL of 1.0 N sodium hydroxide and 20 mL of solvent hexane, insert the stoppers, rotate
the centrifuge tubes for about 15 minutes, and centrifuge
until the supernatants (solvent hexane phase) are clear. With
the aid of separate syringes, transfer the supernatants to
separate 50-mL centrifuge tubes fitted with glass stoppers.
Rinse each syringe with 10 mL of solvent hexane, and add
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1884 Azathioprine / Official Monographs
USP 37
Chromatographic system
(See Chromatography 〈621〉, Thin-Layer Chromatography.)
Mode: TLC
Adsorbent: 0.25-mm layer of microcrystalline cellulose
Application volume: 5 µL
Developing solvent system: Butyl alcohol saturated
with 6 N ammonium hydroxide
Analysis
Samples: Standard solution A, Standard solution B, and
Sample solution
Apply the Samples at points 2 cm from the bottom
edge of a thin-layer chromatographic plate. Allow the
spots to dry, and develop the chromatogram in a
suitable chamber until the solvent front has moved
15 cm from the point of application. Remove the
plate, air-dry, and locate the spots by viewing under
short- and long-wavelength UV light.
Acceptance criteria: 1.0%; any spot from the Sample
solution, other than the principal spot, is not more intense than the spot from the Standard solution B.
USP Monographs
SPECIFIC TESTS
• ACIDITY OR ALKALINITY
Sample: 2.0 g
Analysis: Shake the Sample with 100 mL of water for 15
min, and filter.
Acceptance criteria: 20.0 mL of the filtrate requires for
neutralization NMT 0.10 mL of 0.020 N hydrochloric
acid or NMT 0.10 mL of 0.020 N sodium hydroxide,
using methyl red TS as the indicator.
• LOSS ON DRYING 〈731〉
Analysis: Dry a sample under vacuum at 105° for 5 h.
Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
• USP REFERENCE STANDARDS 〈11〉
USP Azathioprine RS
USP Mercaptopurine RS
ASSAY
• PROCEDURE
Mobile phase: Dissolve 1.1 g of sodium-1-heptanesulfonate in 700 mL of water, and add 300 mL of methanol. Adjust with 1 N hydrochloric acid to a pH of 3.5.
Standard solution: 25 mg of USP Azathioprine RS, in a
50-mL volumetric flask. Add 15 mL of methanol and
0.5 mL of ammonium hydroxide to the flask, swirl, and
sonicate for 2 min. Dilute with methanol to volume.
Transfer 10 mL of this solution to a 50-mL volumetric
flask, and dilute with water to volume.
Sample solution: Agitate the container of Oral Suspension for 30 min on a rotating mixer, remove a 5-mL
sample, and store in a clear glass vial at −70° until analyzed. At the time of analysis, remove the sample from
the freezer, allow it to reach room temperature, and
mix with a vortex mixer for 30 s. Pipet 1.0 mL of the
Sample into a 100-mL volumetric flask, and dilute with
Mobile phase to volume.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm × 25-cm; 5-µm packing L1
Flow rate: 2 mL/min
Injection size: 20 µL
System suitability
Sample: Standard solution
[NOTE—The retention time for the azathioprine peak is
about 4 min.]
Suitability requirements
Relative standard deviation: NMT 1.3% for replicate
injections
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
azathioprine (C9H7N7O2S) in the portion of Oral Suspension taken:
Result = (rU/rS) × (CS/CU) × 100
rU
rS
CS
= peak response from the Sample solution
= peak response from the Standard solution
= concentration of azathioprine in the Standard
solution (µg/mL)
CU
= nominal concentration of azathioprine in the
Sample solution (µg/mL)
Acceptance criteria: 90.0%–110.0%
.
Azathioprine Oral Suspension
DEFINITION
Azathioprine Oral Suspension contains NLT 90.0% and NMT
110.0% of the labeled amount of azathioprine
(C9H7N7O2S).
Prepare Azathioprine Oral Suspension of 50 mg/mL as follows (see Pharmaceutical Compounding—Nonsterile Preparations 〈795〉).
Azathioprine
Vehicle: a mixture of Vehicle for Oral Solution,
(regular or sugar-free), NF and Vehicle for
Oral Suspension, NF (1:1), a sufficient quantity to make
5g
100 mL
If using tablets, comminute them to a fine powder in a suitable mortar, or add Azathioprine powder to the mortar.
Add about 10 mL of the Vehicle, and mix to a uniform
paste. Add the Vehicle in small portions almost to volume,
and mix thoroughly after each addition. Transfer the contents of the mortar, stepwise and quantitatively, to a calibrated bottle. Add sufficient Vehicle to bring to final volume, and mix well.
[CAUTION—Avoid skin contact or inhalation of azathioprine
by using protective gloves and a fume hood or surgical
mask.]
SPECIFIC TESTS
• PH 〈791〉: 3.8–4.8
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Store at room temperature, or in a cold
place.
• LABELING: Label it to state that it is to be well shaken
before use, and to state the Beyond-Use Date.
• BEYOND-USE DATE: NMT 60 days after the day on which
it was compounded
• USP REFERENCE STANDARDS 〈11〉
USP Azathioprine RS
.
Azathioprine Tablets
DEFINITION
Azathioprine Tablets contain NLT 93.0% and NMT 107.0%
of the labeled amount of azathioprine (C9H7N7O2S).
Official from December 1, 2014
Copyright (c) 2015 The United States Pharmacopeial Convention. All rights reserved.