Azathioprine Sodium for Injection

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Azathioprine Sodium for Injection is a sterile solid prepared by the freeze-drying of an aqueous solution of Azathioprine and Sodium Hydroxide.

It contains NLT 93.0% and NMT 107.0% of the labeled amount of azathioprine (C₉H₇N₇O₂S).

2 IDENTIFICATION

A. The principal spot from the Sample solution shows the same R, value as that obtained from Standard solution A in the test for Limit of Mercaptopurine.

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

3.1 PROCEDURE

Solution A: 1.6 g/L of sodium 1-heptanesulfonate in water

Mobile phase: Methanol and Solution A (30:70). Adjust with 1 N hydrochloric acid to a pH of 3.5 ± 0.1.

Standard stock solution: 0.5 mg/mL of USP Azathioprine RS prepared as follows. Transfer USP Azathioprine RS to a suitable volumetric flask. Add 25% of the flask volume of methanol and 1% of ammonium hydroxide to the flask, swirl, and sonicate for 2 min or until dissolved. Dilute with methanol to volume.

Standard solution: 0.1 mg/mL of USP Azathioprine RS in water from the Standard stock solution

Sample solution: Nominally equivalent to 0.1 mg/mL of azathioprine in water from Azathioprine Sodium for Injection

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 3.9-mm × 30-cm; 10-µm packing L1

Flow rate: 1.8 mL/min

Injection volume: 10 µL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2

Relative standard deviation: NMT 1.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of azathioprine (C₉H₇N₇O₂S) in the portion of Azathioprine Sodium for Injection taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of azathioprine from the Sample solution

r_S = peak response of azathioprine from the Standard solution

C_S = concentration of USP Azathioprine RS in the Standard solution (mg/mL)

C_U = nominal concentration of azathioprine in the Sample solution (mg/mL)

Acceptance criteria: 93.0%-107.0%

4 PERFORMANCE TESTS

UNIFORMITY OF DOSAGE UNITS (905): Meets the requirements

5 IMPURITIES

5.1 LIMIT OF MERCAPTOPURINE

Standard solution A: 10 mg/mL of USP Azathioprine RS in dimethylformamide

Standard solution B: 100 µg/mL of USP Mercaptopurine RS in dimethylformamide

Sample solution: 10 mg/mL of Azathioprine Sodium for Injection in dimethylformamide

Chromatographic system

(See Chromatography (621), Thin-Layer Chromatography.)

Mode: TLC

Adsorbent: 0.25-mm layer of microcrystalline cellulose

Application volume: 5 µL for Standard solution A and the Sample solution, and 15 µL for Standard solution B

Developing solvent system: Butyl alcohol saturated with 5 N ammonium hydroxide

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Proceed as directed in the chapter. Apply the Samples at points 2 cm from the bottom edge of a TLC 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: 3.0%; any spot from the Sample solution, other than the principal spot, is not more intense than the spot from Standard solution B.

6 SPECIFIC TESTS

PH (791).

Sample solution: The contents of one container dissolved in 10 mL of water

Acceptance criteria: 9.8-11.0

BACTERIAL ENDOTOXINS TEST (85): It contains NMT 1.0 USP Endotoxin Unit/mg of azathioprine.

WATER DETERMINATION (921), Method I: NMT 7.0%, when the Test Preparation is prepared as directed for a hygroscopic specimen

OTHER REQUIREMENTS: It meets the requirements in Injections and Implanted Drug Products (1).

7 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve as described in Packaging and Storage Requirements (659), Injection Packaging, Packaging for constitution, at controlled room temperature.

USP REFERENCE STANDARDS (11).

USP Azathioprine RS

USP Mercaptopurine RS