Azathioprine

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

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

DOWNLOAD PDF HERE

C₉H₇N₇O₂S  277.26

1H-Purine, 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)thio]-;

6-[(1-Methyl-4-nitroimidazol-5-yl)thio]purine CAS RN^®: 446-86-6; UNII: MRK240IY2L.

1 DEFINITION

Azathioprine contains NLT 98.0% and NMT 102.0% of azathioprine (C₉H₇N₇O₂S), calculated on the dried basis.

2 IDENTIFICATION

Change to read:

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197K (CN 1-MAY-2020)

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: 0.1 mg/mL of Azathioprine prepared as follows. Transfer 50 mg of sample to a 100-mL volumetric flask. Add 25 mL of methanol and 1.0 mL of ammonium hydroxide to the flask, swirl, and sonicate for 2 min or until dissolved. Dilute with methanol to volume.

Transfer 10.0 mL of this solution into a 50-mL volumetric flask, and dilute with water to volume.

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 0.73%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of azathioprine (C₉H₇N₇O₂S) in the portion of Azathioprine 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 = concentration of Azathioprine in the Sample solution (mg/mL)

Acceptance criteria: 98.0%-102.0% on the dried basis

IMPURITIES

3.2 RESIDUE ON IGNITION (281)

NMT 0.1%

3.3 ORGANIC IMPURITIES

Buffer: 2.76 g/L of monobasic sodium phosphate. Adjust with phosphoric acid to a pH of 2.5.

Solution A: Methanol and Buffer (5:95)

Solution B: Methanol and Buffer (60:40)

Mobile phase: See Table 1. Return to original conditions, and re-equilibrate the system.

Table 1

Diluent: 0.8 g/L of sodium hydroxide in water

System suitability stock solution A: 0.2 mg/mL each of USP Azathioprine Related Compound A RS and USP Mercaptopurine RS prepared as

follows. Transfer USP Azathioprine Related Compound A RS and USP Mercaptopurine RS to a suitable volumetric flask. Add 35% of the flask volume of Diluent, and dilute with Buffer to volume.

System suitability stock solution B: 0.1 mg/mL each of USP Azathioprine Related Compound G RS and USP Azathioprine RS prepared as follows. Transfer USP Azathioprine Related Compound G RS and USP Azathioprine RS to a suitable volumetric flask. Add 35% of the flask volume of Diluent, and dilute with Buffer to volume.

System suitability solution: 0.002 mg/mL each of USP Azathioprine Related Compound A RS, USP Mercaptopurine RS, USP Azathioprine Related Compound G RS, and USP Azathioprine RS prepared as follows. Transfer 1 mL of System suitability stock solution A and 2 mL of System suitability stock solution B to a 100-mL volumetric flask. Add 35 mL of Diluent, and dilute with Buffer to volume.

Standard stock solution: 0.1 mg/mL of USP Azathioprine RS prepared as follows. Transfer USP Azathioprine RS to a suitable volumetric flask. Add 35% of the flask volume of Diluent, and dilute with Buffer to volume.

Standard solution: 0.1 µg/mL of USP Azathioprine RS in Buffer from Standard stock solution

Sample solution: 0.1 mg/mL of Azathioprine prepared as follows. Transfer Azathioprine to a suitable volumetric flask. Add 35% of the flask volume with Diluent, and dilute with Buffer to volume.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 240 nm

Column: 4.6-mm × 15-cm; 5-µm packing L11

Column temperature: 30°

Flow rate: 1.0 mL/min

Injection volume: 20 µL

System suitability

Sample: System suitability solution

Suitability requirements

Resolution: NLT 2 between azathioprine related compound A and mercaptopurine; NLT 2 between azathioprine related compound G and azathioprine

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Azathioprine taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of each impurity 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 = concentration of Azathioprine in the Sample solution (mg/mL)

Acceptance criteria: See Table 2. Disregard any impurity peaks less than 0.05%.

Table 2

^a 1-Methyl-4-nitro-1H-imidazol-5-amine.

^b 9H-Purine-6-thiol.

^c 6-[(1-Methyl-4-nitro-1H-imidazol-5-yl)thio]-9H-purin-2-amine.

4 SPECIFIC TESTS

4.1 LOSS ON DRYING (731)

Analysis: Dry under vacuum at 105° for 5 h.

Acceptance criteria: NMT 1.0%

5 ADDITIONAL REQUIREMENTS

5.1 PACKAGING AND STORAGE

Preserve in tight, light-resistant containers.

5.2 USP REFERENCE STANDARDS (11)

USP Azathioprine RS

USP Azathioprine Related Compound A RS

1-Methyl-4-nitro-1H-imidazol-5-amine.

C₄H₆N₄O₂  4642  142.12

USP Azathioprine Related Compound G RS

6-[(1-Methyl-4-nitro-1H-imidazol-5-yl)thio]-9H-purin-2-amine.

C₉H₈N₈O₂NS  292.28

USP Mercaptopurine RS