Azathioprine Tablets

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 Tablets contain NLT 93.0% and NMT 107.0% of the labeled amount of azathioprine (C₉H₇N₇O₂S).

2 IDENTIFICATION

Change to read:

A. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. (USP 1-May-2020)

Add the following:

B. The UV spectrum of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. (USP 1-May-2020)

3 ASSAY

Change to read:

3.1 PROCEDURE

Mobile phase: Dissolve 1.1 g of sodium 1-heptanesulfonate in 700 mL of water, and add 300 mL of methanol. Adjust the solution with 1 N hydrochloric acid to a pH of 3.5. (USP 1-May-2020)

Standard stock solution: 0.5 mg/mL of USP Azathioprine RS prepared as follows. Transfer USP Azathioprine RS to a suitable volumetric flask. Add methanol equivalent to 30% of the flask volume and ammonium hydroxide equivalent to 1% of the flask volume, swirl, and sonicate for 2 min. Dilute with methanol to volume.

Standard solution: 0.1 mg/mL of USP Azathioprine RS in water prepared from the Standard stock solution (USP 1-May-2020)

Sample stock solution: Nominally 0.5 mg/mL of azathioprine prepared as follows. Finely powder NLT 20 Tablets and transfer a portion of the powder to a suitable volumetric flask. Add methanol equivalent to 25% of the flask volume and ammonium hydroxide equivalent to 1% of the flask volume, swirl, and sonicate for 2 min. Dilute with methanol to volume. Allow the excipients to settle. (USP 1-May-2020)

Sample solution: Nominally 0.1 mg/mL of azathioprine from the Sample stock solution in water. Pass a portion of the solution through a suitable filter of 0.45-µm pore size. (USP 1-May-2020)

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm. For Identification B, use a diode array detector in the range of 200-400 nm. (USP 1-May-2020)

Column: 4-mm x 30-cm; 10-µm (USP 1-May-2020) Packing L1

Flow rate: 2 mL/min

Injection volume: 10 µL

System suitability

Sample: Standard solution

Suitability requirements

(USP 1-May-2020)

Tailing factor: NMT 1.5

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of azathioprine (C₉H₇N₇O₂S) in the portion of Tablets 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

Change to read:

4.1 DISSOLUTION (711)

Medium: Water; 900 mL

Apparatus 2: 50 rpm

Time: 30 min

Standard solution: USP Azathioprine RS in Medium

Sample solutions: Pass portions of the solution under test through a suitable (USP 1-May-2020) filter and dilute with Medium, if necessary, to a concentration similar to that of the Standard solution.

Instrumental conditions

Mode: UV

Analytical wavelength: Maximum absorbance at about 280 nm

Analysis

Samples: Standard solution and Sample solutions

Calculate the percentage of the labeled amount of azathioprine (C₉H₇N₇O₂S) dissolved:

Result = (A_U/A_S) x C_S x V x D x (1/L) x 100

A_U = absorbance of the Sample solution

A_S = absorbance of the Standard solution

C_S = concentration of azathioprine in the Standard solution (mg/mL)

V = volume of Medium, 900 mL

D = dilution factor for the Sample solution, if applicable

L = label claim (mg/Tablet)

(USP 1-May-2020)

Tolerances: NLT 75% (Q) of the labeled amount of azathioprine (CH,N,O2S) is dissolved.

4.2 UNIFORMITY OF DOSAGE UNITS (905): Meet the requirements

5 IMPURITIES

Add the following:

5.1 ORGANIC IMPURITIES

Protect the solutions from light.

Mobile phase: Dissolve 1.2 g of sodium 1-heptanesulfonate in 750 mL of water, and add 250 mL of methanol. Adjust the solution with 1 N hydrochloric acid to a pH of 3.0.

System suitability stock solution: 0.25 mg/mL each of USP Mercaptopurine RS, USP Azathioprine Related Compound A RS, and chloromethylnitroimidazole in methanol

System suitability solution: 0.5 mg/mL of USP Azathioprine RS and 0.005 mg/mL each of USP Mercaptopurine RS, USP Azathioprine Related Compound A RS, and chloromethylnitroimidazole prepared as follows. Transfer a suitable amount of USP Azathioprine RS to a suitable volumetric flask. Add methanol equivalent to 20% of the flask volume and sonicate to dissolve. Add a suitable amount of the System suitability stock solution and dilute with Mobile phase to volume.

Standard stock solution: 0.25 mg/mL of USP Azathioprine RS prepared as follows. Transfer a suitable amount of USP Azathioprine RS to a suitable volumetric flask. Add methanol equivalent to 75% of the flask volume, sonicate to dissolve, and dilute with methanol to volume.

Standard solution: 0.005 mg/mL of USP Azathioprine RS in Mobile phase from the Standard stock solution

Sensitivity solution: 0.0005 mg/mL of USP Azathioprine RS in Mobile phase from the Standard solution

Sample solution: Nominally 0.5 mg/mL of azathioprine prepared as follows. Transfer a portion of the powder, from NLT 20 powdered Tablets, to a suitable volumetric flask. Add 20% of the flask volume of methanol, sonicate for 20 min with intermittent shaking, and dilute with Mobile phase to volume. Pass a portion of the solution through a suitable filter of 0.45-µm pore size.

Chromatographic system

(See Chromatography (621), System Suitability)

Mode: LC

Detector: UV 220 nm

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

Column temperature: 30°

Flow rate: 1.1 mL/min

Injection volume: 25 µL

System suitability

Samples: System suitability solution, Standard solution, and Sensitivity solution

Suitability requirements

Resolution: NLT 1.5 between azathioprine related compound A and mercaptopurine; NLT 2.0 between chloromethylnitroimidazole and azathioprine, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each individual degradation product in the portion of Tablets taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of each degradation product 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: See Table 1.

Table 1

^a Process impurity included in the table for identification only. Process impurities are controlled in the drug substance and are not to be reported or included in the total degradation products for the drug product.

^b Di(9H-purin-6-yl)sulfide.

^c C5-Chloro-1-methyl-4-nitro-1H-imidazole.

(USP 1-May-2020)

6 ADDITIONAL REQUIREMENTS

Change to read:

6.1 PACKAGING AND STORAGE:

Protect from light.

Store at controlled room temperature.

(USP 1-May-2020)

Change to read:

6.2 USP REFERENCE STANDARDS (11)

USP Azathioprine RS

USP Azathioprine Related Compound A RS

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

C₄H₆N₄O₂  142.12

USP Mercaptopurine RS

9H-Purine-6-thiol monohydrate.

C₅H₄N₄S.H₂O  170.19  (USP 1-May-2020)