Azathioprine Tablets

General Notices

Action and use

Immunosuppressant.

Definition

Azathioprine Tablets contain Azathioprine.

The tablets comply with the requirements stated under Tablets and with the following requirements.

Content of azathioprine, C9H7N7O2S

92.5 to 107.5% of the stated amount.

Identification

A. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
(1) Shake a quantity of the powdered tablets containing 0.2 g of Azathioprine with 50 mL of 6m ammonia, filter through a glass micro fibre paper (Whatman GF/C is suitable) and use the filtrate.
(2) 0.4% w/v of azathioprine BPCRS in 6m ammonia.
chromatographic conditions
(a) Use as the coating cellulose F254.
(b) Use the mobile phase as described below.
(c) Apply 5 µL of each solution.
(d) Develop the plate to 20 cm.
(e) After removal of the plate, dry it at 50° and examine under ultraviolet light (254 nm).
mobile phase

butan-1-ol saturated with 6m ammonia.

confirmation

The principal spot in the chromatogram obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2).

B. Heat a quantity of the powdered tablets containing 20 mg of Azathioprine with 100 mL of water and filter. To 5 mL of the filtrate add 1 mL of hydrochloric acid and 10 mg of zinc powder and allow to stand for 5 minutes; a yellow colour is produced. Filter, cool in ice, add 0.1 mL of a 10% w/v solution of sodium nitrite and 0.1 g of sulfamic acid and shake until the bubbles disappear. Add 1 mL of 2-naphthol solution; a pale pink precipitate is produced.

TEST

5-Chloro-1-methyl-4-nitroimidazole and 6-mercaptopurine

Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.

(1) Shake a quantity of the powdered tablets containing 0.20 g of Azathioprine with 10 mL of 6m ammonia and filter through a glass micro fibre filter paper (Whatman GF/C is suitable).
(2) 2.0% w/v of azathioprine BPCRS and 0.020% w/v of 6-mercaptopurine in 6m ammonia.
(3) 0.020% w/v of 6-mercaptopurine in 6m ammonia.
(4) 0.020% w/v of 5-chloro-1-methyl-4-nitroimidazole BPCRS in 6m ammonia.
chromatographic conditions
(a) Use as the coating cellulose F254.
(b) Use the mobile phase as described below.
(c) Apply 5 µL of each solution.
(d) Develop the plate to 20 cm.
(e) After removal of the plate, dry it at 50° and examine under ultraviolet light (254 nm).
mobile phase

butan-1-ol saturated with 6m ammonia.

system suitability

The test is not valid unless the chromatogram obtained with solution (2) shows two clearly separated spots.

limits

Any spot in the chromatogram obtained with solution (1):

corresponding to 6-mercaptopurine in the chromatogram obtained with solution (2) is not more intense than the spot in the chromatogram obtained with solution (3);

any spot corresponding to 5-chloro-1-methyl-4-nitroimidazole in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with solution (4).

Assay

Weigh and powder 20 tablets. Shake a quantity of the powder containing 0.15 g of Azathioprine with 20 mL of dimethyl sulfoxide for 15 minutes and dilute to 500 mL with 0.1m hydrochloric acid. Avoid heating or the use of ultrasound. Filter, dilute 25 mL of the filtrate to 1000 mL with 0.1m hydrochloric acid and measure the absorbance of the resulting solution at the maximum at 280 nm, Appendix II B. Calculate the content of C9H7N7O2S taking 628 as the value of A(1%, 1 cm) at the maximum at 280 nm.

Storage

Azathioprine Tablets should be protected from light.