## STP FOR AZATHIOPRINE TABLETS

1.Description: Visual

1.0) Average Weight: Check weight of 20 tablets at randomly and calculate the average weight by formula :

wt of 20 tablets (gm) x 1000 / 20. found avg. wt in mg

3.0) Uniformity of weight:

Weigh 20 tablets selected at random and calculate the average weight. Not more than two of the individual weights deviate from the average weight by more

than the percentage shown in table.

Tablets were weighed individually and the percentage of deviation of its weight from the average weight was determined for each tablet. Formula for calculate the percentage of deviation = (experimental weight – theoretical weight)    x 100%

Theoretical weight

 Average weight of tablets Percentage deviation More than 80mg but Less than 250mg 7.5% 250mg or More 5%

4.0)      Identification Test:

1. Determine by thin-layer chromatography, coating the plate with cellulose F 254.

Mobile phase: A mixture of butan-1-ol saturated with 6 M ammonia.

Test solution: Shake a quantity of the powdered tablets containing 0.2 g of Azathioprine with 50ml of 6 M ammonia, filter through a glass micro fibred paper (such as Whatman GF/C) and use the filtrate.

Reference solution: A 0.4 per cent w/v solution of azathioprine reference solution in 6 M ammonia.

Apply to the plate 5 µl of each solution. After removal of the plate, dry the plate at 50° and examine under ultraviolet light at 254nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.

1. Heat a quantity of the powdered tablets containing 20mg of Azathioprine with 100ml of water and filter. To 5ml of the filtrate add 1ml of hydrochloric acid and 10mg 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 per cent w/v solution of sodium nitrite and 0.1 gm of sulphamic acid and shake until the bubbles disappear. Add 1ml of 2-napthol solution; a pale pink precipitate is produced.

5.0)      Hardness:

The standard method used for tablet hardness testing is compression testing. The tablet is placed between two jaws that crush the tablet. The machine measures the force applied to the tablet and detects when it fractures. Although compressive force is applied to the tablet, along the diameter of the tablet at right angles to the applied force.

6.0)      Disintegration Time

Unless otherwise stated in the individual monograph, introduce one tablet into each tube and add a disc to each tube. The assembly is suspended in the liquid medium in a suitable vessel, preferably a 1-litre beaker. The volume of liquid is such that the wire mesh at its highest point is at least 15mm below the surface of the liquid, and at its lower point is at least 25mm above the bottom of the beaker. At no time should the top of the basket-rack assembly become submerged. There is a thermostatic arrangement for heating the liquid and maintaining the temperature at 37±2°.

If 1 or 2 tablets fail to disintegrate, repeat the test on 12 additional tablets; not less than 16 of the total of 18 tablets tested disintegrate.

If the tablets adhere to the disc and the preparation under examination fails to comply, repeat the test omitting the disc. The preparation complies with the test if all the tablets in the repeat test disintegrate.

7.0)      Dimension of Tablet:

Length, breadth and thickness are determined by vernier in mm

8.0)      Friability:

The test is applicable to compressed tablets and is intended to determine the physical strength of tablets. Tablets with a unit weight equal to or less than 650 mg, take a sample of whole tablets corresponding as near as possible to 6.5 g. For tablets with a unit weight of more than 650mg, take a sample of 10 whole tablets. The tablets should be carefully dedusted prior to testing. Accurately weigh the tablet sample, and place the tablets in the drum. Rotate the drum 100 times and remove the tablets. Remove any loose dust from the tablets as before, and accurately weigh.

The test is run only once unless the results are difficult to interpret or if the weight loss is greater than the target value, in which case, the test is repeated twice and the mean of the three tests is determined.

Formula: initial wt. – after friability wt. x100 / initial wt.

A maximum loss of weight (From a single test or from the mean of three tests) not greater than 1.0 percent is acceptable for most tablets.

9.0)      Dissolution:

Medium: 900ml of water

Time: 30 minutes

Speed: 50 rpm

Limit: NLT 80% of the stated amount of Azathioprine.

Withdraw a suitable volume of the medium and filter, rejecting the first few ml of filtrate. Dilute a suitable volume of the filtrate with the medium, if necessary. Measure the absorbance of the resulting solution at the maximum at about 280nm. Calculate the content of Azathioprine, in the medium from the absorbance obtained a solution of known concentration of azathioprine reference standard in the dissolution medium.

10.0)    Leak test:

The apparatus is used to test for the integrity of packed strips, blisters and Alu-Alu Blister pack containing tablets. Ensure apparatus bath is filled with purified water upto mark indicated and add 0.5% crystal violet solution in water. Samples are placed into the desiccators and the lid is placed in position. The pump starts to produce a vacuum 15inHg inside the desiccators and the vacuum is held for 1 minute. The sample remains at the required vacuum level for given time interval buzzer will sound after time is over and will cut off the vacuum pump. As the package is immersed in a colored dye solution the venting of the desiccators will allow any holes to be penetrated by the dye and the contents of the flexible packaging will also be stained with the same coloring material.

Examine all the strips for any leakage by opening the pockets manually. If anyone pocket shows evidence of leakage, reject the sample, stop the Blister / Strip machine and immediately take corrective action.

11.0)    5-Chloro-1-methyl-4-nitroimidazole and 6-mercaptopurine:

Determined by thin layer chromatography, coating the plate with cellulose F-254.

Mobile phase: A mixture of butan-1-ol saturated with 6 M ammonia.

Test solution: Shake a quantity of the powdered tablets containing 0.2 g of Azathioprine with 10ml of 6M ammonia and filter through a glass micro fibre                  filter paper (such as Whatman GF/C)

Reference solution (a): A solution containing 2.0 per cent w/v of azathioprine reference standard and 0.02 per cent w/v of 6-mercaptopurine in 6                             ammonia.

Apply to the plate 5µl of each solution. After removal of the plate, dry the plate at 50° and examine under ultraviolet light at 254nm. Any spot in the chromatogram obtained with the test solution corresponding to 6-mercaptopurine in the chromatogram obtained with reference solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b). Any spot corresponding to 5-chloro-1-methyl-4-nitroimidazole in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (c).

12.0)    Assay:

Weigh and powder 20 tablets. Shake a quantity of the powder containing about 0.15g of Azathioprine with 20ml of dimethyl sulphoxide for 15 minutes and dilute to 500.0ml with 0.1 M hydrochloric acid, filter. Dilute 25ml of the filtrate to 1000.0ml with 0.1 M HCl. Measure the absorbance of the resulting solution at the maximum at about 280nm using 0.1 M HCl as the blank. Calculate the content of Azathioprine using a solution of Azathioprine reference standard of the same concentration in 0.1 M HCl.

Formula:

Area of Test      STD Wt.(mg)  25            100               50       Potency

—————–X—————-X——-X—————X——–X———–X Average wt.

Area of STD        500             1000      Test Wt.(mg)     5           100

Acceptance criteria: 92.5%-107.5%

13.0)    MICROBIOLOGICAL PURITY

Perform the test according to requirements of IP,

Total aerobic Microbial count (TAMC): NMT 103 CFU/g

Total combined yeasts/Moulds count (TYMC): NMT 102 CFU/g

Pathogens: in 1gm drug.

Escherichia Coli – Should be absent

Pseudomonas aeroginosa – Should be absent

Salmonella – Should be absent

Staphylococcus aureus– Should be absent

Abbreviations:

Wt.: Weight

mg: Miligram

ml: Milileter

STD: Standard

inHg: Inch of Mercury

rpm: Rounds per minute

CFU: Colony forming unit

G.A.A: Glacial acetic acid

HCl: Hydrochloric acid