## STP FOR CLOZAPINE TABLETS

1.0) Description: Visual

2.0) 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.

Weigh the tablets individually and calculate the percentage of deviation for each tablet. By using formula = (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. In the related substances, the principal spot in the chromatogram obtained with the test solution corresponds to the principal spot obtained with the reference solution (a).
2. In the assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution (a).

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 6.5g. 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 acetate buffer pH 4.0 prepared by dissolving 2g of sodium hydroxide in 450ml of water, adjusting the pH to 4.0 with glacial acetic acid and diluting with              water to 1000ml,

Speed: 100rpm

Time: 45 minutes

Limit: NLT 85% of the stated amount of Clozapine.

Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted with the dissolution medium if necessary, at the maximum at about 290nm. Similarly measure the absorbance of a standard solution of known concentration of clozapine reference standard and calculate the content of clozapine.

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 up to 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)    Related substances: Determine by thin-layer chromatography, coating the plate with silica gel GF254.

Mobile phase: A mixture of 75 volumes of chloroform and 25 volumes of methanol.

Solvent mixture: 80 volumes of chloroform and 20 volumes of methanol.

Test solution: Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 125mg of clozapine and dissolve in 25ml of the solvent mixture.

Reference solution (a): A 0.5 per cent w/v solution of clozapine reference standard in the solvent mixture.

Reference solution (b): Dilute 1.0ml of reference solution (a) to 200ml with the solvent mixture.

Reference solution (c): Dilute 1.0ml of reference solution (a) to 250ml with the solvent mixture.

Reference solution (d): Dilute 3.0ml of reference solution (a) to 1000ml with the solvent mixture.

Reference solution (e): Dilute 1.0ml of reference solution (a) to 500ml with the solvent mixture.

Reference solution (f): Dilute 1.0ml of reference solution (a) to 1000ml with the solvent mixture.

Apply to the plate 20µl of each solution. Allow the mobile phase to rise 10cm. Dry the plate in air and examine under Ultraviolet light at 254nm. Compare the intensities of any secondary spots observed in the chromatogram obtained with the test solution with those of the principal spots in the chromatogram obtained with the reference solutions: no secondary spot from the chromatogram obtained with the test solution is larger or more intense than the principal spot obtained with reference solution (b) (0.5 per cent); and the sum of the intensities of all secondary spots obtained with the test solution corresponds to not more than 2.0 per cent.

12.0)    Assay: Determined by liquid chromatography.

Test solution: Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 125mg Clozapine, disperse in 640.0ml of the methanol and add sufficient water to produce 1000ml.

Reference solution (a): Weigh 12.5mg of Clozapine reference standard in 80ml of methanol and dilute to 100.0ml with water.

Reference solution (b): Weigh accurately about 10mg of Clozapine, add 5ml of 0.1 M HCl and heat for 2 hours at 90°, cool, add 15ml of water, dilute with methanol to 100ml and mix. To 10ml of this solution add 10ml of reference solution (a) and mix.

Chromatographic system:

• A stainless steel column 25cm X4.0mm, packed with octylsilane bonded to porous silica (5µm).
• Mobile phase: 800 volumes of methanol, 200 volumes of water and 0.75 volumes of triethylamine,
• Flow rate: 1ml per minute.
• Spectrophotometer set at 257nm,

Inject the reference solution (a). The test is not valid unless the column efficiency is not less than 1500 theoretical plates, the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent.

Inject reference solution (b). The resolution between the clozapine peak and any secondary peak is not less than 1.5.

Inject reference solution (a) and the test solution.

Calculate the content of Clozapine in the tablets.

Alternative Method: By UV Spectrophotometer

Test Solution: Weigh and powder 20 tablets. Weight sample equivalent to 50mg of Clozapine 100ml volumetric flask add 20ml 0.1 M HCl into it and sonicate to dissolve the test sample. Make up to mark with 0.1 M HCl. Filter and dilute 1ml of this solution to 50ml in 0.1 M HCl.

Reference Solution: Weigh 50mg of reference standard of Clozapine in 100ml of volumetric flask. Add 20ml 0.1 M HCl into it and sonicate to dissolve. Make up to mark with 0.1 M HCl. Dilute 1ml of this solution to 50ml with 0.1 M HCl.

Check Absorbance of test as well as reference standard at about 240nm in UV Spectrophotometer.

Calculate the content of Clozapine in the tablets.

Formula:

Abs. of Test      STD Wt.(mg)  1            100               50       Potency

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

Abs. of STD        100               50     Test Wt.(mg)     1           100

Acceptance criteria: 90.0%-110.0%

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

HCl: Hydrochloric acid

Abs. Absorbance