## STP FOR PREGABALIN AND METHYLCOBALAMIN CAPSULES

• Description: Visual
• Average Weight: Check weight of 20 Capsules at randomly and calculate the average weight by formula :                                                                                                     wt of 20 tablets (gm) x 1000 / 20. found avg. wt in mg
• Average Fill: Check weight of 20 Capsules at randomly. And calculate the average weight by above formula and subtract empty weight of capsule from average weight.
• Uniformity of weight:

Weigh 20 capsules 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.

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

Theoretical weight

 Average weight of capsules Percentage deviation 300 mg or less 10.0 % More than 300 mg 7.5%
• Identification Test:
1. In the assay of Pregabalin, the principal peak in the chromatogram obtained with the test solution corresponds to the principal peak in the chromatogram obtained with the reference solution.
2. In the assay of Methylcobalamin, the principal peak in the chromatogram obtained with the test solution corresponds to the principal peak in the chromatogram obtained with the reference solution.

Disintegration Time:

Unless otherwise stated in the individual monograph, introduce one capsule 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 capsules fail to disintegrate, repeat the test on 12 additional capsules; not less than 16 of the total of 18 capsules tested disintegrate.

If the capsules 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 capsules in the repeat test disintegrate.

Dimension of Capsule:

Length and diameter are determined by vernier in mm.

Dissolution:

For pregabalin:

Medium: 500ml of water;

Time: 30 minutes

Speed: 100 rpm

Withdraw a suitable volume of the medium and filter.

Determine by liquid chromatography.

Test solution: Use the filtrate and dilute, if necessary, with the dissolution medium.

Reference solution: Weigh accurately about 15mg of Pregabalin reference standard dissolve and dilute to 20ml with mobile phase.                   Dilute 5ml of this solution to 25ml with dissolution medium.

Chromatographic system:

• A stainless steel column 25cmX 4.6mm, packed with octadecylsilane bonded to porous silica (5µm),
• Mobile phase: a mixture of 90 volumes of a buffer solution prepared by dissolving 3.72g of water, add 2ml of triethylamine and adjusted to pH 6.0 with O.P.A., 6 volumes of methanol and 4 volumes of acetonitrile,
• Flow rate: 1ml per minute,
• Spectrophotometer set at 205nm,
• Injection volume: 20µl

Inject the reference solution and the test solution.

Calculate the content of Pregabalin.

Limit: NLT 75% of the stated amount of Pregabalin.

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.

Uniformity of content:

For capsules containing 10mg or less.

Determined by liquid chromatography.

Test solution: Transfer the whole contents of a capsule in 25ml of volumetric flask, add 20ml of mobile phase and disperse with the aid of ultrasound for about 5 minutes, cool and dilute to volume with mobile phase, and filter. Dilute further with mobile phase, if necessary.

Reference solution: A 0.002 per cent w/v solution of methylcobalamin reference standard in mobile phase. Dilute with mobile phase, if necessary.

Chromatographic system:

• A stainless steel column 25cmX 4.6mm, packed with octadecylsilane bonded to porous silica (5µm),
• Mobile phase: a mixture of 90 volumes of a buffer solution prepared by dissolving 3.72g of water, add 2ml of triethylamine and adjusted to pH 6.0 with O.P.A., 6 volumes of methanol and 4 volumes of acetonitrile,
• Flow rate: 1ml per minute,
• Spectrophotometer set at 205nm,
• Injection volume: 20µl

Inject the reference solution and test solution.

Calculate the content of methylcobalamin in each capsule.

Assay: Determined by liquid chromatography.

For Pregabalin:

Test solution: Weigh accurately a quantity of the mixed contents of 20 capsules containing about 75mg of pregabalin in 100ml volumetric flask add 50ml of mobile phase and disperse with the aid of ultrasound for 10 minutes and dilute to volume with mobile phase and filter.

Reference solution: A 0.075 per cent w/v solution of pregabalin reference standard in the mobile phase.

Chromatographic system:

• A stainless steel column 25cmX 4.6mm, packed with octadecylsilane bonded to porous silica (5µm),
• Mobile phase: a mixture of 90 volumes of a buffer solution prepared by dissolving 3.72g  of disodium hydrogen phosphate in 1000 ml water, add 2ml of triethylamine and adjusted to pH 6.0 with O.P.A., 6 volumes of methanol and 4 volumes of acetonitrile,
• Flow rate: 1ml per minute,
• Spectrophotometer set at 205nm,
• Injection volume: 20µl

Inject reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Inject the reference solution and the test solution.

Calculate the content of Pregabalin in the capsules.

For Methylcobalamin:

Note: All the test for methylcobalamin shall be carried out in dark environment.

Test solution: Weigh accurately a quantity of the mixed contents of 20 capsules containing about 0.75mg of Methylcobalamin in 25ml volumetric flask, add 20ml of mobile phase and disperse with the aid of ultrasound for about 5 minutes, cool and dilute to volume with mobile phase and filter.

Reference solution: 0.003 per cent w/v solution of methylcobalamin reference standard in mobile phase.

Chromatographic system:

• A stainless steel column 25cmX 4.6mm, packed with octadecylsilane bonded to porous silica (5µm),
• Column temperature: 40°
• Sample temperature: 15°
• Flow rate: 1ml per minute,
• Spectrophotometer set at 266nm,
• Mobile phase: a mixture of a solution containing 5.80 g of disodium hydrogen phosphate in 810ml of water, adjusted to pH 3.5 with citric acid, and add 3.76g of sodium 1-hexane sulphonate sodium salt and add 190ml acetonitrile,
• Injection volume: 20µl

Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 –per cent.

Inject the reference solution and the test solution.

Calculate the content of Methylcobalamin in the capsules.

Acceptance criteria: 90.0%-110.0%

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:

mg: Miligram

ml: Milileter

inHg: Inch of Mercury

rpm: Rounds per minute

CFU: Colony forming unit

w/v: Weight/Volume

v/v: Volume/Volume

O.P.A.: orthophosphoric acid