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.
| Average weight of tablets | Percentage deviation |
| More than 80mg but Less than 250mg | 7.5% |
| 250mg or More | 5% |
- Identification Test:
Extract a quantity of the powdered tablets containing 50 mg of Methylprednisolone with 100ml of chloroform, filter and evaporate the filtrate to dryness. The residue complies with the following tests.
- Determine by infrared absorption spectrophotometry. Compare the spectrum with that obtained with methyprednisolone reference standard or with the reference spectrum of methylprednisolone.
- Determine by thin- layer chromatography, coating the plate with silics gel G.
Solvent mixture: A mixture of 90 volumes of acetone and 10 volumes of formamide.
Mobile phase: A mixture of 30 volumes of toluene and 10 volumes of chloroform.
Test solution: Dissolve 25mg of the residue in 10ml of the solvent mixture.
Reference solution (a): Dissolve 25mg of methylprednisolone acetate reference standard in 10ml of the solvent mixture.
Reference solution (b): Mix equal volumes of the test solution and reference solution (a).
Place the dry plate in a tank containing a shallow layer of the solvent mixtue, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of mobile phase in the direction in which the aforementioned treatment was done.
Apply to the plate 2 µl of each solution. Allow the mobile phase to rise 12cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120° for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120° for further 10 minutes, allow to cool and examine in daylight and under ultraviolet light at 365nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot.
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. For tablets with an average weight of 0.65 gm or less take a sample of whole tablets corresponding to about 6.5gm and for tablets with an average weight of more than 0.65gm take a sample of 10 tablets.
Dedust the tablets carefully and weigh accurately the required number of tablets. Place the tablets in the drum and rotate it 100 times (25 rpm for 4 minutes). Remove the tablets, remove any loose dust from them and weigh them accurately. 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:
Apparatus: Basket
Medium: 900ml of water
Time: 45minutes
Speed: 100 rpm
Limit: NLT 75% of the stated amount of Methylprednisolone.
Withdraw a suitable volume of the medium and filter. Measure the absorbance of a layer of suitable thickness of the filtered solution at the maximum at about 246nm. Calculate the content of Methylprednisolone taking 400 as the specific absorbance at 246nm.
Calculation:
For 4 mg
Sample value 1000 900
—————– x ———- x ———– x 100 = Found Result in %
400 100 4
For 8 mg
Sample value 1000 900
—————– x ———- x ———– x 100 = Found Result in %
400 100 8
For 16 mg
Sample value 1000 900
—————– x ———- x ———– x 100 = Found Result in %
400 100 16
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) Related substance: Determined by liquid chromatography.
Solvent mixture: A filtered mixture of 72 volumes of water, 25volumes of tetrahydrofuran and 3 volumes of glacial acetic acid.
Test solution: Extract a quantity of the powdered tablets containing 25mg of methylprednisolone with the solvent mixture and dilute to 25ml with the solvent mixture. Filter and centrifuge, if necessary.
Reference solution: A 0.001 per cent w/v solution of methylprednisolone referenc standard in the solvent mixture.
Chromatographic system:
- A stainless steel column 20cmX4.6 mm, packed with octadecylsilane bonded to porous silica (3 to 10µm),
- Mobile phase: a mixture of 149 volumes of water, 40 volumes of tetrahydrofuran, 10 volumes of dimethylsulfoxide and 1 volume of butanol,
- Flow rate: 1ml per minute,
- Spectrophotometer set at 254nm,
- Injection volume: 10µl
Inject the reference solution. The column efficiency is not less than 800 theoretical plates and relative standard deviation for replicate injections is not more than 5.0 per cent.
Inject the reference solution and the test solution. In the chromatogram obtained with the test solution, the area of any secondary peak is not more than the area of the principal peak in the chromatogram obtained with the reference solution (1.0 per cent). The sum of the areas of all the secondary peaks is not more than twice the area of the principal peak in the chromatogram obtained with the reference solution (2.0 per cent).
12.0) Uniformity of content:
Determine by liquid chromatography using the chromatographic system and the reference solution described in the assay.
To one tablet add 0.5ml of water (in the case of tablets containing 10mg or less) or 1.0ml of water (in the case of tablets containing more than 10mg). Allow the tablet to stand for about 2 minutes, then swirl to disperse the tablet. Add 5.0ml of the internal standard used in the assay for each mg of methylprednisolone, shake for 15 minutes, filter and centrifuge. Use the filtrate as the test solution.
Calculate the content of Methylprednisolone in the tablet.
13.0) Assay: Determined by liquid chromatography.
Internal standard solution: Weigh a suitable quantity of prednisone in a 3 per cent w/v solution of glacial acetic acid in chloroform to obtain a solution having a known concentration of about 0.2mg per ml of prednisone.
Test solution: Weigh and powder 20 tablets. Weigh a quantity of the powder containing about 10mg of methylprednisolone transfer to a suitable container and add 2.5ml of water. Swirl to form slurry. Add 50.0ml of the internal standard solution, and shake for 15 minutes. Filter and centrifuge a portion of the filtrate if necessary and use this as the test solution.
Reference solution: Weigh a suitable quantity of methylprednisolone reference standard in the internal standard solution to obtain a solution having a known concentration of about 0.2mg per ml of methylprednisolone.
Chromatographic system:
- A stainless steel column 25cmX4 mm, packed with porous silica particles (3 to 10µm)
- Mobile phase: a mixture of 475 volumes of butyl chloride, 475 volumes of water-saturate butyl chloride, 70 volumes of tetrahydrofuran, 35 volumes of methanol, and 30 volumes of glacial acetic acid,
- Flow rate: 1ml per minute,
- Spectrophotometer set at 254nm,
- Injection volume: 10µl
Inject reference solution. The resolution between methylprednisolone and prednisone is not less than 4.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent.
Inject reference solution and test solution. The relative retention time with reference to methylprednisolone for prednisone is about 0.7.
Calculate the content of Methylprednisolone in the tablets.
Alternative Method: By UV Spectrophotometer
Test Solution: Weigh and powder 20 tablets. Weight sample equivalent to 0.1g of methylprednisolone and dissolve in sufficient ethanol (95 per cent) and mix well. Dissolv the sample by ultrasonication and by shaking the sample. Filter the sample and dilute 2.0ml of this solution to 100.0 ml with ethanol. And mix well. Determine the absorbance of the resulting solution at maximum at about 243nm. Calculate the content of Methylprednisolone taking 395 as the specific absorbance at 243nm.
Formula for calculation:
Absorbance of TEST 1000 100 100
—————————-X———-X——————-X———X Avg. Wt.
395 100 Test wt (mg) 2
Acceptance criteria: 90.0%-110.0%
14.0) MICROBIAL ANALYSIS
A.) Total microbial count
B.) Pathogen detection
A.) TOTAL MICROBIAL COUNT
- Pour plate method by serial dilution:
Take 2 test tubes and label them A & B and 6 petriplates (3 for SCDA &3 for SDA) and label the petriplates as 10ˉ¹, 10ˉ², 10ˉ³.
Pour 9ml buffer solution (NaCl / Peptone water) in each test tube.
Crush the tablets in pestle motar and make it in powder form.
Take 99ml of Soyabean Casein Digest Medium (Tryptone Soya Broth) in borosilicate glass bottle and add 1 gm of tablet powder sample to it to make 1:100 dilution and mix well.
Pipette out 1ml of this dilution with the help of sterile micropipette and open the lids of empty sterile petriplate labeled 10ˉ¹ and dispense into the middle of each petriplate aseptically and then close the lid of petriplate. Transfer 1ml of tablet sample sample to the test tube labeled A to make 1:10 dilution and mix well. Take 1ml of sample from test tube A and dispense aseptically into the petriplate labeled 10ˉ². Transfer 1ml of sample from test tube A to test tube B and mix well. Then take 1ml of sample from test tube B and dispense the sample into the petriplate labeled 10ˉ³. Remove the cotton plug from the flask of molten agar (SCDA & SDA) and pass the rim of opened flask through the flame of Bunsen burner. Open the lid of petriplate containing the sample and pour 20-25ml sterile Soyabean Casein Digest Agar (for TAMC) and Sabouraud Dextrose Agar (for TYMC) cooled at 45ºC into the petriplates. Close the lid of petriplates and mix thoroughly by gently swirling the plates & allow solidifying the media at room temperature.
Use aseptic technique throughout the procedure
Invert and incubate the SCDA plates at 30- 35ºC for 3-5 days (for TAMC) and SDA plates at 20-25ºC for 5-7 days (for TYMC). Also incubate the Soyabean Casein Digest Medium containing capsule sample for 24 hours at 30- 35ºC temperature for pathogen detection test.
After incubation examine the SCDA plates for total bacterial count and SDA plates for total yeast and mold count. Take maximum from the two plates and calculate the no. of CFU/ml of water sample.
B.) PATHOGEN DETECTION
- Detection of Escherichia coli:
Streak a loopful culture of enriched pre-incubated Soyabean Casein Digest Medium containing capsule sample on the surface of sterile MacConkey Aagr plate. Incubate the plates at 30-35 for 18-72 hours. After incubation presence of brick red colonies on MacConkey agar indicate the presence of E.Coli. Further confirmation is done by gram’s staining.
- Detection of Salmonella:
Transfer 0.1 ml of enriched Soyabean Casein Digest medium to 10 ml of Rappaport Vassiliadis
Salmonella enrichment broth and incubate at 30-35 for 24-48 hours. Growth of green colonies with blank centre and in 48 hours the colonies become uniformly black colonies surrounded by a dark zone and metallic sheen indicates the possibility of presence of Salmonella.
If subcultured on plate Xylose-Lysine Deoxycholate Agar and incubate at 30-35 for 24-48 hours. Well developed red colonies with or without black centre indicates the possibility of presence of Salmonella. Further confirmation is done by gram’s staining.
- Detection of Pseudomonas aeruginosa:
Streak a loopful culture of pre-incubated Soyabean Casein Digest Medium (containing capsule sample) on the surface of Cetrimide Agar medium and incubate at 30-35 for 18-72 hours. A greenish colored colony indicates the possibility of presence of P.aeruginosa. Carry out the further conformation by gram’s staining. Further confirmation done by oxidase test.
- Detection of Staphylococcus aureus:
Streak a loopful culture of pre-incubated Soyabean Casein Digest medium (containing capsule sample) on the surface of Mannitol Salt Agar plates. Incubate the plates at 30-35 for 18-72 hours. Examine the plates after incubation. If upon examination of incubated plates none of them contains yellow colonies with yellow zones the sample meets the requirements for the absence of Staphylococcus aureus. If growth occurs further confirm by streak the colonies on the surface of Baired Parker agar plates black, shinny colonies surrounded by clear zones indicates the presence of Staphylococcus aureus. Further confirm by gram’s staining.
- Detection of Asperigillus brasiliensis:
Streak a loopful culture of enriched fluid Soyabean Casein Digest medium on the surface of Sabooraud Dextrose Agar plates. Incubate the plates at 20-25 for 18-72 hours.
Growth of black color colonies may indicate the presence of Asperigillus brasiliensis.
Further confirm by microscopic examination.
- Test for Candida albicans:
Streak a loopful culture of enriched fluid Soyabean Casein Digest medium on the surface of Sabooraud Dextrose Agar plates. Incubate the plates at 20-25 for 18-72 hours.
Growth of white colonies may indicate the presence of Candida albicans.
Abbreviations:
Wt.: Weight
mg: Miligram
ml: Milileter
STD: Standard
inHg: Inch of Mercury
RPM: Rounds per minute
E.coli: Escherichia coli
P.: Pseudomonas
TAMC: Total aerobic microbial count
SCDA: Soyabean casein digest medium
TYMC: Total yeast and mold count
SDA: Sabourand dextrose agar
CFU: Colony forming unit
NaCl: Sodium Chloride
