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.
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% |
- Identification Test:
Extract a quantity of the powdered tablets containing 10mg of Methotrexate with sufficient 0.1 M sodium hydroxide to produce 100ml, filter and dilute 1ml of the filtrate to 100ml with 0.1 sodium hydroxide.
When examined in the range 230nm to 380nm, the resulting solution shows absorption maxima at about 258nm, 303nm and 371nm.
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: Paddle
Medium: 900ml of 0.1 M Hydrochloric acid.
Time: 45minutes
Speed: 50 rpm
Limit: NLT 80% of the stated amount of Methotrexate.
Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc having an average pore diameter not greater than 1.0µm, rejecting the first few ml of the filtrate. Measure the absorbance of the filtrate at maximum at about 306nm. Calculate the content of Methotrexate taking 430 as the specific absorbance at 306nm.
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.
Test solution: Disperse a quantity of the powdered tablets containing about 10mg of Methotrexate in 70ml of the mobile phase with the aid of ultrasound for 5 minutes and dilute to 100.0ml with mobile phase, filter.
Reference solution (a): A 0.0002 per cent w/v solution of methotrexate reference standard in the mobile phase.
Reference solution (b): A 0.0003 per cent w/v solution of methotrexate impurity C reference standard in the mobile phase.
Reference solution (c): A solution containing 0.0003 per cent w/v each of methotrexate reference standard and methotrexate impurity D reference standard in the mobile phase.
Chromatographic system:
- A stainless steel column 25cmX4.6 mm, packed with base deactivated octadecylsilane bonded to porous silica (5µm)( such as hypersil BDS)
- Mobile phase: a mixture of 70 volumes of acetonitrile and 93 volumes of citro-phosphate buffer solution prepared by dissolving 7.8gm of citric acid and 17.9gm of disodium hydrogen orthophosphate, dilute to 1000ml with water,
- Flow rate: 1.2ml per minute,
- Spectrophotometer set at 265nm,
- Injection volume: 20µl
Inject reference solution(c). The test is not valid unless the resolution between the peaks due to methotrexate and methotrexate impurity D is not less than 2.0.
Inject reference solutions (a), (b) and the test solution. Run the chromatogram 3 times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any peak corresponding to methotrexate impurity C is not more than the area of the principal peak in the chromatogram obtained with solution (b) (3.0 per cent). The area of any peak corresponding to methotrexate impurity B is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent). The area of any peak corresponding to methotrexate impurity E is not more than 1.5 times the area of principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent). The area of any other secondary peak is not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent). The sum of the areas of any other secondary peak is not more than 5 times the area of principal peak in the chromatogram obtained with reference solution (a) (1.0 per cent). Ignore any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.02 per cent).
12.0) Uniformity of content:
Carry out the test as described in the assay, using the following solutions.
Test solution: Crush one tablet and mix with 100ml of the mobile phase with the aid of ultrasound, centrifuge and use the supernatant liquid.
Reference solution (a): A 0.025 per cent w/v solution of methotrexate reference standard in the mobile phase.
Reference solution (b): A solution containing 1.0025per cent w/v each of methotrexate reference standard and folic acid in the mobile phase.
Calculate the content of Methotrexate in the tablet.
13.0) Assay: Determined by liquid chromatography.
Test solution: Weigh and powder 20 tablets. Weigh a quantity of the powder containing about 2.5mg of methotrexate and mix with 100.0ml of the mobile phase with the aid of ultrasound, centrifuge and use the supernatant liquid.
Reference solution (a): A solution containing 0.0025 per cent w/v each of methotrexate reference standard and folic acid in the mobile phase.
Reference solution (b): A 0.0025 per cent w/v solution of methotrexate reference standard in the mobile phase.
Chromatographic system:
- A stainless steel column 25cmX4.6 mm, packed with octadecylsilane bonded to porous silica (5µm)
- Mobile phase: a mixture of 92 volumes of phosphate buffer pH 6.0 and 8 volumes of acetonitrile.
- Flow rate: 1.4ml per minute,
- Spectrophotometer set at 302nm,
- Injection volume: 20µl
Inject reference solution (a). The resolution between the peaks due to methotrexate and folic acid is not less than 5.0.
Inject reference solution (b). The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.
Inject reference solution (b) and the test solution.
Calculate the content of Methotrexate in the tablets.
Formula for calculation:
Area of test STD wt. 5 100 Potency
—————-–X————-X——-X—————–X———-XAverage weight
Area of STD 100 50 Weight of test 100
Alternative Method: By UV Spectrophotometer
Test Solution: Weigh and powder 20 tablets. Weight sample equivalent to 5mg of methotrexate in 100ml volumetric flask add some 0.1 M NaOH into it and sonicate to dissolve the test sample. Make up to mark with 0.1M NaOH. Filter and dilute 5ml of this solution to 25ml in same solvent.
Reference Solution: Weigh 50mg of reference standard of methotrexate in 100ml of volumetric flask. Add 20ml of 0.1 M NaOH into it and sonicate to dissolve. Dilute 1ml of this solution to 50ml with same solvent.
Check Absorbance of test as well as reference standard at 303nm in UV Spectrophotometer.
Calculate the content of Methotrexate.
Formula for calculation:
Absorbance of TEST STD wt. (mg) 1 100 25 Potency
—————————-X——————–X———X————–X———X———–X Avg. Wt.
Absorbance of STD 100 50 Test wt (mg) 5 100
Acceptance criteria: 90.0%-110.0%
