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————–X Average 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%

HPLC Method