Methotrexate Tablets

Action and use

Dihydrofolate reductase inhibitor; cytostatic.

Definition

Methotrexate Tablets contain Methotrexate or methotrexate sodium prepared by the interaction of Methotrexate with Sodium Hydroxide.

The tablets comply with the requirements stated under Tablets and with the following requirements.

Content of methotrexate, C₂₀H₂₂N₈O₅

95.0 to 105.0% of the stated amount.

Identification

A. Extract a quantity of the powdered tablets containing 10 mg of Methotrexate with sufficient 0.1m sodium hydroxide to produce 100 mL, filter and dilute 10 mL of the filtrate to 100 mL with 0.1m sodium hydroxide. The light absorption of the resulting solution, Appendix II B in the range 230 to 400 nm, exhibits three maxima, at 258, 303 and 371 nm.

B. In the Assay, the chromatogram obtained with solution (1) shows a peak with the same retention time as the principal peak in the chromatogram obtained with solution (2).

TESTS

Disintegration

Maximum time, 30 minutes, Appendix XII A1.

Dissolution

Comply with the requirements for Monographs of the British Pharmacopoeia in the dissolution test for tablets and capsules, Appendix XII B1.

test conditions

(a) Use Apparatus 2, rotating the paddle at 50 revolutions per minute.

(b) Use 900 mL of 0.1m hydrochloric acid, at a temperature of 37°, as the medium.

procedure

(1) After 45 minutes withdraw a 10 mL sample of the medium and measure the absorbance of the filtered sample, suitably diluted with the dissolution medium if necessary, at the maximum at 303 nm, Appendix II B, using 0.1m hydrochloric acid in the reference cell.

(2) Measure the absorbance of a suitable solution of methotrexate EPCRS in the dissolution medium using 0.1m hydrochloric acid in the reference cell.

determination of content

Calculate the total content of methotrexate, C₂₀H₂₂N₈O₅, in the medium from the absorbances obtained and from the declared content of C₂₀H₂₂N₈O₅ in methotrexate EPCRS.

Related substances

Carry out the method for liquid chromatography, Appendix III D, using the following solutions.

(1) Disperse a quantity of the powdered tablets containing 10 mg of Methotrexate in 70 mL of the mobile phase with the aid of ultrasound for 5 minutes and dilute to 100 mL with the mobile phase. Mix and filter through a glass fibre filter (Whatman GF/C is suitable).

(2) Dilute 1 volume of solution (1) to 100 volumes with the mobile phase and further dilute 1 volume to 5 volumes with the mobile phase.

(3) Dissolve 6 mg of methotrexate impurity C EPCRS in 0.1 mL of dilute ammonia R1 and 0.5 mL of the mobile phase and dilute to 100 mL with the mobile phase. Dilute 1 volume of the resulting solution to 20 volumes with the mobile phase.

(4) 0.0003% w/v each of methotrexate EPCRS and methotrexate impurity D EPCRS.Dissolve 6 mg each of methotrexate EPCRS and methotrexate impurity D EPCRS in 0.1 mL of dilute ammonia R1 and 0.5 mL of the mobile phase and dilute to 100 mL with the mobile phase. Dilute 1 volume of the resulting solution to 20 volumes with the mobile phase.

(5) Dilute 1 volume of solution (2) to 10 volumes with the mobile phase.

chromatographic conditions

(a) Use a stainless steel column (25 cm × 4.6 mm) packed with base-deactivated octadecylsilyl silica gel for chromatography (5 µm) (Hypersil BDS 5 µm is suitable).

(b) Use isocratic elution and the mobile phase described below.

(d) Use an ambient column temperature.

(e) Use a detection wavelength of 265 nm.

(f) Inject 20 µL of each solution.

(g) Allow the chromatography to proceed for three times the retention time of methotrexate.

mobile phase

7 volumes of acetonitrile and 93 volumes of a citro-phosphate buffer solution prepared by dissolving 7.8 g of citric acid and 17.9 g of anhydrous disodium hydrogen orthophosphate in sufficient water to produce 1000 mL.

The approximate retention times relative to methotrexate are: impurity A, 0.2; impurity B, 0.3; impurity C, 0.4; impurity D, 0.8; impurity E, 2.3.

system suitability

The test is not valid unless, in the chromatogram obtained with solution (4), the resolution between the peaks due to methotrexate and methotrexate impurity D is at least 2.0.

limits

In the chromatogram obtained with solution (1):

the area of any peak corresponding to impurity C is not greater that the area of the principal peak in the chromatogram obtained with solution (3) (3%);

the area of any peak corresponding to impurity B is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with solution (2) (0.5%);

the area of any peak corresponding to impurity E is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with solution (2) (0.3%);

the area of any other secondary peak is not greater than the area of the principal peak in the chromatogram obtained with solution (2) (0.2%);

the sum of the areas of any other secondary peaks is not greater than 5 times the area of the principal peak in the chromatogram obtained with solution (2) (1%).

Disregard any peak with an area less than the principal peak in the chromatogram obtained with solution (5) (0.02%).

Assay

Carry out the method for liquid chromatography, Appendix III D, using the following solutions.

(2) 0.01% w/v of methotrexate EPCRS in the mobile phase.

(3) 0.0003% w/v each of methotrexate EPCRS and methotrexate impurity D EPCRS.Dissolve 6 mg each of methotrexate EPCRS and methotrexate impurity D EPCRS in 0.1 mL of dilute ammonia R1 and 0.5 mL of the mobile phase and dilute to 100 mL with the mobile phase. Dilute 1 volume of the resulting solution to 20 volumes with the mobile phase.

chromatographic conditions

The chromatographic conditions described under Related substances may be used but using 302 nm as the detection wavelength.

system suitability

The test is not valid unless, in the chromatogram obtained with solution (3), the resolution between the peaks due to methotrexate and methotrexate impurity D is at least 2.0.

determination of content

Calculate the content of C₂₀H₂₂N₈O₅ in the tablets using the declared content of C₂₀H₂₂N₈O₅ in methotrexate EPCRS.

Storage

Methotrexate Tablets should be protected from light.

Labelling

When the active ingredient is methotrexate sodium, the quantity is expressed in terms of the equivalent amount of Methotrexate.