Separation and identification of impurities in parenteral methotrexate dosage forms

Eleven lots of parenteral methotrexate (MTX) dosage forms from two manufactures have been analyzed and found to be 93.4% +/- 2.1% (SD) pure by a new procedure employing a DEAE-cellulose column adapted for high performance liquid chromatography instrumentation. In addition to 4-amino-10-methylpteroic acid and N10-methylpteroylglutamic acid, which were previously identified contaminants, and benzyl alcohol, which was added as a preservative, we have also isolated and identified 2,4-diamino-6-methylpteridine, 2,4-diamino-6-bromomethylpteridine, and p-methylaminobenzoic acid. Another impurity has tentatively been identified as 4-amino-10-methylpteroic acid amide. The individual impurities vary from 0.1% to 4.8% of the ultraviolet absorbing material which elutes from the column. Additional impurities were detected by gas chromatography-mass spectrometry. Since "high-dose" therapy may involve the administration of up to 20 g of MTX/day, the effect of the impurities present in these dosage forms on the course of the patient's therapy should be determined.