ANALYSIS OF METHOTREXATE IN HUMAN-PLASMA BY HIGH-PRESSURE LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

Abstract

The antitumor agent methotrexate in human plasma at a concentration as low as 0.01 .mu.g/ml can be assayed with the use of high-pressure liquid chromatography and a fluorescence detection system. Methotrexate is oxidized stoichiometrically to 2,4-diaminopteridine-6-carboxylic acid, a fluorescent product that is separable from other fluorescent materials in plasma with the use of an octadecylsilane (reversed phase) column. The detector response is linear over the range of 0.01-10 .mu.g/ml. Neither folic acid nor citrovorum factor interferes with the analysis. N-{{4-{[2,4-Dihydroxy-6-pteridyl)methyl]-amino}benzoyl}}glutamic acid may be used as an internal standard, since it can be extracted from plasma and oxidized like methotrexate. The procedure is rapid (about 30 min) and should be a useful method for monitoring methotrexate plasma concentrations.