Analysis of methotrexate in human plasma by high-pressure liquid chromatography with fluorescence detection

Methotrexate in human plasma at a concentration as low as 0.01 microgram/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 to 10 microgram/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.