Determination of methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid in biological fluids by liquid chromatography with fluorimetric detection.

A high-performance liquid chromatographic method involving post-column cleavage and fluorimetric detection has been developed for the determination of methotrexate and its metabolites in biological fluids. The cleavage is based on photooxidative reaction of methotrexate and its metabolites to highly fluorescent products. The photoreaction occurs during the flow of eluate containing a certain amount of hydrogen peroxide through a PTFE capillary irradiated by UV light. The method allows the determination of methotrexate, 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid in plasma, urine and ascitic fluid samples at concentrations as low as 2 X 10(-8)M.