Column liquid chromatography of methotrexate and its metabolites using a post-column photochemical reactor and fluorescence detection.

On irradiation with short-wavelength UV light in the presence of hydrogen peroxide, methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid are cleaved into highly fluorescent products. This reaction can be used for the sensitive and selective detection of the compounds in biological fluids, following reversed-phase high-performance liquid chromatographic separation. Study of the effect of the mobile phase composition and irradiation time on fluorescence signal intensity showed that a residence time of ca. 3 s in the on-line photochemical reactor was best. The detection limit for methotrexate was 0.4 ng, for 7-hydroxymethotrexate 1.0 ng and for 2,4-diamino-N10-methylpteroic acid 0.6 ng. The addition of dimethylformamide to the mobile phase enhanced the selectivity of separation.