An assay for methotrexate and its metabolites in serum and urine by ion-pair high-performance liquid chromatography.

Abstract A high-performance liquid chromatographic assay for methotrexate and its metabolites, 7-hydroxy-methotrexate, 4-amino-4-deoxy-N 10 -methyl-pteroic acid and 7-hydroxy-4-amino-4-deoxy-N 10 -methyl-pteroic acid in the range 10 μ/l to 50 mg/l (2.2 × 10 −8 to 1.1. × 10 −4 M) has been developed using l -tryptophyl- l -glutamic acid as internal standard. Extraction was performed using an anion exchange resin (Dowex 1-X2) with subsequent ion-pair chromatography of the appropriate eluent fraction. The method has been found to be sensitive and precise for the analysis of both serum and urine, and may also be used for the quantitation of polyglutamyl metabolites.