High-performance liquid chromatographic determination of methotrexate in plasma

A procedure for the determination of methotrexate in human plasma is reported. The analysis involved extraction of methotrexate as an ion pair in ethyl acetate. Reconstituted residue was analyzed using reverse phase C-18 column and a mobile phase consisting of acetate buffer (87%), methanol (6.5%), and acetonitrile (6.5%). The methotrexate recovery range was 95-97%. Theophylline was used as internal standard with a recovery of 96%. The intraday coefficient of variation for the assay ranged from 1.8-3.0%, while interday variation coefficient range was 3.5-3.7%. The method is selective, reproducible, and covers a wide range of methotrexate concentrations in patient's plasma.