Reversed-phase high-pressure liquid chromatographic determination of serum methotrexate and 7-hydroxymethotrexate

A reversed-phase high-pressure liquid chromatographic method for determining methotrexate and 7-hydroxymethotrexate in human serum is presented. A mobile phase of acetate buffer (0.2 M, pH 5.5 with 0.03 M ethylenediaminetetraacetate), methanol, and acetonitrile (85.3:8.4:6.3), passed through a mu Bondapak C18 column at 1.5 ml/min produced excellent resolution of sharp, symmetrical bands. An improved extraction process, using a sample preparation cartridge, resulted in analytical recoveries in excess of 90% for methotrexate and 70% for 7-hydroxymethotrexate, permitting the determination of serum concentrations of 2.20 and 2.13 x 10(-7) M, respectively, using only 200 microliter of serum. UV detection at 313 nm provided adequate sensitivity for each component. While the reproducibility for 7-hydroxymethotrexate was approximately equal to previous methods, that for methotrexate was greatly improved. Serum methotrexate data at selected time points following high dose methotrexate therapy are presented.