High-presssure liquid chromatographic determination of methotrexate and its major metabolite, 7-hydroxymethotrexate, in human plasma.

A rapid high-pressure liquid chromatographic assay is described for the analysis of methotrexate (MTX) and its major metabolite, 7-hydroxymethotrexate (7-OH-MTX), in plasma samples from patients following high-dose therapy. The method involves an ion-pair extraction of MTX from plasma with chromatography on a microparticulate anion-exchange column using ultraviolet detection at either 254 or 315 nm. Maximum sensitivity achieved was 1 X 10(-7) M. In addition to being specific for parent MTX, this procedure was used to detect and quantitate significant concentrations of 7-OH-MTX in several patients. Comparison to a protein-binding method indicated a greater degree of specificity for the chromatographic procedure. The procedure can be used to allow therapeutic monitoring of plasma MTX and metabolite for patients receiving high-dose MTX therapy as well as to determine the purity of formulations of MTX used clinically. In addition, the procedure is applicable to the support of pharmacokinetic studies of MTX in animals and man.