Evaluation of enzyme immunoassay, radioassay, and radioimmunoassay of serum methotrexate, as compared with liquid chromatography.

We evaluated three commonly used clinical methods for measuring serum methotrexate: enzyme immunoassay (EMIT), radioassay, and radioimmunoassay. Because potentially interfering compounds can be resolved by liquid chromatography, this method was selected as the comparison method. Patients' serum samples, taken during 24 h after 6-h high-dose infusions, contained methotrexate in concentrations ranging from 10(-7) to 10(-4) mol/L. Chromatograms revealed substantial amounts of 7-hydroxymethotrexate in all samples, actually exceeding methotrexate by 12 and 24 h. Nevertheless, analysis of variance revealed no significant differences in results by any of the four methods, nor did results by the three test methods differ significantly after being adjusted by analysis of covariance with liquid chromatography as the covariate. Evidently any of the four techniques is suitable for monitoring serum methotrexate for 24 h after high-dose therapy.