EVALUATION OF CLINICAL ASSAYS FOR MEASURING HIGH-DOSE METHOTREXATE INPLASMA

Four routine assays commonly used for monitoring plasma methotrexate ( MTX) during high-dose therapy were validated by HPLC as the comparison method, MTX and its main metabolite, 7-hydroxymethohexate (7-OHMTX), were analyzed by HPLC with postcolumn derivatization and fluorometric detection, About 200 clinical plasma samples from 13 children with acu te lymphoblastic leukemia who received 5-8 g/m(2) MTX as 24-h infusion s were analyzed, The fraction of measured concentrations of MTX that w ere within 75-125% of the values obtained by HPLC were 64.5% for enzym e inhibition assay, 56.4% for fluorescence polarization immunoassay wi th polyclonal antibodies (FPIA1; Abbott), 58.9% for FPIA2 (with monocl onal antibodies; Abbott), and 46.4% for enzyme-multiplied immunoassay (Emit(TM); Syva). All nonchromatographic procedures were subject to in terferences from MTX plasma metabolites or endogenous substances, The interference from 7-OHMTX was, however, somewhat less pronounced for F PIA2 (monoclonal) than for FPIA1 (polyclonal).