Comparison of a new high-performance liquid chromatography method with fluorescence polarization immunoassay for analysis of methotrexate

A simple high-performance liquid chromatography (HPLC) method for the determination of methotrexate (MTX) in biological fluids is described. The assay is rapid, the time required for analysis is less than 30 min, and it is sensitive, up to 0.01 microgram/ml, which is three times below the toxic MTX concentration. Fifty plasma samples drawn from acute lymphocytic leukemia (ALL) patients were used to compare this method with that of fluorescence polarization immunoassay (FPIA). A good correlation (r = 0.979) was obtained between the results of the two analyses. FPIA constantly overestimates the concentration in samples collected during elimination and underestimates those collected during infusion. The difference between the means of the two methods was 29% and 13% for the elimination and infusion samples, respectively. The means of the peak height ratio of the metabolite to MTX in the HPLC chromatograms were 3.39 and 0.33 during elimination and infusion, respectively. The results therefore indicate that HPLC is more specific when tracing the washout of MTX concentration. Because of this specificity and simplicity, the method is recommended for therapeutic drug monitoring. The stability of MTX in human saliva was investigated in this study. MTX was found to be stable at room temperature and at -20 degrees C for a minimum of 3 h and 3 weeks, respectively.