Development and validation of a high-performance liquid chromatography method for the determination of methotrexate

Abstract

Methotrexate is an anticancer drug belonging to the antimetabolite group. Its toxic potential and the inter- and intra-individual variability in its pharmacokinetics make it necessary to adjust therapeutic protocols based on plasma concentrations. In this work, we describe a method for the determination of methotrexate using High-Performance Liquid Chromatography (HPLC), developed in our laboratory and suitable for routine use with UV detection. We also outline the optimized operating conditions for this assay, including temperature and elution flow rate, mobile phase composition and pH, and extraction methods. Our technique validation procedures indicate that the imprecision of reproducibility and repeatability is 3.65% and 3%, respectively. Linearity was confirmed for concentrations ranging from 0.03 to 500 µmol/l with a correlation coefficient of 99%. The detection limit is 0.02 µmol/l. Finally, we compared two methotrexate assay techniques: High-Performance Liquid Chromatography and Fluorescence Polarization Immunoassay (FPIA). The method we developed is thus simple, fast, reliable, reproducible, and especially well-suited for the technical conditions of a routine clinical pharmacology laboratory.