Fluorometric Determination of Methotrexate in Serum by High- Performance Liquid Chromatography Using In-Line Oxidation with Hydrogen Peroxide

摘要

A fluorometric method has been developed for the determination of methotrexate in serum by high-performance liquid chromatography. Methotrexate after deproteinization with 2M perchloric acid was separated by reversed-phase chromatography with a neutral mobile phase (pH 7.0, 8% acetonitrile in 25mM phosphate buffer) containing 25mM hydrogen peroxide as a fluorogenic reagent, and was fluorometrically detected (Ex 379nm and Em 457nm) via in-line oxidation at high temperature (160°C). The calibration curve could be made linear over the range 1×10-6-1×10-5M by injecting a volume of 20μl of deproteinized serum. The detection limit (signal-to-noise ratio=3) for methotrexate in serum was 2×10-8M using a 50-μl aliquot of deproteinized serum. Comparison with the fluorescence polarization immunoassay gave a good correlation coefficient of 0.949.