Rapid determination of methotrexate in plasma by high-performance liquid chromatography with on-line solid-phase extraction and automated precolumn derivatization.

A high-performance liquid chromatographic system, combining solid-phase extraction and automated precolumn derivatization is described for the routine determination of methotrexate in human plasma. The sample extraction and elution onto the analytical column were performed automatically and concomitantly using the column-switching technique and a protein-coated precolumn. Cerium (IV) trihydroxyhydroperoxide (CTH) was introduced as a packed oxidant before the analytical column for the conversion of methotrexate into highly fluorescent products. The oxidative-cleavage of methotrexate occurs during the flow of 0.04 M phosphate buffer (pH 3.5) containing plasma sample through CTH column with a flow rate of 0.5 mL/min at 40 degrees C. The fluorescent products were transferred to the protein-coated precolumn, which was then flushed with the same buffer for clean-up and enrichment from plasma sample. The trapped substances were desorbed from the precolumn and eluted onto the ODS/TM analytical column by isocratical elution with a mobile phase containing 0.05 M phosphate buffer, pH 6.6 and acetonitrile (90-10, v/v) for subsequent separation. The fluorescent products were detected fluorimetrically at excitation and emission wavelengths of 367 and 463 nm, respectively. The complete analysis was achieved within 15 min per sample. The calibration graph was linear in the range of 50-500 ng/mL of methotrexate and there was no interference from endogenous components.