On-line precolumn derivatization for HPLC determination of methotrexate using a column packed oxidant

A high-performance liquid chromatographic method involving on-line precolumn oxidative cleavage and fluorimetric detection was developed for the determination of methotrexate in plasma. Plasma samples were subjected to protein precipitation followed by solvent purification and then injection into the chromatographic system. Cerium (IV) trihydroxyhydroperoxide (CTH) was introduced as a packed oxidant before analytical column for the conversion of methotrexate into highly fluorescent 2,4-diaminopteridine derivatives. The oxidative cleavage of methotrexate occurs during the flow of 0.04 M phosphate buffer (pH 3.5) containing the drug through CTH column with a flow-rate of 0.2 mL/min at 40 degrees C. The separation was performed on a reversed-phase column (ODS/TM) using a mobile phase consisting of phosphate buffer (0.05 M, pH 6.6) and acetonitrile (90:10 v/v). The fluorescent products were monitored fluorimetrically at emission and excitation wavelengths of 463 and 367 nm, respectively. Validation of accuracy and precision were satisfactory for both within- and between-run assays. All coefficients of variance were less than 4% and mean relative errors were within 1.11% to 7.83%. The average recovery was found to be 93.74% to 98.11%. The proposed method is highly sensitive, specific and applicable to biological fluids.