Flow injection method for the determination of methotrexate with a column-packed oxidizing agent

A highly sensitive flow injection technique involving oxidative cleavage and spectrofluorimetric detection was developed for the determination of methotrexate. Cerium(IV) trihydroxyhydroperoxide (CTH) was introduced as a packed reactor for the construction of a derivatization column which is used in a flowing system for conversion of methotrexate into a highly fluorescent derivative. The oxidative cleavage of methotrexate occurs during the flow of carrier stream containing the drug through the CTH column. The optimum composition of the carrier stream was found to be 0.04 mol l–1 phosphate buffer (pH 3.5). The flow rate was 0.5 ml min–1 and the column temperature was 40 °C. The fluorescent product was monitored spectrofluorimetrically at excitation and emission wavelengths of 367 and 463 nm, respectively. The proposed method was successfully applied to the determination of methotrexate in pharmaceutical preparations.