On-line coupling of derivatization with pre-concentration to determine trace levels of methotrexate

Abstract

A new simple, sensitive and precise green analytical procedure using an automated packed-reactor derivatization technique coupled with on-line solid-phase enrichment (SPEn) has been developed and evaluated to determine trace levels of methotrexate (MTX). The method was based on injection of MTX into a flowing stream of phosphate buffer (0.04M, pH 3.4), carried through the packed oxidant reactor of Cerium (IV) trihydroxyhydroperoxide for oxidative cleavage of the drug into highly fluorescent product, 2,4-diaminopteridine-6-carboxylic acid, followed by SPEn on a head of short ODS column (10mm×4.6mm i.d., 5μm particle size). The flow rate was 0.25mL/min and packed reactor temperature was 40°C. The trapped product was back-flush eluted from the ODS column to the detector by column-switching with an environmentally friendly mobile phase consisting of ethanol and phosphate buffer (0.04M, pH 3.4) in the ratio of 5:95 (v/v). The eluent was monitored at emission and excitation wavelengths of 460 and 360nm, respectively. The calibration curve was linear over the concentration range of 1.25–50ng/mL with a detection limit of 0.08ng/mL. The method was successfully applied to determine MTX in pharmaceutical formulations with mean percentage recovery ranging from 99.48 to 99.60.