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

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 (0.04 M, 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 (10 mm x 4.6 mm i.d., 5 mu m particle size). The flow rate was 0.25 mL/min and packed reactor temperature was 40 degrees 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 (0.04 M, pH 3.4) in the ratio of 5:95 (v/v). The eluent was monitored at emission and excitation wavelengths of 460 and 360 nm, respectively. The calibration curve was linear over the concentration range of 1.25-50 ng/mL with a detection limit of 0.08 ng/mL. The method was successfully applied to determine MTX in pharmaceutical formulations with mean percentage recovery ranging from 99.48 to 99.60. (C) 2013 Xi'an Jiaotong University. Production and hosting by Elsevier B.V. All rights reserved.