Abstract
A simple, sensitive and precise green high-performance liquid chromatographic method including on-line pre-column oxidation combined by column switching with a short Hypersil ODS analytical column (100 mm × 4.0 mm i.d.) for enrichment and separation was developed and validated to determine low levels of methotrexate (MTX). The method was based on oxidative cleavage of MTX into highly fluorescence products, 2,4-diaminopteridine-6-carboxaldehyde and the corresponding 2,4-diaminopteridine-6-carboxylic acid, during the flow of phosphate buffer (0.04 M, pH 3.4) containing the analyte through the packed reactor of cerium (IV) trihydroxyhydroperoxide (CTH) at a flow-rate of 0.2 mL/min and 40℃. The fluorescent products were enriched on the head of ODS analytical column for the final separation. The separation was performed at room temperature using an environmentally friendly mobile phase consisting of ethanol and phosphate buffer (0.04 M, pH 3.4) in the ratio of 10:90 (v/v). The eluent was monitored at emission and excitation wavelengths of 463 and 367 nm, respectively. The method was successfully applied, without any interference from the excipients, for the determination of drug in tablets and vials with a detection limit of 0.06 ng/mL from 500 ?L of sample MTX.
Highlights
Methotrexate (MTX, 2,4-diamino-4-deoxy-N10-methylpteroglutamic acid) is one of the most widely used anticancer drugs and acts as an antimetabolite of folic acid
A simple, sensitive and precise green high-performance liquid chromatographic method including on-line pre-column oxidation combined by column switching with a short Hypersil ODS analytical column (100 mm × 4.0 mm i.d.) for enrichment and separation was developed and validated to determine low levels of methotrexate (MTX)
Many methods have been developed for the determination of MTX, including spectrofluorimetry [2,3,4], immunoassay [5], capillary electrophoresis [6,7] and highperformance liquid chromatography (HPLC) with UV detection [8,9,10,11,12,13]
Summary
Methotrexate (MTX, 2,4-diamino-4-deoxy-N10-methylpteroglutamic acid) is one of the most widely used anticancer drugs and acts as an antimetabolite of folic acid In high doses it is used in treatment of some solid tumors and leukemia. Several methodologies have been developed for the oxidation of MTX to a highly fluorescent product, 2,4-diaminopteridine-6-carboxaldehyde and the corresponding 2,4-diaminopteridine-6-carboxylic acid These methods were based on photo-oxidative irradiation at 254 nm [14,19,20,23,24], electrochemical oxidation [16,17], oxidation with permanganate [4,18,25] and hydrogen peroxide [22,23,24]. Flow injection analysis (FIA) of MTX in pharmaceutical formulations has been reported [26,27] These methods are based on the oxidation of the MTX into highly fluorescent product 2,4-diaminopteridine-6-carboxylic acid by on-line electrochemical oxidation and acidic potassium permanganate; respectively. Cerium (IV) trihydroxyhydroperoxide (CTH) has been introduced as a packed reactor in a flowing system for conversion of JASMI
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