Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector

Abstract

A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of L -ascorbic acid, aminothiols, and methionine in biological matrices is presented. Reduced forms of the analytes are extracted from sample matrices with 10% m-phosphoric acid solution(aqueous). To determine the total vitamin C, the total aminothiols, and the total methionine, samples are treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution(aqueous) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes are separated on a C18 (250 × 4.6 mm, 5 μm) analytical column using methanol-0.05% trifluoroacetic acid solution(aqueous) (05:95 v/v, containing 0.1 mM 1-octane sulfonic acid as the ion-pairing agent) as the isocratic mobile phase that is pumped at a flow rate of 1.5 ml/min at room temperature. The column eluents are monitored at a voltage of 0.85 V. These analytes are efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard.