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 was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10% meta-phosphoric acid solution (aqueous). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were 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 were separated on a C 18 (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 sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min −1 at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity ( r 2 > 0.999 in the range of 0.2–10,000 ng mL −1 for all the analytes), recovery (>96%), precision (%RSD ≤ 2.0), and sensitivity (on column limit of detection: 250–400 fg and limit of quantification: 0.8–1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.