Simultaneous determination of the endogenous free α-lipoic acid and dihydrolipoic acid in human plasma and erythrocytes by RP-HPLC coupled with electrochemical detector

Abstract

A highly sensitive, precise, and accurate reversed-phase high performance liquid-chromatography/ electrochemical detection method for simultaneous determination of the endogenous free α-lipoic acid and dihydrolipoic acid in biological matrices is presented. The two analytes are extracted from samples with acetonitrile–10% m -phosphoric acid solution (aqueous) (50:50 v/v). To determine the total lipoic acid, samples are treated with tris(2-carboxyethyl)phosphine solution in phosphate buffer: pH 2.5 with 85% o -phosphoric acid prior to deproteination. The two analytes are separated on a C 18 (150 × 4.6 mm, 5 μm) analytical column using acetonitrile-50 mM phosphate buffer: pH 2.5 with 85% o -phosphoric acid (35:65 v/v) as the isocratic mobile phase pumped at a flow rate of 2.0 ml/min at the column oven temperature of 35 °C. The column eluents are monitored at a potential of 0.9 V. These analytes are efficiently resolved in <7 min.