Simultaneous Determination of the Endogenous Free α-Lipoic Acid and Dihydrolipoic Acid in Human Plasma and Erythrocytes by RP-HPLC with Electrochemical Detection

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 was developed and validated. The two analytes were extracted from the samples with acetonitrile/10% metaphosphoric acid solution(aqueous) (50/50 v/v). To determine the total lipoic acid, samples were treated with tris(2-carboxyethyl)phosphine solution in phosphate buffer, pH 2.5 with 85% orthophosphoric acid prior to deproteination. The two analytes were separated on a C18 (150 × 4.6 mm, 5 μm) analytical column using acetonitrile-50 mM phosphate buffer, pH 2.5 with 85% orthophosphoric acid (35/65 v/v) as the isocratic mobile phase pumped at a flow rate of 2.0 mL min−1 at the column oven temperature of 35 °C. The column eluents were monitored at a potential of 0.9 V. These analytes were efficiently resolved in <7 min. The present method was sufficiently robust and specific for simultaneous determination of the two analytes and demonstrated acceptable values for linearity (r 2 = 0.999 in the range of 0.1–500 and 0.25–1,000 ng mL−1 for α-lipoic acid and dihydrolipoic acid, respectively), recovery (>97%), precision (RSD% <2), and sensitivity (on column limit of detection, 150 and 375 fg for α-lipoic acid and dihydrolipoic acid, respectively and limit of quantification: 0.5 and 1.25 pg for α-lipoic acid and dihydrolipoic acid, respectively), indicating that the proposed method was more sensitive, precise, economical, and versatile, and has higher throughput than the previously reported methods for simultaneous determination of the two analytes.