Determination of lipoic acid in human plasma by high-performance liquid chromatography with ultraviolet detection

Abstract

This paper describes the development and validation of an HPLC method for the determination of protein bound and total lipoic acid in human plasma. The essential steps in the total lipoic acid assay include reduction of disulfide bridge with tris(2-carboxyethyl)phosphine, derivatization via thiol group with 1-benzyl-2-chloropyridinium bromide and HPLC analysis of S-pyridinium derivative. Protein-bound lipoic acid is first separated from free lipoic acid with the use of liquid extraction, converted to its reduced counterpart then processed as total lipoic acid. The method is reproducible, precise and accurate. The inter- and intraday related standard deviation varied from 1.5% to 11.5% and from 1.8% to 19.6%, respectively, while recovery is in the range of 80.0–106.0% and 80.4–110.8%, respectively. The mean concentration of total lipoic acid in healthy donors after supplementation with 600 mg and 1200 mg was 0.67 ± 0.40 μmol L−1 (137.6 ± 82.1 μg L−1) and 1.57 ± 0.34 μmol L−1 (323.34 ± 70.07 μg L−1), respectively. Keywords: Derivatization, Determination, HPLC, Lipoic acid, Thiol, Plasma