Determination of lipoic acid and dihydrolipoic acid in human plasma and urine by high-performance liquid chromatography with fluorimetric detection

A highly sensitive method for the determination of ol-lipoic acid (LA) and dihydrolipoic acid (DHLA) in human plasma and urine has been developed, Sam ples were acidified and extracted with organic solvent, and the free sulfhy dryls of DHLA protected as the dicarboxyethylate by treatment with ethylchl oroformate. The free carboxylic function of LA and the SH-protected DHLA we re converted into their amide derivatives with the strong fluorophore 2-(4- aminophenyl)-6-methylbenzothiazole in the presence of a coupling agent and a base catalyst. The resulting fluorescent amides of both LA and DHLA were separated on a reversed-phase column (Ultrasphere C-8) using simple isocrat ic elution with acetonitrile-water (80:20) and detected fluorimetrically (e xcitation 343, emission 423 nm), The method is highly sensitive, reproducib le, and is easily applied for the simultaneous determination of LA and DHLA in biological samples. (C) 2000 Elsevier Science B.V. All rights reserved.