Determination of lipoic acid in human plasma by HPLC-ECD using liquid-liquid and solid-phase extraction: Method development, validation and optimization of experimental parameters.

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001-10μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250μl) was carried out with a simple one step liquid-liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40°C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30°C on Discovery HS C18 RP column (250mm×4.6mm, 5μm) was achieved in 6min using 0.05M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5ml/min using electrochemical detector in DC mode at the detector potential of 1.0V. The limit of detection and limit of quantification of lipoic acid were 200pg/ml and 1ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50pg/ml, respectively. The absolute recoveries of lipoic acid with liquid-liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.