Analysis of reduced and oxidized lipoic acid in biological samples by high-performance liquid chromatography.

Using HPLC with electrochemical detection at a dual Hg/Au electrode, both reduced and oxidized lipoic acid can be measured in biological samples after addition of lipoic acid. The method does not detect bound lipoic acid, which must be liberated by strong acid or base hydrolysis. The detection limit for this HPLC method is 0.01 nmol of dihydrolipoate and 0.05 nmol of lipoate. Baseline separation of lipoate and dihydrolipoate is achieved on a 10 cm octadecyl column. The analysis is rapid (8 min/sample) and uses a single HPLC pump with isocratic mobile phase. The method has been adapted to study cellular and whole animal reduction of lipoate, membrane transport of lipoate and dihydrolipoate, and subcellular enzymes that reduce lipoate.