Liquid chromatographic determination of polythiols based on pre-column excimer fluorescence derivatization and its application to alpha-lipoic acid analysis.

We developed an LC method for the sensitive and selective fluorometric determination of polythiols. This method employs pre-column intramolecular excimer-forming fluorescence derivatization with N-(1-pyrene)iodoacetamide followed by LC separation. Polythiols were converted to the corresponding dipyrene-labeled derivatives, and the derivatives afforded intramolecular excimer fluorescence (440-540 nm). After the optimization using dithiothreitol and dimercaprol as model polythiols, alpha-lipoic acid (LA) and alpha-lipoamide were determined with high sensitivity and selectivity. The detection limits for polythiols were 0.6-3.5 fmol on column. Furthermore, this method could be successfully applied to the determination of LA in commercial dietary supplements and in human urine.