Liquid chromatographic determination of polythiols based on pre-column excimer fluorescence derivatization and its application to α-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, α-lipoic acid (LA) and α-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.