Determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7-tetramethyl-8-bromomethyl-difluoroboradiaza-s-indacene

Altered levels of thiols in biological fluids are considered to be an important indicator for several diseases. In this article, 1,3,5,7-tetramethyl-8-bromomethyl-difluoroboradiaza-s-indacene is proposed as a fluorescent derivatization reagent for the determination of thiols including glutathione, cysteine, N-acetylcysteine, and homocysteine by HPLC. Under the optimized derivatization and separation conditions, a baseline separation of all the four derivatives has been achieved using isocratic elution on an RP C-8 column within 26 min. With fluorescence detection at 505 and 525 nm for the excitation and emission, respectively, the LODs (S/N = 3) are from 0.2 nM (glutathione) to 0.8 nM (cysteine). The feasibility of this method in real samples has been evaluated by the determination of thiols in human plasma from the healthy persons and hypertensive patients with recoveries of 92-105.3%.