Reversed-phase liquid chromatography method for the determination of total plasma thiols after derivatization with 1-benzyl-2-chloropyridinium bromide.

A high-performance liquid chromatography method was developed for simultaneous detection and quantitation of total cysteine, glutathione, homocysteine and cysteinylglycine in human plasma. The two key steps in the analysis are reduction of disulfides and treatment with 1-benzyl-2-chloropyridinium bromide, which rapidly and quantitatively reacts with thiol groups to form stable S-pyridinium derivatives with intense UV absorption. The derivatives are well separated on a Zorbax SB C(18) column using reversed-phase high-performance liquid chromatography and monitored at 315 nm. The calibration graphs were linear over concentration ranges covering most experimental and clinical cases with a regression coefficients better than 0.999. The detection and quantitation limits for all analytes were 0.2 and 0.5 micromol/L, respectively. The recoveries were 99.25-101.68%. The intra- and interassay imprecisions were 0.88-4.24 and 1.68-5.14%, respectively. The method was applied for plasma samples donated by apparently healthy volunteers.