Determination of N-Acetylcysteine and Main Endogenous Thiols in Human Plasma by HPLC with Ultraviolet Detection in the Form of Their S-Quinolinium Derivatives

A new, sensitive, repeatable, and robust high performance liquid chromatography assay for the determination of total N-acetylcysteine, cysteine, cysteinylglycine, glutathione, and homocysteine in human plasma has been developed. The thiol concentrations were measured using precolumn specific derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate, followed by high performance liquid chromatographic reversed phase separation with spectrophotometric detection. The oxidized and protein bound forms of thiols were converted into their reduced forms employing a reductive agent tris(2-carboxyethyl) phosphine. The chromatographic separation was accomplished in 12.5 min; the within run and between run imprecision were all less than 10%. The elution profile was as follows: 0-4 min, 11% B; 4-8 min, 11-30% B; 8-12 min, 30-11% B. The calibration graphs, obtained with the use of normal plasma spiked with growing amounts of analytes, were linear over the concentration ranges, covering most experimental and clinical cases (1-32 mu M for homocysteine and glutathione, 1-320 mu M for N-acetylcysteine and cysteine, and 1-64 mu M for cysteinylglycine). For all analytes, recoveries between 91.2 and 108.6%, were observed.