Determination of reduced, protein-unbound, and total concentrations of N-acetyl-L-cysteine and L-cysteine in rat plasma by postcolumn ligand substitution high-performance liquid chromatography

A high-performance liquid chromatographic assay was developed for the quant itative determination of the sulfur-containing amino acids N-acetyl-L-cyste ine (NAC) and L-cysteine (Cys) in rat plasma. The thiols were separated by reverse-phase ion-pair chromatography, and the column eluent was continuous ly mixed with an iodoplatinate-containing solution. The substitution of sul fur of the thiol compound with iodide was quantitatively determined by meas uring changes in the absorption at 500 nm. The low-molecular-weight disulfi des and mixed disulfide conjugates of thiols with proteins were entirely re duced to the original reduced compounds by dithiothreitol. By reducing thes e two types of disulfides separately during sample pretreatment, the reduce d, protein-unbound, and total thiol concentrations could also be determined . Validation testing was performed, and no problems were encountered. The l imit of detection was approximately 20 pmol of thiol on the column. The pre sent method was used to measure the plasma concentrations of NAC and Cys in the rat after a bolus intravenous administration of NAG, focusing on disul fide formation. The binding of NAC to protein through mixed disulfide forma tion proceeds in a time-dependent and reversible manner. Moreover, this "st able" covalent binding might limit total drug elimination, while the unboun d NAC is rapidly eliminated. Consequently, the analytical method described in this study is very useful for the determination of plasma NAC and Cys, i ncluding disulfide conjugates derived from them. (C) 2001 Academic Press.