Methodologies for the analysis of reduced and oxidized N-acetylcysteine in biological systems

A rapid and sensitive assay is described for both reduced and oxidized N-acetylcysteine in biological matrices. Free, reduced N-acetylcysteine is derivatized, along with any endogenous free thiols, in situ by treatment of the samples with the membrane-permeable, thiol-reactive agent monobromobimane. The N-acetylcysteine-monobromobimane adduct thus formed is analysed by high performance liquid chromatography with fluorescence detection. Oxidized N-acetylcysteine is released from disulfides by in situ treatment of the samples with dithiothreitol, rendering the total N-acetylcysteine content of the system available for derivatization and analysis. The conditions of derivatization ensured 100 per cent recovery of N-acetylcysteine as the monobromobimane adduct, and calibration curves were linear over the range 0.1 microM to 1.0 mM N-acetylcysteine. The precision of the assay procedures was 97 per cent over this range. These assay procedures have been applied to studies of the pharmacokinetics of N-acetylcysteine following single oral and intravenous administrations of the drug to a single human volunteer.