High-performance liquid chromatography assay for N-acetylcysteine in biological samples following derivatization with N-(1-pyrenyl)maleimide.
N-Acetylcysteine is a thiol antioxidant with expanding clinical importance. A sensitive, rapid method for determining reduced N-acetylcysteine (NAC) concentration in biological samples has been developed which uses a modified reversed-phase high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl)maleimide (NPM). The NAC-NPM adduct was analyzed by HPLC with fluorescence detection. The calibration curve for NAC was linear over the range 8-2500 nM and the coefficient of variation obtained for the within-run precision and the between-run precision for 0.5 mM NAC was 1.5% and 2.7%, respectively. Relative recovery of NAC from biological materials ranged between 86% and 96% and the limit of quantitation from biological samples was 32 nM. These results suggest practical advantages relative to other widely-accepted methods of NAC measurement.