Improved method for the determination of N-acetylcysteine in human plasma by high-performance liquid chromatography

An improved method for the determination of N-acetylcysteine by paired-ion reversed-phase high-performance liquid chromatography has been developed. Following incubation with dithiothreitol to release bound N-acetylcysteine, free N-acetylcysteine and the internal standard N-acetylpenicillamine were derivatised with 2,4-dinitro-1-fluorobenzene. The samples were then deproteinised by ultrafiltration. The dinitrophenyl derivatives were extracted from acidified ultrafiltrate into diethyl ether and purified by using a back-extraction step. They were then separated from naturally occurring plasma components and reagent impurities by high-performance liquid chromatography, utilising an Ultrasphere ODS (5 microns) reversed-phase column and detection at 360 nm.