Determination of cysteine, glutathione and N-acetylcysteine in plasma by ion-pair reversed-phase liquid chromatography and post-column derivatization.

The thiol and oxidized forms of cysteine, glutathione and N-acetylcysteine in plasma were determined by ion-pair liquid chromatography and post-column derivatization. The thiol forms were measured after direct injection of deproteinized plasma. The oxidized forms, present either as a dimer or oxidized with other small thiols, were assayed in deproteinized plasma after reduction with dithiothreitol. The total amounts, including the fraction bound to plasma proteins via disulphide bonds, were determined after reductive cleavage in plasma with dithiothreitol. The compounds were separated by ion-pair liquid chromatography on a reversed-phase column (C18) and were detected by fluorimetry after post-column derivatization. The endogenous plasma levels of all forms of cysteine, glutathione and N-acetylcysteine, except for the thiol form of N-acetylcysteine, were above the quantification limits. The quantification limit of N-acetylcysteine as a thiol, was 0.15 microM. The precision was better than 12% for the endogenous concentrations.