Determination of biologically active low-molecular-mass thiols in human blood I. Fast qualitative and quantitative, gradient and isocratic reversed-phase high-performance liquid chromatography with photometric and fluorescence detection

The fast isocratic and gradient reversed-phase high-performance liquid chro matographic methods employing photometric and/or fluorescence detection are described for the precise reproducible simultaneous measurement of total h omocysteine, cysteine, and glutathione in human blood. Sample preparation i nvolves conversion of disulfides to free thiols with triphenylphosphine, pr ecipitation of proteins with sulfosalicylic acid, and conjugation of thiols with monobromobimane. The aminothiol assay is optimized by reduction and d erivatization step conditions (pH, temperature and time of reactions), as w ell as by chromatographic conditions to obtain reliable quantitative result s within the concentration range corresponding to the levels of these thiol s in human blood in norm and pathology. Its sensitivity allows the detectio n of aminothiol quantities >2 pmol. (C) 2000 Elsevier Science B.V. All righ ts reserved.