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 chromatographic methods employing photometric and/or fluorescence detection are described for the precise reproducible simultaneous measurement of total homocysteine, cysteine, and glutathione in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of thiols with monobromobimane. The aminothiol assay is optimized by reduction and derivatization step conditions (pH, temperature and time of reactions), as well as by chromatographic conditions to obtain reliable quantitative results within the concentration range corresponding to the levels of these thiols in human blood in norm and pathology. Its sensitivity allows the detection of aminothiol quantities >2 pmol.