Determination of biologically active low-molecular-mass thiols in human blood III. Highly sensitive narrow-bore isocratic reversed-phase highperformance liquid chromatography with fluorescence detection

The fast isocratic narrow-bore reversed-phase high-performance liquid chrom atographic method employing fluorescence detection is described for the pre cise reproducible simultaneous measurement of total homocysteine, cysteine and glutathione in human blood. Sample preparation involves conversion of d isulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of thiols with monobromobimane. Optimized sample preparation conditions as well as chromatographic conditio ns allowed to obtain reliable quantitative results within the concentration range corresponding to the levels of these thiols in human blood in norm a nd pathology. The detection limit was similar to 70 amol for all labeled am inothiols. The proposed method for these compounds analysis includes simple sample preparation, high selectivity, good linearity (r(2)>0.998), high re producibility (within-run precision for derivatized aminothiol peaks area R SD<1.8% for three times consequently injected sample); high reliability and the small sample volume (1 <mu>l) required for analysis make it suitable f or clinical studies. (C) 2001 Elsevier Science B.V, All rights reserved.