Determination of biologically active low-molecular-mass thiols in human blood II. High-performance capillary electrophoresis with photometric detection

A new high-performance capillary electrophoresis assay for aminothiols in h uman blood, including homocysteine, a marker of several human metabolism di sorders, has been developed. Sample preparation involves conversion of disu lfides to free thiols with triphenylphosphine, precipitation of proteins wi th sulfosalicylic acid, and conjugation of the thiols with monobromobimane. Derivatized thiols were separated in a sodium phosphate buffer using a fus ed-silica capillary (65 cmx50 mum I.D.) at 30 degreesC. With the electric f ield of 250 V cm(-1), separation of homocysteine, glutathione and cysteine occurred at less than 10 min. Detection at 250 or 234 nm was used to confir m the monobimane-thiols peaks. The detection Limit was similar to5 nmol/ml for all labeled aminothiols. The proposed method for these compounds' analy sis included simple sample preparation, high selectivity, good linearity (r (2)>0.999), high reproducibility (within-run precision for derivatized amin othiol peaks area RSD<5% for three times consequently injected sample); hig h reliability and the small volumes required for analysis made it suitable for clinical studies. (C) 2000 Elsevier Science B.V. All rights reserved.