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 human blood, including homocysteine, a marker of several human metabolism disorders, has been developed. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of the thiols with monobromobimane. Derivatized thiols were separated in a sodium phosphate buffer using a fused-silica capillary (65 cm×50 μm I.D.) at 30°C. With the electric field 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 confirm the monobimane–thiols peaks. The detection limit was ∼5 nmol/ml for all labeled aminothiols. The proposed method for these compounds’ analysis included simple sample preparation, high selectivity, good linearity (r2>0.999), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<5% for three times consequently injected sample); high reliability and the small volumes required for analysis made it suitable for clinical studies.