Determination of reduced and oxidized homocysteine and related thiols in plasma by thiol-specific pre-column derivatization and capillary electrophoresis with laser-induced fluorescence detection

A new sensitive and rapid capillary electrophoresis (CE) assay for measurin g reduced and oxidized thiols in human plasma has been developed. To preven t oxidation of the thiols, whole blood was immediately centrifuged after co llection and the plasma proteins were precipitated with perchloric acid. Th e reduced thiols in the supernatant were derivatized quantitatively at 25 d egrees C, pH 7.5 with a fluorescent reagent, fluorescein-5-maleimide (FM). The total plasma concentration of thiols, including the fraction coupled to proteins, was assayed after an initial reduction of the disulfide linkage in plasma with dithiothreitol. The separation of FM-thiols was performed in an acetonitrile/10 mM sodium phosphate-50 mM SDS buffer [25:75 (v/v); pH 7 .0] using a fused-silica capillary (57 cmX75 mu m I.D.) at 45 degrees C. A 3-mW argon-ion laser (lambda(ex) 488 nm/lambda(em) 520 nm) was employed for FM-thiol detection. With the electric field of 530 V/cm, the time needed f or the separation of FM-homocysteine, FM-glutathione and FM-N-acetylcystein e was less than 8 min. The lower Limit of detection was 3 mu M for the tota l thiols and 10 nM for the reduced thiols. The method was applied to the de termination of homocysteine levels in plasma from patients with end-stage r enal disease. (C) 1999 Elsevier Science B.V. All rights reserved.