Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection

The procedure for measurement of different forms of four plasma thiols cysteine, cysteinylglycine, glutathione and homocysteine is proposed. The analytes are derivatized with thiol-specific ultraviolet labeling reagent, 2-chloro-1-methylquinolinium tetrafluoroborate, and separated from each other, reagent excess and plasma matrix constituents by reversed-phase high performance liquid chromatography with detection at 355 nm. Oxidized forms are converted to their thiol counterparts by reductive cleavage with sodium borohydride prior to derivatization step. In order to circumvent the loss of reduced fraction of thiols due to oxidation during sample preparation, the derivatization reagent is added to whole blood immediately after collection and before separation of plasma from erythrocytes. The method measures total thiols, total free thiols, protein-bound thiols and reduced thiols. The method is linear within the physiological and pathological ranges of thiols and is applied for plasma samples donated by apparently healthy volunteers.