Determination of glutathione in blood via capillary electrophoresis with pH-mediated stacking

A new approach has been developed for the direct determination of reduced (glutathione [GSH]) and oxidized (glutathione disulfide [GSSG]) GSH in whole blood by means of capillary electrophoresis. Its features include GSH-stabilizing sample preparation, the use of an internal standard, and pH-mediated stacking. Blood stabilized with acid citrate and K(3)EDTA was treated with acetonitrile with N-ethylmaleimide, and then the analytes were extracted with diethyl ether. The total analysis time was 8 min using a 50-mu m (i.d.) and 32.5-cm (eff. length) silica capillary. The background electrolyte was 0.075-M citrate Na pH 5.8 with 200-mu M cetyltrimethylammonium bromide and 5-mu M sodium dodecyl sulfate, and the separation voltage was -14 kV. The quantification limit (S/N = 15) of the method was 1.5 mu M for GSSG. The accuracy levels of GSH and GSSG analysis were 104% and 103%, respectively, and between-run precision levels were 2.6% and 3.2%, respectively. Analysis of blood samples from healthy volunteers (N = 24) showed that the levels of GSH and GSSG and the GSH/GSSG ratio in the whole blood were 1.05 +/- 0.14 mM, 3.9 +/- 1.25 mu M, and 256 +/- 94, respectively. Thus, the presented approach can be used in clinical and laboratory practice.