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

Abstract 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‐µm (i.d.) by 32.5‐cm (eff. length) silica capillary. The background electrolyte was 0.075‐M citrate Na pH 5.8 with 200‐µM cetyltrimethylammonium bromide and 5‐µM sodium dodecyl sulfate, and the separation voltage was −14 kV. The quantification limit (S/N = 15) of the method was 1.5 µM 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 µM, and 256 ± 94, respectively. Thus, the presented approach can be used in clinical and laboratory practice.