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 K3EDTA 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.