Determination of glutathione in blood via capillary electrophoresis with pH-mediated stacking |
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Authors: | Alexander Vladimirovich Ivanov Mikhail Aleksandrovich Popov Valery Vasil'evich Aleksandrin Lubov Mikhailovna Kozhevnikova Alexey Aleksandrovich Moskovtsev Maria Petrovna Kruglova Silina Ekaterina Vladimirovna Stupin Victor Aleksandrovich Aslan Amirkhanovich Kubatiev |
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Institution: | 1. Department of Molecular and Cell Pathophysiology, Institute of General Pathology and Pathophysiology, Moscow, Russia;2. Department of Cardiac Surgery, M.F. Vladimirsky Moscow Regional Research and Clinical Institute, Moscow, Russia;3. Department of Human Pathology, I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University), Moscow, Russia;4. Department of Hospital Surgery №1, Pirogov Russian National Research Medical University (RNRMU), Moscow, Russia |
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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 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. |
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Keywords: | blood capillary electrophoresis glutathione stacking |
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