14 BODOR, NEČASOVÁ, PECHOVÁ AND MASÁR Table 1: Electrolyte systems. Electrolyte Parameter ES1 ES2 LE1 Leading ion Chloride Concentration [mmol/l] 10 10 Counter ion 6-Aminocaproate Concentration [mmol/l] 12 12 EOF supressor Methylhydroxy- ethylcellulose Concentration [% w/V] 0.1 0.1 pH 3.4 3.4 LE2 Leading ion Chloride Concentration [mmol/l] 5 2 Counter ion 6-Aminocaproate Concentration [mmol/l] 6 2.4 EOF supressor Methylhydroxy- ethylcellulose Concentration [% w/V] 0.1 0.1 pH 3.8 3.9 TE Terminating ion Caproate Concentration [mmol/l] 20 20 Counter ion 6-Aminocaproate Concentration [mmol/l] 15 15 EOF supressor Methylhydroxy- ethylcellulose Concentration [% w/V] 0.1 0.1 pH 4.7 4.7 using built-in peristaltic pumps. Between analyses a rela- tively short rinsing procedure (ca. 1 min) with electrolyte solutions was used. 2.1 Samples The bovine blood samples were collected using PVC tak- ing set with an integrated needle HEMOS (Gama Group, České Budějovice, Czech Republic), and immediately transferred to the test tube containing K3-EDTA. The samples after dilution, required for hemolysis, were fil- tered prior to the analysis using a syringe filter with a glass fiber membrane and a pore size of 1 µm. During the dilution step a thiol-masking agent and NaOH were added to each sample. 3. Results and Discussion 3.1 Separation conditions The combination of two columns with different IDs and the employment of a column-switching technique is ben- eficial for the CITP determination of analytes present in the multicomponent sample at low concentrations and/or at different concentration levels. The first (wider) cap- illary allowed the separation of sample constituents in- jected at a relatively high volume (37 µl). Typical macro- constituents, e.g. chloride and ethylenediaminetetraacetic acid (EDTA), migrated out of the separation path through Figure 1: A scheme of the electrophoresis system. Au- tosampler: S – sample loop, SP – syringe pump, 6wV – 6-way valve. Interface: during the injection the septums are pierced by the needles (N). Interface*: during the sep- aration the autosampler is disconnected from the analyzer. Separation and electrolyte unit: V1-V4 – pinch valves, E1- E3 – driving electrodes, P1-P3 – peristaltic pumps, IM – injection module, W – waste, D1-CD, D2-CD, D2-UV – contactless conductivity and absorbance detection cells, M – membrane, BF – bifurcation, TE, LE1, LE2 – ter- minating and leading electrolytes. a bifurcation block, and as a result they were removed from the separation compartment. During this stage of the separation the driving current flowed between electrodes E1 and E3 (Fig. 1). The very small isotachophoresis (ITP) zones of ana- lytes created in the first column were insufficient for their concentrations to be determined. As a result of switching the direction of the driving current through both columns (by connecting electrodes E2 and E3, Fig. 1), the sepa- rated constituents were transferred to the second column. The signal from D1-CD was used to determine an appro- priate time to switch the current (Fig. 1). In the second (narrower) capillary the ITP zones of analytes were prolonged. In addition, due to the low GSSG concentration in the samples of blood (not in ex- cess of tens of µmol/l), their ITP zone length was fur- ther extended by a reduction in the concentration of lead- ing ions (ES2, Table 1) in the second column (Fig. 2). A higher degree of sensitivity in ES2 is also evident from the parameters of regression equations for the analytes (Table 2). Hungarian Journal of Industry and Chemistry CAPILLARY ISOTACHOPHORESIS OF GLUTATHIONE 15 Figure 2: Isotachopherograms from the separations of GSSG and GSH performed in the electrolyte systems ES1 (a) and ES2 (b). The isotachopherograms were recorded by D2-CD (Fig. 1). The concentrations of GSSG and GSH in the injected sample were both 25 µmol/l. 3.2 Stabilization of glutathione The stability of GSH and its oxidation to GSSG during the period between the collection of the sample and its analysis is the main source of systematic errors. In the sample of bovine blood a much higher concentration of GSSG and a lower concentration of GSH than expected was measured. To avoid this problem, a thiol-masking agent was used, namely iodoacetic acid (IA) [18]. The sub- stitution reaction between IA and GSH formed S- (carboxymethyl)glutathione (GS-MC). Under the ITP separation conditions used, GS-MC migrated in front of GSSG (Fig. 3). The optimum con- ditions for the reaction between IA and GSH were de- termined by the ITP separations of reaction mixtures at different periods after the reagents were mixed. These ex- periments were conducted with both model and real sam- ples. Under neutral and slightly alkaline conditions the reaction was very slow. An excess of IA (2 mmol/l) and the presence of NaOH (4 mmol/l) in the reaction mix- ture led to the fast (less than 20 mins.) and quantitative conversion of GSH to GS-MC (Figs. 3 and 4) without an Table 2: Parameters of regression equations. Analyte Range a b R 2 [µmol/l] [s l/µmol] [s] GSSG1 5-50 0.67 1.45 0.999 7 GSSG2 2-25 1.66 2.01 0.9992 GSH1 10-50 0.48 0.25 0.9999 GSH2 10-50 1.24 0.60 0.9994 GSH (GS-MC)2 40-120 1.98 -6.60 0.9969 1 Electrolyte system ES1 and 2 ES2 used for data evaluation. Regres- sion equation: Y = aX + b. Figure 3: Isotachopherograms from the separation of the reaction mixture present in the electrolyte system ES2. The isotachopherograms were recorded by D2–CD. The mixture contained 15 µmol/l of GSSG, 80 µmol/l of GSH, 2 mmol/l of IA, 4 mmol/l of NaOH, and 10 % of TE. The sample was injected 5 mins (dot), 15 mins (dash) and 20 mins (solid) after the reagents were mixed. increase in the concentration of GSSG. For the purpose of a quantitative analysis, IA and NaOH was added to the sample of bovine blood immedi- ately after its collection (during the sample dilution step required for hemolysis). Blood samples that had been di- luted by a factor of ten were directly analyzed after their filtration through a syringe filter with a pore size of 1 µm. The concentrations of GSSG and GSH that were measured in the diluted blood samples were 4.4 µmol/l and 63.4 µmol/l, respectively. The average concentrations Figure 4: Dependence of the ITP zone length on the reac- tion time of the mixture containing 15 µmol/l GSSG, 80 µmol/l GSH, 2 mmol/l IA, 4 mmol/l NaOH and 10 % TE. 46(1) pp. 13-17 (2018) 16 BODOR, NEČASOVÁ, PECHOVÁ AND MASÁR calculated from eight repetitive measurements of iden- tical samples were in good agreement with those deter- mined by enzymatic methods. The degrees of precision of the method, expressed by the relative standard deviation (RSD) values of the measured concentrations of GSSG and GSH, were 10.3% and 4.4%, respectively. 4. Conclusion The sensitive and simultaneous determination of GSH and GSSG concentrations in entire samples of bovine blood is facilitated by the capillary isotachophoretic method developed. The simple and rapid preparation of blood samples, that only involves the masking of thiol group of GSH and the dilution and filtration of the sam- ple, increases the accuracy of the GSSG concentration measured. 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