44 TROŠKA, MANDŽÁKOVÁ, HRADSKI, ŠEVČÍK AND MASÁR Figure 1: A scheme of the CC microchip with conduc- tivity detection. C-LE = the first (ITP) separation chan- nel (volume of 4.5 µL) filled with a leading electrolyte; C-BE = the second (CZE) separation channel (volume of 4.3 µL) filled with a background electrolyte; C-TE = the third channel (volume of 0.8 µL) filled with a terminat- ing electrolyte; C-S = sample injection channel (volume of 9.9 µL); D1, D2 = Pt conductivity sensors; BF = bi- furcation section; BE, LE, TE, S = inlets for the back- ground, leading, terminating and sample solutions to the microchip channels, respectively; W = an outlet channel for the waste container. CC technology employed on the microchip with contact conductivity detection enables online capillary zone elec- trophoresis (CZE) to be coupled with the isotachophore- sis (ITP) sample pretreatment. Samples of urine and saliva were simplified by solid-phase microextraction (SPME) prior to the ITP-CZE separations on the mi- crochip. SPME based on silver- and barium-form resins provided a high degree of compatibility with MCE and a high level of selectivity, whilst simultaneously remov- ing a huge amount of chloride and sulfate in the analyzed samples of body fluids. 2. Experimental ITP-CZE separations were carried out on a poly(methyl methacrylate) microchip (Fig.1) with the CC tech- nique and integrated conductivity detection sensors (IonChipTM 3.0, Merck, Darmstadt, Germany). Chemicals used for the preparation of electrolyte solutions and model samples were obtained from Sigma-Aldrich (Bratislava, Slovakia), Fluka Chemika- Biochemika (Buchs, Switzerland), Serva Electrophoresis GmbH (Heidelberg, Germany) and Erba Lachema s.r.o. (Brno, Czech Republic). Samples of urine and saliva were collected from volunteers. Before the analysis, the sam- ples were homogenized and analyzed after being diluted appropriately with deionized water, and pretreated by SPME to remove chloride and sulfate. Solid-phase ex- traction (SPE) microcolumns of 0.5 mL in volume con- taining silver- and barium-form resins (Alltech, Grace Davison Discovery Sciences, Deerfield, Illinois, USA) were used for this purpose. 3. Results and Discussion ITP-CZE separations were performed in a hydrodynami- cally closed separation system with suppressed electroos- Figure 2: Electropherogram from the ITP-CZE separation of Hcy in the presence of discrete spacers. The sample in- jected onto the microchip: 100 µmol/L Apm, 15 µmol/L Hcy, 150 µmol/L Thr in a 50 % terminating electrolyte. Apm = aminopimelate; Hcy = homocysteine; Thr = threo- nine; G = conductivity. motic flow. These working conditions effectively reduce the fluctuations in the total migration velocity of the ana- lytes which is crucial to obtain reproducible results, espe- cially on the microchip with short separation paths [21]. In the ITP separations, a leading electrolyte with a pH of 9.1 and glycine as a terminating electrolyte were used. In the CZE step of the ITP-CZE technique, the pH of the background electrolyte was 9.8. The ITP realized in the first separation channel on the microchip (Fig. 1) preconcentrates the analyte and other constituents of the sample for the CZE separation step. On the other hand, the close migration configuration of the constituents in the ITP stage of the ITP-CZE tech- nique can be a limiting factor for achieving the required resolution of the analyte from matrix constituents, espe- cially on the microchip with short separation channels. In such a case, discrete spacers (DSs) are effectively used to define the fraction of the sample transferred to the second CZE channel when the CC technique is employed. Then, the constituents of the sample migrating outside of the mobility interval defined by the DSs are removed from the separation system. In this way, ITP works as an on- line sample clean-up technique prior to the CZE. In our case aminopimelate and threonine were used as the front and rear DSs, and the undesirable constituents of the sam- ple were electrophoretically removed from the separation system by column switching prior to the CZE separation realized in the second channel (Fig. 2). A 1.4 µmol/L limit of detection (LOD) for Hcy was obtained by the ITP-CZE technique on the microchip. A relatively large volume of the sample that was in- jected onto the microchip (9.9 µl) contributed to achiev- ing the low value of LOD when a universal and relatively low-sensitive conductivity detector was used. Under the working and separation conditions employed, the repro- ducible migration velocities (the relative standard devi- Hungarian Journal of Industry and Chemistry DETERMINATION OF HOMOCYSTEINE BY MICROCHIP ELECTROPHORESIS 45 Figure 3: Electropherograms from ITP-CZE analyses of urine samples after SPME pretreatment with various ad- ditions of Hcy. Sample injected on the microchip: urine diluted twice, 400 µmol/L Apm, 200 µmol/L Thr in 50 % terminating electrolyte with (a) 0; (b) 10; (c) 20; (d) 40 µmol/L Hcy. Apm = aminopimelate; Hcy = homocysteine; Thr = threonine; imp. = impurity; G = conductivity. ations (RSDs) of migration times were between 0.5 and 1.2 %) and determinations of trace concentrations of Hcy (RSDs of peak areas were 1.2 %) were achieved. Considering the high concentration levels of chloride and sulfate in urine and saliva samples, these interfer- ing anions were removed before ITP-CZE analysis from real samples. 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