Transient pseudo-isotachophoretic stacking in analysis of plasma for homocysteine by capillary zone electrophoresis

An analytical procedure enabling routine analysis of human plasma for total homocysteine has been developed and validated. The method includes reduction of homocysteine disulfides to thiol with tris 2-carboxyethylphosphine, derivatization of the thiol with 2-chloro-1-methylquinolinium tetrafluoroborate, separation of homocysteine 2-S-quinolinium derivative from those of plasma endogenous and exogenous thiol derivatives by capillary zone electrophoresis, and quantitation with the use of ultraviolet detection based on acetonitrile stacking. Method performance characteristics, for example recovery, calibration, precision, limit of detection, and limit of quantitation, are presented. The procedure was applied to analysis of plasma samples donated by apparently healthy volunteers.     

Nonaqueous capillary electrophoresis of imatinib mesylate and related substances

In the present study, nonaqueous capillary electrophoretic separation of imatinib mesylate (IM) and related substances, N-(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-pyrimidinamine (PYA), N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-((piperazin-1-yl)methyl) benzamide (NDI) and 4-chloromethyl-N-(4-methyl-3-((4-(pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) benzamide (CPB) was developed. The influential factors affecting separation, including type and concentration of the electrolyte, applied voltage, and buffer modifier were investigated. Baseline separation of the studied analytes was obtained using a buffer of 50 mM Tris and 50 mM methanesulfonic acid in methanol at a apparent pH (pH*) of 1.65. To enhance the sensitivity, large-volume sample stacking was employed for online concentration. The strongest analytical signal with a suitable separation was achieved when the injection time was 100 s. The linearity ranges of PYA and NDI were 0.100-2.50 μg mL(-1) , and that of CPB was 0.125-2.50 μg mL(-1) , with good coefficients (r(2) > 0.9948). The relative standard deviations of intra- and interday were satisfactory. Under the optimized conditions, seven batches of the synthesized samples were analyzed and CPB was detected in two batches. Owing to its simplicity, effectiveness, and low price, the developed method is promising for quality control of IM.     

Direct analysis of inorganic anions in samples with high salt content by capillary zone electrophoresis

Trace-level inorganic anions in seawater are separated efficiently by capillary zone electrophoresis using direct UV detection. The carrier electrolyte is 50 mM borate at pH 9.3 and contained 1.5M NaCl. This buffer solution is adopted to prevent interference from high concentrations of the chloride ion in seawater. No electro-osmotic flow reverser is used to shorten the analysis time. The experimental conditions such as the concentration of NaCl in the carrier electrolyte, capillary inner diameter, applied current, and temperature are optimized. Linear plots are obtained in the concentration range of 0.1 to 20 microg/mL. The quantitation limits of the anions are in the order of 0.02 to 0.1 microg/mL. The proposed method may be applicable to the determination of inorganic anions in other environmental samples and effluents of a power plant.     

Determination of sodium tripolyphosphate in meat samples by capillary zone electrophoresis with on-line isotachophoretic sample pre-treatment

The usefulness of zone capillary electrophoresis (CZE) in combination with isotachophoresis (cITP) as on-line preconcentration technique was examined for analysis of tripolyphosphate (STPP) in meat and meat products. The mean concentrations of STPP in different types of meat products varied from 39 mg P₂O₅/100 g to 219 mg P₂O₅/100 g, these values are below the legal requirements. The detection (LOD) and quantification (LOQ) limits for STPP in extracted solutions were 0.80 mg P₂O₅/dm³ and 2.69 mg P₂O₅/dm³, respectively. Obtained results were compared with the Kjeldahl method. Accuracy (97.4-98.3%) was determined using recovery assay based on standard additions method. Precision was evaluated by within-day R.S.D. (1.40-2.19%), between-days R.S.D. (3.00-3.82%) and demonstrates the benefit of using this procedure for the routine analysis of STPP in meat and their products. The F-Snedecor test was employed to compare the precision of the used methods and calculated F-test values (4.00, 6.13) were less than the theoretical (6.39).     

Analysis of ephedrine in ephedra callus by acetonitrile modified capillary zone electrophoresis

A simple method has been developed for the quantitative determination of ephedrine in ephedra callus. The dependence of effective mobility of ephedrine on pH was investigated, and a simulated equation was obtained. The separation was performed in an uncoated capillary and detected at 185 nm. A new Tris–NaOH–H₃PO₄ run buffer was used and the pH was adjusted to 3.20. To increase the solubility of hydrophobic analytes and improve the separation efficiency, 15% acetonitrile was used in the buffer as a modifier. The content of ephedrine in an ephedra callus sample and an ephedra herba sample were determined with this method, and the result was satisfactory.     

Strategies for the determination of cefazolin in plasma and microdialysis samples by short-end capillary zone electrophoresis.

Capillary zone electrophoresis was employed to determine cefazolin, a first-generation cephalosporin antibiotic, in plasma and microdialysis samples from patients. To shorten the analysis, the samples were injected from the short end of the capillary, resulting in a separation time of < 3 min. Due to a high ionic strength of the biological matrices it was necessary to optimize the stacking conditions. For microdialysis samples a 1:10 dilution with water before injection was sufficient to obtain good peak shape. For plasma samples a protein removal step was required to obtain clean electropherograms and a good peak shape. Acetonitrile was used as precipitant resulting in an enhanced sample stacking in comparison to water dilution. The disadvantage of using acetonitrile was severe evaporation loss making quantitation impossible. A self-sealing film was used to seal each individual sample vial to suppress evaporation during long-term sequences. The calibration curves for spiked plasma and cefazolin in Ringer's solutions were linear in the range from 2-500 and 2.5-100 microg/mL, respectively. Limits of detection were 1.0 and 2.0 microg/mL in plasma and microdialysis samples, respectively. The assay was successfully applied to plasma and microdialysis samples obtained in vivo from the interstial space fluid of subcutaneous adipose and muscle tissue of patients undergoing cardiac surgery.     

Investigation of the factors that induce analyte peak splitting in capillary electrophoresis

Peak splitting has a detrimental effect on analyses by capillary electrophoresis. Many papers have reported it and several mechanisms have been proposed to explain the phenomenon. We investigated the electrophoretic behavior of an amphoteric analyte, levodopa, in phosphate buffer and observed a peak splitting phenomenon at moderate sample concentrations and under general analytical conditions, even without organic solvent. The dependence of effective mobility on pH was taken into account and pKa values of 2.30, 8.11, and 9.92 were obtained for levodopa. Then, we constructed pH-dependent distribution diagrams of levodopa and phosphate species present in aqueous solution and proposed that the most relevant factors contributing to peak splitting are the presence of ionizable groups in the analyte molecule and the occurrence of ionization, yielding charged species which interacted with buffer electrolyte species in a definite pH range to form complexes. This result is different from those presented in the literature and broadens our understanding of amphoteric analyte peak splitting.     

Elimination of the artefact peaks in capillary electrophoresis determination of glutamate by using organic solvents in sample preparation

Focusing on the demand from the food industry for fast and reliable alternative methods to control the quality of food products, we present in this paper a method for amino acid separation and glutamic acid quantification in complex matrices employing capillary electrophoresis with capacitively coupled contactless conductivity detection. We demonstrate by simulation and experimentally the use of organic solvents in sample preparation to prevent peak splitting and increase stacking in capillary electrophoretic separations of amino acids. Additionally, we obtained results for glutamic acid quantification comparable to those obtained via traditional methods used at industrial sites. We tested premium and low‐cost samples with large variations in their glutamic acid content, which demonstrated the wide range of applicability of the method presented herein. The results of the proposed capacitively coupled contactless conductivity detection based capillary electrophoresis method agreed with those obtained by an enzymatic detector and ultra high performance liquid chromatography coupled to tandem mass spectrometry, considering a confidence level of 95%.     

Determination of purines and pyrimidines in beer samples by capillary zone electrophoresis

Ten purine and pyrimidine bases were separated using capillary zone electrophoresis (CZE) with direct UV detection at 254 nm as well as mass spectrometric (MS) detection using an electrospray ionization (ESI) interface. For this purpose a carrier electrolyte composition compatible with both methods of detection containing 300 mM diethylamine (DEA) was selected. Limits of detection were in the range between 0.1 and 0.3 mg l⁻¹ and calibration plots were found to be linear over at least two orders of magnitude. The applicability of the developed method for the analysis of real samples was demonstrated for some beer samples. A series of “Lager” beer samples from different breweries in Europe as well as a number of completely different types of beers were investigated with respect to their content in the selected purine and pyrimidine bases using the developed CE method with UV detection at 254 nm.     

Determination of the beta-blocker atenolol in plasma by capillary zone electrophoresis

A capillary zone electrophoretic method was optimised for the determination of the beta-blocker atenolol in plasma. Separation was performed in an uncoated silica capillary of 58.5 cm (effective length 50 cm) x 75 microm I.D., and detection was at 194 nm. The effects of the buffer (concentration and pH), the injection time, the voltage applied and the plasma clean-up procedure were studied. The determination of atenolol was achieved in less than 3 min, using an electrolyte of 50 mM H3BO3-50 mM Na2B4O7 (50:50, v/v) pH 9, injected hydrodynamically for 4 s at 50 mbar and applying a voltage of +25 kV. This method was applied to the determination of atenolol in plasma of nine hypertensive patients (male and female, aged from 39 to 73 years). Atenolol concentrations found vary from 30 to 585 ng/ml.     

Simultaneous determination of weakly ionizable analytes in urine and plasma samples by transient pseudo-isotachophoresis in capillary zone electrophoresis

A rapid method for the simultaneous determination of several non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma and urine was developed using transient pseudo-isotachophoresis (ITP) in capillary zone electrophoresis (CZE). The influence of different parameters on resolution and preconcentration efficiency, such as background electrolyte (BGE) composition, sample injection, sample matrix composition, and pH, were studied to optimize the transient pseudo-ITP performance. Optimized conditions were a BGE consisting of 100 mM Na₂B₄O₇ in 10% aqueous MeOH solution and hydrodynamic injection of the sample at 50 mbar for 90 s. The sample was prepared in a solution mixture of 1% NaCl/ethanol (30:70 v/v) at pH 10. Our results show that this simple strategy offers improved sensitivity compared to conventional CZE analysis, reaching a 45-fold preconcentration factor. The detection limits (LODs) were as low as 0.07 mg/L for standard samples with good repeatability (values of relative standard deviation, %RSD < 11%). The method was applied to the analysis of NSAIDs in biological samples. Validation for human plasma and urine samples demonstrated good linearity, low detection limits, and satisfactory repeatability values.     

二维毛细管区带电泳/胶束电动毛细管色谱分离尿样中的药物及其对映体

建立了毛细管区带电泳(CZE)/胶束电动毛细管色谱(MEKC)二维毛细管电泳分离平台,CZE毛细管和MEKC毛细管通过一段带微孔的聚四氟乙烯(polytetrafluoroethylene, PTFE)套管固定。样品在CZE毛细管中分离后进入MEKC毛细管进一步分离,在二维转换过程中采用动态pH连接-胶束扫集法避免第一维分离区带在接口处扩散。将该方法成功用于鼠尿样品中4种药物及其对映体的分离,各组分的理论塔板数为(2.8～4.3)×104/m,检出限为0.015～0.052 mg/L,实际样品中峰面积和迁移时间的相对标准偏差(n=7)分别为1.7%～3.8%和1.3%～4.6%。方法重现性好、灵敏度和分离度高、峰容量大,适用于尿样中多种药物组分及其对映体的同时分离检测。     

Determination of magnesium,calcium, sodium,and potassium in blood plasma samples by capillary zone electrophoresis

A capillary zone electrophoretic assay has been developed and validated for analysis of magnesium, calcium, sodium, and potassium in blood plasma samples. Optimum results were obtained with 20 mmol L⁻¹ imidazole (pH 2.8) and 0.5 mmol L⁻¹ oxalic acid containing 5% methanol, capillary temperature 25°C, applied voltage 30 kV, hydrodynamic injection time 3 s, and a poly(vinyl alcohol)-coated capillary (i.d. 50 μm, total length 64.5 cm and effective length 56 cm). Indirect detection was performed at 214 nm. Cadmium was used as internal standard. The migration times of magnesium, calcium, sodium, and potassium were 4.25, 3.79, 3.96, and 2.79 min, respectively. The method was applied to the determination of magnesium, calcium, sodium, and potassium in blood plasma samples. The results were compared with those from atomic absorption spectrophotometry and no statistically significant difference was found (P>0.05). This study was supported by the Turkish Republic, Prime Ministry State Planning Organization (Project Number: 98K121730)     

A capillary zone electrophoresis for determination of thiolic peptides in biological samples

A new method to improve the analyses of thiolic peptides (cysteine, γGlu-Cys, glutathione, phytochelatins and desglycyl-phytochelatins) derivatized with monobromobimane (mBrB) in complex biological samples by CZE is described. The method involves a SPE using Sep-Pak Light C18 Cartridges after derivatization and a later CZE analysis. Elution of mBrB-thiols was achieved with 10 mM HCl + 70% methanol v/v in deionised water. Electrophoretic parameters, such as BGE pH and concentration, different organic additives (methanol and trifluoroethanol), applied voltage and capillary length were studied in order to establish suitable analytical conditions. Optimum separation of the mBrB-thiolic peptides was obtained with 100 mM sodium borate buffer at pH 7.60. The electrophoretic conditions were +15 kV, capillary length of 90 cm from inlet to detector (98 cm total length, 50 μm ID), samples were loaded into the capillary by hydrodynamic injection (50 mbar, 20 s) and detection was performed at 390 nm. The improved method showed good reproducibility, linearity and sensitivity. The LODs and LOQs estimated using a standard of GSH were 1.41 and 4.69 μM respectively.     

Capillary zone electrophoresis of basic drugs in non-aqueous acetonitrile with buffers based on a conventional pH scale

Summary The pK _a ^* values of 10 nitrogen-containing basic drugs in non-aqueous acetonitrile were determined from the pH^* dependence of their electrophoretic mobilities. The pH^* scale in the organic solvent was established using background electrolytes with known conventional pK _a ^* values, making further calibration with reference pH electrodes unnecessary. In acetonitrile the pK _a ^* values of analytes (or their conjugated cation acids, BH⁺, respectively) were 5.2±8.9 pK units>those in water. The observed change in pK _a ^* values of cationic analytes was, however, much less than the known respective change for neutral acids type HA. From the pK _a ^* values and the actual mobilities, it is possible to predict pH^* conditions to enable separation of analytes, and this was demonstrated for two pairs of common drugs.     

Influence of solvent on temperature and thermal peak broadening in capillary zone electrophoresis

The present paper deals with the role of the solvent on thermal peak broadening. One main solvent property that determines the magnitude of the temperature gradient due to the generation of Joule heat in capillary zone electrophoresis is the thermal conductivity. As organic solvents have lower thermal conductivity than water (methanol and acetonitrile, e.g., nearly by a factor of 3) it can be hypothesized that the temperature gradient inside the capillary is more pronounced in organic solvents compared to an aqueous solution. On the other hand, the temperature dependence of the ion mobility (which is responsible for the velocity profile and thus for thermal peak broadening) is smaller in organic solvents. To get insight into the thermal effect of the solvent, first the temperature of a solution in a cylindrical tube was calculated utilizing the heat balance equation. It was shown that the two theoretical models most common in the literature (based on the analytical solution or on an assumption of the parabolic temperature profile in the tube, respectively) give the same results. The latter model was chosen for the further calculations, adding a quadratic term to express the electric conductivity as a function of the temperature. The temperature at the inner capillary wall and center as function of the capillary dimensions and the electric power was computed for electrolytes with a given conductivity at 25.0 degrees C with water, methanol, and acetonitrile as solvents. Capillary cooling systems used were circulating liquid cooling, enforced air-cooling, and natural convection in still air. The mean temperature (averaged over the cross section) resulting from Joule heating was compared with experimentally determined temperatures established upon application of an electric field; the latter temperature was derived from the measurement of the electric conductance of the background electrolyte solution and its (measured) temperature dependence. All investigations were carried out with solutions of the same initial electric conductivity (about 0.5 S.m(-1) at 25.0 degrees C). Agreement is found for natural convection conditions, and the deviation between theoretical and experimental results for the forced air and circulated liquid cooling systems can be related to the poorly defined thermal conditions of the capillaries in commercial instrumentation (with a part in a thermostated cassette and a part outside). For given conditions the temperature gradients in the organic solvents exceed largely those in water, independent of the type of cooling. As a consequence, the thermal plate height is significantly larger in organic solvents, at least under conditions where the deviation from the Nernst-Einstein limiting case is not too high. However, even for the maximum applicable field strengths the thermal plate height contributions are negligible compared to longitudinal diffusion in all solvents.     

Application of capillary isotachophoresis and capillary zone electrophoresis to the determination of inorganic ions in food and feed samples

The purpose of this review is to summarise critically the possibilities of capillary isotachophoresis and capillary zone electrophoresis for the determination of inorganic ions in food and feed samples. This article covers papers published since 1977.     

Determination of the carboxylic acids in acidic and basic process samples by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) with indirect UV detection was used in developing a method for the simultaneous determination of inorganic anions, aliphatic and heterocyclic organic acids in various processed samples. The analytes were determined simultaneously in 10 min using an electrolyte containing 20 mM 2,3-pyrazine dicarboxylic acid, 65 mM tricine, 2 mM BaCl₂, 0.5 mM cetyltrimethylammonium bromide, and 2 M urea at pH 8.06. Linear plots for the analytes were obtained in the concentration range of 2–150 mg L⁻¹. Relative standard deviations (RSDs) of peak areas during a 3-day analysis period varied from 5.5% for glycolate to 9.5% for oxalate. RSDs of migration times varied between 0.4% and 1.1%. The detection limit (at S/N 3) was 1 mg L⁻¹ for all the analytes studied. The proposed method was successfully demonstrated for the determination of carboxylic acids in eight oxygen treated samples of commercial softwood and hardwood kraft lignin and two red wine samples of Pinot Noir grapes. In the kraft lignin samples the concentrations of carboxylic acids correspond to the oxidation time. The acid concentrations of wine varied considerable.     

Nonaqueous capillary electrophoresis method for the analysis of gleevec and its main metabolite in human urine

The viability of nonaqueous capillary electrophoresis (NACE) was investigated for determination of gleevec and its main metabolite in human urine using a fused-silica capillary. Baseline separation of the studied solutes was obtained using a nonaqueous solution composed of 12 mM ammonium acetate and 87.6 mM acetic acid in methanol-acetonitrile (ACN) (80:20, v:v) providing analysis time shorter than 3 min. Different aspects including stability of the solutions, linearity, accuracy and precision were studied in order to validate the method in the urine matrix. Detection limits of 24 microg L(-1) for gleevec and its metabolite were obtained. A robustness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments. The developed method is simple, rapid and sensitive and has been used to determine gleveec and its metabolite at clinically relevant levels in human urine. Before NACE determination, a solid-phase extraction (SPE) procedure with a C18 cartridge was necessary. Real determination of these analytes in two patient urines were done.     

Simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by capillary zone electrophoresis

A capillary electrophoretic method for the simultaneous determination of sulfamethoxazole and trimethoprim in plasma was developed. Sulfamethoxazole and trimethoprim extracted from human plasma with ethyl acetate were analyzed at 20 kV and 25 degrees C using 15 mm phosphate buffer (pH 6.2) as the electrolyte. The detection was by UV at 220 nm. The run time was 8.0 min and the limit of quantification was 10.00 microg/mL for sulfamethoxazole and 2.00 microg/mL for trimethoprim. The recovery was >99% for both compounds. This method enabled the detection of sulfamethoxazole and trimethoprim in plasma of patients after oral ingestion of their combined formulation. The present simple and rapid method is applicable to drug monitoring in immunocompromised patients who are taking the combined formulation of these compounds for the treatment or prophylaxis of Pneumocystis carinii pneumonia.