Rapid capillary zone electrophoresis method for the determination of metal cations in beverages

A rapid and reliable capillary zone electrophoresis method for the determination of inorganic cations was developed. The complete separation of K⁺, Ba²⁺, Ca²⁺, Na⁺, Mg²⁺, Mn²⁺, Ni²⁺, Cd²⁺, Li⁺ and Cu²⁺ can be achieved in 4 min with a simple electrolyte composed by 10 mM imidazole as the carrier buffer and background absorbance provider and acetic acid as the complexing agent (pH 3.60). Injection was performed hydrostatically by elevating the sample at 10 cm for 30 s. The running voltage was +25 kV at room temperature. Indirect UV-absorption detection was achieved at 185 nm. The detection limit was in the range between 0.06 mg/l (Mg²⁺) and 0.57 mg/l (K⁺) and the quantification limits ranged from 0.10 mg/l (Ni²⁺) to 0.80 mg/l (Cu²⁺). The calibration graphs were linear in the concentration range from the quantification limit till at least 1 g/l in K⁺, 10 mg/l in Ba²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺ and Cd²⁺, 40 mg/l in Na⁺ and 12 mg/l in Li⁺ and Cu²⁺. The repeatability, intraday and interday analysis were ≤1.55% and ≤3.64% for migration time and ≤3.38% and ≤3.63% for peak area. The method developed has been applied to several beverage samples with only a simple dilution and filtration treatment of the sample. The proposed method is simple, fast, cheap and it is achieved with common products in either laboratory. For these reasons, it is a very useful method for routine analysis.     

Analysis of recombinant monoclonal antibodies by capillary zone electrophoresis

Analytical platforms that characterize charge heterogeneity in therapeutic proteins, such as mAbs, are important tools that can be used to define quality attributes. CZE separates protein moieties close to their native state and is a valuable physicochemical analytical method that can be used in parallel with other orthogonal methods for characterization and comparability. In this study, custom conditions for the analysis of charge heterogeneity of two mAbs were developed with regard to critical parameters in the BGE, running conditions, and sample treatment. The method application was tested for up to four mAbs and one mAb fragment. The electropherograms showed specific profiles and contrasting levels of basic and acidic isoforms with respect to the main isoform. Issues that surround this method, such as peak tailing and capillary lifetime, are summarized. Using this method, the identities of rituximab and trastuzumab were confirmed, based on the correspondence between the biosimilars and reference products, noninterference of the sample matrix, and the ability to separate spiked samples of related mAbs. The RSD of the isoform content and migration time for the method repeatability were less than 2 and 1%, respectively.     

Development and validation of a capillary zone electrophoresis method for the quantitative determination of anthocyanins in wine

A capillary zone electrophoresis (CZE) method is proposed for the quantitative determination of anthocyanins in wine as an alternative to high-performance liquid chromatography. The CZE separation was carried out using a 46 cm (effective length)×75 μm I.D. fused-silica capillary at 10 °C and a 50 mM sodium tetraborate buffer at pH 8.4 with 15% of methanol as modifier. A voltage of 25 kV and a hydrodynamic injection of 300 mbar s were used. The electropherograms were recorded at 599 nm. It was found that SO₂ (antibacterial and antioxidant agent added to wine during its production) increased the absorbance of anthocyanins at 599 nm in a basic medium. Therefore, a concentration of 250 mg/l of SO₂ was added to the samples and the calibration solution before the analysis in order to avoid errors by this matrix effect. The analytical response was linear (R=0.998) between 10 and 700 μg/ml of malvidin-3-O-glucoside. The limit of detection and the reproducibility (as a relative standard deviation, n=11) were 1 μg/ml and 1.5%, respectively. Finally, the CZE method was validated by the analysis of synthetic wine samples (errors less than 8%) and by the comparison of the results obtained in the analysis of different monovarietal wines by CZE with those obtained by the standard HPLC method. In this comparison, a good correlation (R=0.998) with a slope of 1.005±0.044 and an intercept of −0.752±6.690 was obtained for malvidin-3-O-glucoside.     

Rapid inorganic ion analysis using quantitative microchip capillary electrophoresis

Rapid quantitative microchip capillary electrophoresis (CE) for online monitoring of drinking water enabling inorganic ion separation in less than 15 s is presented. Comparing cationic and anionic standards at different concentrations the analysis of cationic species resulted in non-linear calibration curves. We interpret this effect as a variation in the volume of the injected sample plug caused by changes of the electroosmotic flow (EOF) due to the strong interaction of bivalent cations with the glass surface. This explanation is supported by the observation of severe peak tailing. Conducting microchip CE analysis in a glass microchannel, optimized conditions are received for the cationic species K+, Na+, Ca2+, Mg2+ using a background electrolyte consisting of 30 mmol/L histidine and 2-(N-morpholino)ethanesulfonic acid, containing 0.5 mmol/L potassium chloride to reduce surface interaction and 4 mmol/L tartaric acid as a complexing agent resulting in a pH-value of 5.8. Applying reversed EOF co-migration for the anionic species Cl-, SO42- and HCO3- optimized separation occurs in a background electrolyte consisting of 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 10 mmol/L HEPES sodium salt, containing 0.05 mmol/L CTAB (cetyltrimethylammonium bromide) resulting in a pH-value of 7.5. The detection limits are 20 micromol/L for the monovalent cationic and anionic species and 10 micromol/L for the divalent species. These values make the method very suitable for many applications including the analysis of abundant ions in tap water as demonstrated in this paper.     

Development of a capillary zone electrophoresis method for caseinoglycomacropeptide determination

Caseinoglycomacropeptide (CGMP) is a polypeptide of 64 amino acid residues, derived from the C-terminal part of bovine κ-casein. A sensitive and selective capillary zone electrophoresis method has been developed and validated for the analysis and quantitation of CGMP. Separation is carried out at 30 kV, using an uncoated fused-silica capillary and 20 mM sodium citrate buffer at acidic pH 3.5. The described method allows the separation of various CGMP subcomponents. The validation data proves that the method has the requisite selectivity, sensitivity, reproducibility and linearity for CGMP assay and for quality control during CGMP manufacturing (batch-to-batch reproducibility).     

New method for standardization of electropherograms obtained in capillary zone electrophoresis

A new method for standardization of electropherograms obtained by capillary zone electrophoresis was proposed, where the migration time axis was replaced by the effective mobility axis. The mobility increase due to temperature increase by Joule heating and the relaxation effect of the potential gradient were eliminated successfully by introducing a temperature coefficient for mobility expression and a delay time, respectively. The precision of the mobility evaluated by the proposed conversion methods was evaluated for a model sample. By using the conversion method, almost the same electropherograms could be obtained even from the electropherograms originally obtained by using different hardware conditions.     

Polypyrrole-coated capillaries for capillary zone electrophoresis

Fused-silica capillaries are permanently coated by silanization with 3-{[3-(N-pyrrole)-2-hydroxypropyl]amino}propyltriethoxysilane followed by oxidative polymerization of the pyrrole moieties with iron (III) or peroxodisulfate in the presence of chloride, perchlorate, or dextransulfate as anions. This approach allows to modulate the EOF in its magnitude as well as in its direction. With the small anions chloride and perchlorate, the EOF is reversed below pH 5 while with the large dextransulfate polyanions (DS) the EOF is relatively constant over the pH range from 2.5 to 9.4. This can be of advantage at low pH, at which the EOF of uncoated capillaries is close to zero. Application for separation of some herbicides is shown. The lifetime of PP-modified capillaries is satisfactory: the decrease in EOF is less than 3% during 80 analyses (160 min) and less than 5% over three months of storage. The reproducibility of capillary modification is about 5% (RSD of EOF).     

Fraction collector for capillary zone electrophoresis

An instrument is described which is capable of collecting fractions from a capillary zone electrophoresis apparatus. The fraction collector is characterized in terms of discretely collecting the separated components of a multi-component sample. In addition, the fraction collector permits the study of the effect of capillary zone electrophoresis on the biological activity of alpha-chymotrypsin.     

Split-flow injector for capillary zone electrophoresis

A simple construction of a split-flow injector eliminating some common problems connected with the use of such devices is described. It consists of a low-pressure pump, an injection valve and a delivery tube in which the separating capillary inlet is fixed. The sample is injected without moving the separating capillary inlet and without interrupting the applied voltage. The grounded electrophoretic electrode is close to the injection valve so that all metal parts of the injector are kept at a sufficiently low potential. Minimum length and small internal diameter of delivery tube minimizes additional sample zone broadening. The effects of some experimental parameters, such as the position of the separation capillary inlet with respect to the background solution flow direction and background solution flow-rate are experimentally studied. The injector was tested primarily for the electrokinetic injection.     

Development and validation of a method for quantitative determination of econazole nitrate in cream formulation by capillary zone electrophoresis

A simple, fast, inexpensive and reliable capillary zone electrophoresis (CZE) method for the determination of econazole nitrate in cream formulations has been developed and validated. Optimum conditions comprised a pH 2.5 phosphate buffer at 20 mmol L(-1) concentration, +30 kV applied voltage in a 31.5 cm x 50 microm I.D. capillary. Direct UV detection at 200 nm led to an adequate sensitivity without interference from sample excipients. A single extraction step of the cream sample in hydrochloric acid was performed prior to injection. Imidazole (100 microg mL(-1)) was used as internal standard. Econazole nitrate migrates in approximately 1.2 min. The analytical curve presented a coefficient of correlation of 0.9995. Detection and quantitation limits were 1.85 and 5.62 microg mL(-1), respectively. Excellent accuracy and precision were obtained. Recoveries varied from 98.1 to 102.5% and intra- and inter-day precisions, calculated as relative standard deviation (RSD), were better than 2.0%. The proposed CZE method presented advantageous performance characteristics and it can be considered suitable for the quality control of econazole nitrate cream formulations.     

Rapid determination of momordicoside in Momoradica charantia seeds by capillary zone electrophoresis

A novel method is described for the rapid determination of momordicoside in bitter melon based on capillary zone electrophoresis. Electrophoresis conditions, including the pH value and concentration of running buffer, as well as the voltage applied were optimized. The contents of momordicoside in 52 samples originating from different agricultural areas in China were determined. The results showed an excellent linearity in the concentration range of 0.50–2.51 mg mL^?1 for all samples (R ²?>?0.9999), with a recovery of 96.0% and a relative standard deviation of 1.4%. The method is characterized by rapidity, accuracy, the need for small samples only, and is suitable for analyses at a large scale.     

Contemporary chiral simulators for capillary zone electrophoresis

For separation of enantiomers in presence of a chiral selector, data obtained with the 1D dynamic simulators SIMUL5complex and GENTRANS are compared to data predicted by PeakMaster 6, a recently released generalized model of the linear theory of electromigration. Four electrophoretic systems with stereoisomers of weak bases were investigated. They deal with the estimation of input data for complexation together with the elucidation of the origin of observed system peaks, the interference of analyte and system peak migration, the change of enantiomer migration order as function of the selector concentration and the inversion of analyte migration direction in presence of a multiply negatively charged selector. For all systems, data predicted with PeakMaster 6 are in agreement with those of the dynamic simulators and simulation data compare well with experimental data that were monitored with setups featuring conductivity and/or UV absorbance detection along the capillary. SIMUL5complex and GENTRANS provide the full dynamics of any buffer and sample arrangement and require very long execution time intervals. PeakMaster 6 is restricted to conventional CZE, is based on an approximate solution of the transport equations, provides data for realistic experimental conditions within seconds and represents a practical tool for an experimentalist.     

高效毛细管区带电泳法快速测定尿液中的肌酐

建立了一种快速测定尿中肌酐浓度的高效毛细管电泳方法。利用非涂层石英毛细管(64.5 cm×50μm i.d),以pH 2.5,0.1 mmol/L H3PO4作为电泳缓冲液,检测波长191 nm,用0.05 Pa压力进样4 s,在电压16 kV快速分离尿液中的肌酐,采用外标法定量。肌酐的迁移时间约为5.5 min,肌酐浓度在34.5~8840μmol/L范围内呈良好的线性(r2=0.999)。平均日内精密度为2.5%,日间精密度为3.0%。回收率94.1%~99.0%。与全自动生化分析仪碱性苦味酸速率法相比有良好的相关性(r=0.990,n=56)。高效毛细管电泳法测定尿肌酐可应用于临床样品的检测。     

Formamide as solvent for capillary zone electrophoresis

A comprehensive investigation of a number of aspects when using formamide as background electrolyte solvent in capillary zone electrophoresis was presented. It included (i) the change of the ion mobility with ionic strength, (ii) the influence of the ionic strength on diffusion coefficients, and (iii) on the separation efficiency expressed by the maximum reachable plate numbers (when only longitudinal diffusion contributed to zone broadening), (iv) the effect of the solvent on pKa values (taken from the literature) of neutral and cation acids, (v) the establishment of the a pH scale in formamide by dissolving acids with known pKa values and their salts at defined proportion (thus circumventing the problem of calibrating the pH meter), (vi) the agreement between the experimentally derived and the theoretical dependence of the effective mobility on pH, (vii) the uptake of water of this hygroscopic solvent from the humidity of the environment and its consequence to the ion mobilities, pKa values, and the chemical stability of the solvent (e.g., hydrolysis), and finally (viii) the use of conductivity and indirect UV absorption to enable detection of analytes below the optical cutoff of formamide.     

毛细管区带电泳法快速测定人血血清蛋白

人血血清蛋白电泳分析是临床上诊断多种疾病的常用生化指标 ,也是临床实验室检查的常规项目。目前采用的方法有醋酸纤维薄膜电泳和琼脂糖电泳 ,尤以前者为主。由于醋酸纤维薄膜电泳法操作繁琐 ,每一步骤均需手工完成 ,影响因素较多 ,初学者往往不易掌握 ,且测定的重复性较差。高效毛细管电泳是近几年来迅速发展起来的分离分析技术 [1,2 ]。用该技术分析人血清蛋白国内外虽有报道 ,但由于人血清中蛋白质组分甚为复杂 ,采用不同的分离方法和条件可出现不同数量的组分峰 ( 6、7个甚至 1 0个以上组分 ) ,与目前临床上常用的醋酸纤维薄膜电泳图谱…     

Rapid method for the determination of amino acids in serum by capillary electrophoresis

Ion-pair and hydrophilic interaction chromatographies are considered to be complementary methods of choice for analyzing intact glucosinolates from broccoli. Ion-pair chromatography resolves non-polar glucosinolates, such as those containing indole moieties, while hydrophilic interaction chromatography is superior for separating polar glucosinolates, such as glucoraphanin and glucoiberin. Reversed-phase separations using hydrophilic endcapped C18-bonded silica and a 50 mM ammonium acetate-methanol gradient mobile phase resolve both polar and non-polar glucosinolates negating the need for switching columns.     

A new quantitative method for circulating DNA level in human serum by capillary zone electrophoresis with laser-induced fluorescence detection

We present a method for the quantification of circulating DNA in serum by capillary zone electrophoresis (CZE) with laser-induced fluorescence detection (LIF). The serum was digested by proteinase to release free DNA. SYBR Gold was utilized as DNA intercalating dye, fluorescein as internal standard (ISTD). CZE-LIF was applied for the separation and quantification of total circulating DNA. Good linearity (R = 0.9992) in the low range of DNA concentrations (0.5-40 ng/mL) and a detection limit of 0.5 ng/mL for DNA (S/N = 3) were obtained. Our data demonstrated that CZE-LIF system has a good linearity with excellent sensitivity and satisfactory reproducibility in the quantification of circulating DNA in serum. This method was successfully used for the quantification of circulating DNA levels in serum. We observed that the circulating DNA levels in certain cancer patients were significantly higher than that in healthy individuals. Compared to current methods, our protocol does not need the extraction of DNA from serum. Our preliminary results have illustrated that CZE-LIF system is simple, rapid, and sensitive, and it is well suitable for large-scale quantification of circulating DNA levels in clinical diagnosis.     

Separation of nicotine metabolites by capillary zone electrophoresis and capillary zone electrophoresis/mass spectrometry

The use of capillary zone electrophoresis (CZE) and capillary zone electrophoresis/mass spectrometry (CZE/MS) has been demonstrated, in principle, for the separation of nicotine and nicotine metabolites. The buffer system developed for separation and detection by CZE/UV was modified for use in CZE/MS analysis. Several of the metabolites are isobaric and tandem mass spectrometric (MS/MS) techniques have been used to differentiate such analytes.     

Stability-indicating assay using capillary zone electrophoresis for an azaphenothiazine in an ointment formulation

A stability-indicating assay using a capillary zone electrophoresis method is reported for the determination of isothipendyl in an ointment formulation. Sample preparation consisted of a simple dissolution of the ointment in an internal standard (3-aminobenzoic acid hydrochloride) solution. Separation was carried out in a short fused silica capillary using a pH 2.5 phosphate buffer as electrolyte. After introduction of the sample solution into the capillary, the compounds were separated as cations and detected within 3 min. The procedure was validated following the guidelines of the International Conference on Harmonisation. The method was found to be specific in relation to potential degradation products and excipients. Linearity determined between 50 and 150% of the target concentration was satisfactory (r² = 0.999). Recovery studies from an analytical placebo spiked with the analyte at five concentration levels (50-150% of the target concentration) on each of 3 days ranged from 103.5 to 99.2%. The repeatability of the entire procedure (n = 7) was better than 1.0%. The detection limit was estimated to be approximately 0.5 mg l⁻¹.