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.     

Evaluation of capillary electrophoresis for the analysis of nicotine and selected minor alkaloids from tobacco

Summary Difficulties encountered in the gas or liquid chromatographic analysis of nicotine and other alkaloids in tobacco are largely due to the ionic character of these compounds. The potential of using capillary electrophoresis (CE) as an alternative analytical tool to eliminate these problems was evaluated. Parameters including electroosmotic flow, ionic forms of the analytes, buffer composition and applied voltage were studied using nicotine as a model compound. Ionic forms and electrophoretic mobility, as well as UV absorbance, of nicotine were controlled by varying the pH of an aqueous buffer solution. Thus the separation was optimized based on the characters of alkaloids and the nature of capillary electrophoresis. For tobacco samples in which nicotine accounts for more than 98% of the total alkaloid content, a quick method for the determination of nicotine in an aqueous tobacco extract within 100 seconds can be achieved.     

Analysis of lignin degradation products by capillary electrophoresis

Summary A method for the quantitative analysis of phenolic lignin degradation products by capillary electrophoresis (CE) with on-column UV detection has been developed. The liquid biomass solutions contain low molecular hemicellulosic sugars and phenolic lignin degradation products with various degrees of polymerization. Special attention has been paid to the monomeric phenolic components of lignin degradation fragments, e.g. derivatives of phenolic acids, aldehydes, and alcohols. Uncoated fused silica capillaries and borate-phosphate buffer systems at moderate pH conditions were used in order to separate the compounds of interest. To provide validation of the method, the same samples were analyzed independently by HPLC using gradient elution on a RP-C18 column. As sugar degradation fragment, furan-2-carboxylic acid was detected.     

Determination of hydroxycarboxylic acids in food products by capillary electrophoresis

A procedure for the capillary-electrophoretic determination of lactic, malic, tartaric, and citric acids in food products was developed. The use of 3-nitrobenzoic acid as a light-absorbing component of the running buffer was proposed for the indirect photometric detection of these substances. This considerably increased the determination sensitivity (up to n × 10 ??g/L), as compared with currently available analogs. The composition of the running buffer was optimized: 3-nitrobenzoic acid, 10 mM; cetyltrimethylammonium bromide, 0.5 mM; EDTA, 0.1 mM; and monoethanolamine, to pH 5.3. The procedure was tested with the samples of food products: fruits, juices, nectars, wines, beer, etc. The accuracy of the analytical results was confirmed by the standard addition method.     

毛细管电泳电化学检测法测定烟草中的多元酚

采用毛细管电泳电化学检测法同时测定了烟草中的多元酚,即芦丁、绿原酸,槲皮素和咖啡酸。考察了工作电极的氧化电位、运行缓冲溶液浓度和pH值,分离电压和进样时间对分离和检测的影响。在优化条件下,以300μm直径的碳圆盘电极为工作电极,检测电位为+0.9 V(vs.SCE),在50 mmol/L硼酸盐(pH 8.4)的运行缓冲液中,被测物浓度与峰电流在三个数量级范围内呈良好线性,检出限为2×10-7或5×10-7g/mL。方法有着良好的重现性,被测组分的迁移时间和峰高的相对标准偏差(RSDs)小于4%(n=7)。单次测定可在16 min内完成,已用于实际样品多元酚的测定,样品处理简单,无须预富集。     

Determination of tobacco alkaloids in single plant cells by capillary electrophoresis

A new capillary electrophoresis system with direct UV detection for the analysis of the tobacco alkaloids nicotine, nornicotine and anabasine in plant microsamples was developed. An electrolyte containing a high concentration of citric acid to provide good buffer capacity at pH 3.6 was found to be most suitable in terms of sensitivity and separation efficiency. At this low pH the tobacco alkaloids are present in cationic form, showing high mobility and increased UV absorption. This system was used for the analysis of nicotine in single epidermal leaf cells of tobacco plants. Only vacuolar concentrations of nicotine were determined, as the vacuole occupies >95% of the entire volume in epidermal cells. The procedure of sample acquisition and preparation for nicotine analysis of vacuolar samples in the pl range is shown. The results indicate a gradient of nicotine from the leaf base to the tip with higher concentrations present in the cells at the tip. Compared to simultaneously measured bulk leaf samples containing all types of cells, tissues and compartments, the concentrations in epidermal cells are much higher. As nicotine is the major defence substance against insects in tobacco and the epidermis is the most exposed leaf tissue this result is physiologically plausible.     

Analysis of dyestuff degradation products by capillary electrophoresis.

Capillary electrophoresis (CE) with two different detection methods, mass spectrometry (MS) and UV absorption spectroscopy with photodiode array detection (UV/DAD), was used for the analysis of the degradation products of dyestuffs. We have studied wet oxidation with solid catalyst as the treatment method of wastewaters containing dyestuffs. When the Orange II (C.I. Acid Orange 7) solution was used as the model wastewater, treated solution contained unknown highly polar degradation products. We were able to determine the molecular masses of some products by CE-MS. From this clue, we tried to identify these products by CE-UV/DAD. By means of the comparison of the migration time and UV spectra of standard samples, three degradation products were identified. The separation of degradation products was successful within 15 min.     

Quantitation of oxidized and reduced glutathione in plasma by micellar electrokinetic capillary electrophoresis.

A method for the separation of reduced (GSH) and oxidized (GSSG) glutathione was optimized in terms of buffer concentration, sodium dodecyl sulfate concentration, buffer pH, detection wavelength, run voltage and injection volume. The method demonstrated good linearity (r2 > 0.999) and reproducibility (internal standard corrected peak area RSD < 2.3%) in the range of interest (16-81 microM GSH and 8-40 microM GSSG). A detection limit of less than 1 microM GSH and GSSG was obtained using a high sensitivity flow cell. When the optimized method was applied to plasma samples, concentrations of 1.6 microM GSH and 0.8 microM GSSG were easily detected without the need for derivatization. The on-capillary detection was calculated to be 38.6 fmol of GSH and 18.3 fmol of GSSG.     

Determination of parabens in pharmaceutical and cosmetic products by capillary electrophoresis

A validated capillary electrophoresis method for the determination of parabens-methyl, ethyl, propyl, and butyl is described. The optimum results were obtained by the use of a run buffer containing 20 mM borate buffer (pH 9.0) with 10% methanol and by applying a voltage of 20 kV, a low hydrodynamic injection of 10 s, and detecting the signals at 200 nm. Moxifloxazin was used as an internal standard. All parabens were separated within 10 min. The method showed a good repeatability, linearity, and sensitivity. It was applied to the determination of parabens in pharmaceutical and certain cosmetic products. The text was submitted by the authors in English.     

Quantitation of insulin injection by high-performance liquid chromatography and high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was evaluated as a potential technique for the regulatory analysis of commercial dosage forms of insulin. A comparison was made to a liquid chromatographic analysis presently being proposed as an official monograph in the United States Pharmacopeia. The salient points of this comparison were accuracy, precision and ease of use. Both authentic (i.e. single blind, spiked) samples and commercial pharmaceutical formulations (injections) were examined. Chromatographic analyses of both commercial formulations and authentic samples were characterized by good precision, with accuracy being supported by results from authentic (spiked) samples. Conventional HPCE (by which is meant a non-micellar electrolyte used with an uncoated, unmodified fused-silica capillary) achieved reasonable accuracy, but less than impressive precision, when applied to authentic samples. When used for commercial formulations, this type of HPCE did not produce a level of accuracy suitable for regulatory purposes, even with the use of an internal standard.     

Separation of miRNA and its methylation products by capillary electrophoresis

Methylation is a crucial step in plant microRNA biogenesis. To improve our understanding of the methylation process and its regulation, a rapid and convenient high-throughput method should be sought to help with the study of reaction kinetics and assist the search for chemical inhibitors of the methyltransferase, HEN1. In this short communication, we report a pioneering work that used capillary electrophoresis (CE) to separate the miRNA and its methylation product. Capillary zone electrophoresis (CZE) with UV-absorption detection and a reduced running temperature achieved good separation of miR173/miR173* and miR173-m/miR173*-m with a detection limit of around 1 microM. To enhance detection sensitivity, capillary gel electrophoresis (CGE) coupled with laser-induced fluorescence (LIF) detection was also tested, and base-line separation of nanomolar duplex RNA samples was achieved using 4% polyvinylpyrrolidone (PVP) as the sieving matrix and SYBR Green II RNA gel stain for on-column labeling. Although further study is needed to investigate if the separation is sequence dependent, our study demonstrated, for the first time, that CE could be an effective and rapid method in monitoring the miRNA methylation process.     

Quantitation of branched-chain amino acids in ascites by capillary electrophoresis with light-emitting diode-induced fluorescence detection

Branched-chain amino acids (BCAAs) are one of the important biomarkers for monitoring liver disease such as hepatitis or hepatoma. In this communication, we present the determination of the concentrations of BCAA in ascites by CE light-emitted diode-induced fluorescence (LEDIF) using 1.5% m/v poly(ethylene oxide) (average M(v) : ~8?000?000?g/mol) that was prepared in 10?mM sodium tetraborate solution (pH 9.3). Naphthalene-2,3-dicarboxaldehyde was used to derivatize 15 amino acids (AAs) to form naphthalene-2,3-dicarboxaldehyde (NDA)-AA derivatives prior to CE analysis. The separation of 15 NDA-AA derivatives was accomplished within 15?min, with RSD values of <5.8% (within-day) and 7.4% (between-days) with respect to their migration times. The limits of detection for the tested BCAAs ranged from 10.6 to 10.9?nM. We determined the concentrations of three BCAAs--leucine, isoleucine and valine--in ascites by applying a standard addition method, with recovery percentages ranging from 93.9 to 111%. The results obtained from this CE-LEDIF method is in good agreement with those by a gold standard method using an AA analyzer. We have found that the concentrations of the three BCAAs in ascites obtained from patients suffering from liver diseases were lower than those from healthy individuals. Our approach is highly efficient, sensitive, and cost-effective, which holds great potential for the diagnosis of liver diseases.     

Quantitation of talinolol and other beta-blockers by capillary electrophoresis for in vitro drug absorption studies

A capillary zone electrophoresis method is described for the enantioseparation of talinolol using heptakis(2,3-diacetyl-6-sulfo)-beta-cyclodextrin (HDAS-beta-CD) as a chiral selector. After liquid-liquid extraction of talinolol from physiological solution, electrokinetic injection was employed to improve the sensitivity. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A baseline separation of the talinolol enantiomers was achieved in less than 10 min using 100 mM phosphate solution as background electrolyte and pH 3.5, at the presence of 3.0 mM HDAS-beta-CD and at 20 degrees C. In addition, this analytical condition proved to be useful for the enantioseparation of a number of other beta-blocking agents such as alprenolol, atenolol, bisoprolol, celiprolol, metipranolol, oxprenolol, and sotalol. For determining talinolol, the method could be validated in terms of precision, accuracy and linearity, and was found to be suitable in determination of talinolol enantiomers in highly diluted samples obtained from in vitro experiments.     

Analysis of acrylamide in food products by in-line preconcentration capillary zone electrophoresis

Two in-line preconcentration capillary zone electrophoresis (CZE) methods (field amplified sample injection (FASI) and stacking with sample matrix removal (LVSS)) have been evaluated for the analysis of acrylamide (AA) in foodstuffs. To allow the determination of AA by CZE, it was derivatized using 2-mercaptobenzoic acid. For FASI, the optimum conditions were water at pH > or = 10 adjusted with NH3 as sample solvent, 35 s hydrodynamic injection (0.5 psi) of a water plug, 35 s of electrokinetic injection (-10 kV) of the sample, and 6s hydrodynamic injection (0.5 psi) of another water plug to prevent AA removal by EOF. In stacking with sample matrix removal, the reversal time was found to be around 3.3 min. A 40 mM phosphate buffer (pH 8.5) was used as carrier electrolyte for CZE separation in both cases. For both FASI and LVSS methods, linear calibration curves over the range studied (10-1000 microg L(-1) and 25-1000 microg L(-1), respectively), limit of detection (LOD) on standards (1 microg L(-1) for FASI and 7 microg L(-1) for LVSS), limit of detection on samples (3 ng g(-1) for FASI and 20 ng g(-1) for LVSS) and both run-to-run (up to 14% for concentration and 0.8% for time values) and day-to-day precisions (up to 16% and 5% for concentration and time values, respectively) were established. Due to the lower detection limits obtained with the FASI-CZE this method was applied to the analysis of AA in different foodstuffs such as biscuits, cereals, crisp bread, snacks and coffee, and the results were compared with those obtained by LC-MS/MS.     

Separation and determination of cations in beverage products by capillary zone electrophoresis

Cation determination is important for quality control of beverage products. To determine a large group simultaneously, a capillary electrophoresis procedure is developed with indirect UV at 214 nm in a three-complex buffer system (10 mM N,N-dimethylbenzylamine (DBA), 8 mM lactic acid and 2 mM 18-crown-6) with good mobility matching with desired cations. Under optimized conditions with pH adjusted to 4.65, a baseline separation is achieved for 14 cations (Rb(+), NH(4)(+), K(+), Ca(2+), Na(+), Mg(2+), Mn(2+), Co(2+), Fe(2+), Cd(2+), Cr(3+), Ni(2+), Zn(2+) and Cu(2+)) within 7 min using an uncoated silica column. To cover ng/l to mug/l range, both hydrostatic and electrokinetic sampling are studied, showing working ranges within (0.05-50)/(0.005-2) microg/l and detection limits (13-78)/(1.4-10) ng/l, respectively with satisfactory repeatability (RSD 0.31-0.47% for migration time, and 3.0-4.0% for peak height measurement). Agreeable results with established inductively coupled plasma-atomic emission spectrometry method have been obtained for orange juice and tea samples.     

Analysis of bovine whey proteins in soybean dairy-like products by capillary electrophoresis

The simultaneous separation of bovine whey proteins [alpha-lactalbumin and beta-lactoglobulin (A+B)] and soybean proteins was performed, for the first time, by capillary electrophoresis. Different experimental conditions were tested. The most suitable consisted of 0.050 M phosphate buffer (pH 8) with 1 M urea and 1.2 mg/ml methylhydroxyethylcellulose, UV detection at 280 nm, 15 kV applied voltage, and 30 degrees C temperature. Quantitation of bovine whey proteins in a commercial powdered soybean milk manufactured by adding bovine whey to its formulation was performed using the calibration method of the external standard. Direct injection of a solution of the powdered soybean milk only enabled quantitation of alpha-lactalbumin in the commercial sample. Detection of beta-lactoglobulin (A+B) required acid precipitation of the solution of the sample in order to concentrate bovine whey proteins in the supernatant prior to the analysis of this protein in the whey obtained. Since alpha-lactalbumin could also be quantitated from the injection of the whey, the simultaneous determination of alpha-lactalbumin and beta-lactoglobulin (A+B) was possible upon acid precipitation of the powdered soybean milk solution. Detection limits obtained were 14 microg/g sol. for alpha-lactalbumin and 52 microg/g sol. for beta-lactoglobulin (A+B) which represent protein concentrations about 60 microg/100 g sample for alpha-lactalbumin and 100 microg/100 g sample for beta-lactoglobulin (A+B).     

Determination of pesticides in environmental materials and food products by capillary electrophoresis

A survey of electrophoresis methods for the determination of pesticides from different classes in environmental materials and food products is presented. The main sample preparation procedures, the cleaning of extracts, and the use of various versions of capillary electrophoresis are considered.     

Direct chiral resolution of lactic acid in food products by capillary electrophoresis

Chiral resolution of native DL-lactic acid was performed by capillary electrophoresis using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Various factors affecting chiral resolution, migration time, and peak area of lactic acid were studied. The running conditions for optimum separation of lactic acid were found to be 90 mM phosphate buffer (pH 6.0) containing 240 mM 2-hydroxypropyl-beta-cyclodextrin with an effective voltage of -30 kV at 16 degrees C, using direct detection at 200 nm. In order to enhance the sensitivity, sample injection was done under a pressure of 50 mbar for 200 s. On-line sample concentration was accomplished by sample stacking. With this system, D- and L-lactic acids in food products were analyzed successfully.     

Profiling of erythropoietin products by capillary electrophoresis with native fluorescence detection

The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal‐to‐noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE‐Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO‐α and EPO‐β) based on the recorded glycoform pattern. The developed CE‐Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.