Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (≈90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins (Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin.     

Enantiomeric analysis of rivastigmine in pharmaceuticals by cyclodextrin-modified capillary zone electrophoresis

Chiral separation method development is usually very time-consuming due to the diversity in chemical structures of pharmaceutical drug substances as well as the suitable separation conditions and the problem to choose the appropriate chiral selector. This paper shows capillary zone electrophoresis (CZE) which was developed for chiral separation of a basic compound - rivastigmine (RIV) using 30 cm × 50 μm i.d. polyacrylamide (PAA)-coated fused-silica capillary (effective length 20 cm), amine-modified phosphate buffer of pH 2.5 and sulfated-β-CD (S-β-CD) as chiral selector. Other selected native or derivatized cyclodextrins (CDs) were also tested: β-CD (5, 30 mM), carboxymethyl-β-CD (5, 30 mM), dimethyl-β-CD (15 mM), hydroxypropyl-β-CD (5, 30 mM), hydroxypropyl-α-CD (5, 30 mM) and hydroxypropyl-γ-CD (5, 30 mM). Complete enantiomeric separation of RIV was achieved at 20 kV, 18 °C and detection at 200 nm within 8 min with R.S.D. for the absolute migration time reproducibility of less than 2.1%. Rectilinear calibration range was 5.0-500.0 μM of each enantiomer (r = 0.9994-0.9995). The CZE method proposed was used for the control of chiral purity of pharmaceutically active S-RIV and for the analysis of Exelon caps preparation.     

Analysis of underivatized cellodextrin oligosaccharides by capillary electrophoresis with direct photochemically induced UV‐detection

This work focuses on the development of a CE method allowing, for the first time, the simultaneous separation of the underivatized first seven cellodextrin oligomers (glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose), with a view to analyze the hydrolysates obtained after partial acid depolymerization of nitrocellulose, and eight carbohydrates (ribose, xylose, fructose, mannose, galactose, maltose, lactose, and sucrose), which might be potential interfering compounds in explosives samples. Separation was achieved with a highly alkaline BGE containing sodium chloride and direct mid‐UV‐absorbance detection was performed after photo‐oxidation in the detection window. EOF was reversed to speed up the analysis using a dynamic capillary coating by hexadimethrine bromide. A central composite design was carried out to determine the effects of BGE conductivity and sodium hydroxide concentration on resolutions between neighboring peaks, and analysis time. A desirability analysis on modeled responses was applied to maximize resolutions and to minimize analysis time. The simultaneous analysis in 20 min total runtime of the 15 carbohydrates plus internal reference (naphthalene sulfonate) was carried out at 25°C with a BGE composed of 77.4 mM NaOH and 183 mM NaCl to adjust the conductivity at the optimum value. Finally, the resolution robustness was checked. This new method should also be of interest to monitor food and nonfood crop products.     

On-line solution stability determination of pharmaceuticals by capillary electrophoresis

Summary Good agreement between the impurity levels in a batch of a related impurity of ranitidine were obtained by CE and HPLC. A solution of the impurity was positioned on the CE autosampler and analysed sequentially. The extent of degradation was monitored by loss of main peak and the formation of two principal degradation products. It was found that after 9.25 hours only 2% area/area of the original impurity remained. Buffering of the sample solution to pH 7 was shown to minimise this degradation.Unattended in-situ stability testing of an solution of the impurity in water was performed by CE.     

Pesticide analysis by capillary electrophoresis

In this work, a critical and updated revision of the current situation of the analysis of pesticides by Capillary Electrophoresis (CE) is presented. The review has been written in two main sections. The first one presents a thorough revision of the various offline and on-line sample preconcentration procedures that have been used in conjunction with CE to analyze these compounds. The second part reviews the various detection strategies (i.e., UV, LIF, MS, and electrochemical) and CE modes that have been applied to the analysis of pesticides. Future trends that can be expected from this hot research area are also discussed.     

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.     

Fluorophore-assisted carbohydrate electrophoresis as detection method for carbohydrate-protein interactions

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straight-forward, sensitive method for determining the presence and relative abundance of individual (oligo) saccharide in a(n) (oligo) saccharide mixture. The single terminal aldehydes of (oligo) saccharides were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling is not biased by (oligo) saccharide length. Therefore, band fluorescence intensity is directly related to the relative abundance of individual (oligo) saccharide moieties in heterogeneous sample. In the same time, it also indicates that FACE can be used to investigate the interactions of carbohydrates and proteins.     

毛细管电泳免疫分析法研究癌胚抗原与抗体相互作用

采用毛细管电泳免疫分析法研究癌胚抗原和抗体相互作用.探讨了缓冲体系、癌胚抗原和抗体的配比、进样时间,进样电压等因素对分离检测的影响.结果表明分离电压为14 kV,进样时间为10 s, 在pH值为5.92的Tris-乙酸缓冲体系（TAE）中, 癌胚抗原及其复合物得到较满意的分离.     

On-line pre-concentration of atrazine by antibody immobilization capillary electrophoresis

Antibody coupled capillary electrophoresis was employed in the pre-concentration and detection of atrazine at the parts per billion (ppb) level. A 1000-fold increase in detection has been demonstrated in the use of IgG anti-atrazine monoclonal antibodies for the analysis of atrazine in well water by capillary zone electrophoresis (CZE) with UV-VIS detection. These results were confirmed by an enzyme linked immunosorbent assay (ELISA) specific for atrazine.     

Determination of terbinafine in pharmaceuticals and dialyzates by capillary electrophoresis

A capillary electrophoresis method has been developed for the separation and determination of terbinafine (TER) in various pharmaceutically relevant matrices. Capillary zone electrophoresis (CZE) separation and UV absorbance photometric detection were carried out in a 160 mm capillary tube with a 300 μm i.d., hydrodynamically (membrane) closed. The influences of pH, carrier cation and counterion on migration parameters of TER were studied and the following conditions were selected: a 20 mmol l⁻¹ glycine running buffer adjusted to pH 2.7 with acetic acid, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC) as an electro-osmotic flow (EOF) suppressor, a 250 μA driving current, and 20 °C. The optimized separation conditions were convenient for the determination of TER in commercial tablets and spray and in dialyzates. Here, the dialysis was used to investigate in vitro permeation of TER through the skin from the gel. The samples of dialyzates were examined with and without simple extraction procedure and the results were compared. A permeation profile of the drug present in the gel of given composition was obtained analyzing pretreated samples. The proposed electrophoretic method was successfully validated. It was suitable for the simple, sensitive, rapid and highly reproducible assay of TER. CZE analysis was completed within 5.5 min. The detection limit of TER was 1.73 μmol l⁻¹ at a 224 nm detection wavelength. The intra- and inter-laboratory precisions over the concentration range 6.0-60.0 μmol l⁻¹ were between 0.32-0.69% and 1.04-1.44% including R.S.D. of migration times and peak areas, respectively. The mean absolute recoveries of drugs from samples were found to be 98.34 (tablets) and 99.47% (spray). It is suggested that there are potentialities to determine TER present in unpretreated complex samples, as CZE in a hydrodynamically closed separation system may be easily on-line combinable with purification and preconcentration CE modes (e.g., isotachophoresis, ITP).     

Enantiomeric analysis of methotrexate in pharmaceuticals by cyclodextrin-modified capillary electrophoresis

A capillary electrophoresis for the chiral separation of racemic methotrexate (rac-MTX) was developed and validated. The two enantiomers were separated by using fused-silica capillary and a running buffer containing phosphate and hydroxypropyl-β-cyclodextrin (HP-β-CD). Several parameters were studied, including concentration and pH of phosphate buffer, separation voltage, and type and concentration of CD. The quantitative ranges were 12.5-200.0 μM for each enantiomer. The intra- and inter-day relative standard deviations (R.S.D.) and relative errors (R.E.) (n=5) were all <5%. The detection limits were found to be about 4 μM (S/N=3, injection 5 s) at 280 nm. All recoveries were greater than 93%. This method was applied to the assay of l-MTX in pharmaceuticals.     

A technique for capillary joining in capillary electrophoresis

Summary Aspects of cracking and joining capillaries have been investigated. Capillary coupling was achieved using various methods. The most successful used hydrofluoric acid-etched capillaries to form male and female ends which were then joined together. This type of joint was used to connect sections of capillary of similar and different internal diameters with minimal loss in resolution, peak width and number of theoretical plates. (Uridine and hypoxanthine was used as a test mixture). For hypoxanthine on a 50 m/50 m etched joined capillary 10 cm from the detector window the number of theoretical plates was 96.6% of that for hypoxanthine on an unbroken capillary. Following the relative success of capillary joining, a coupled capillary flowcell (50 m/200 m) was produced and evaluated.     

Protein separation by capillary gel electrophoresis: A review

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples.     

Quantitative and selective analysis of lactose by capillary electrophoresis

Summary A CE method has been validated for the analysis of batches of lactose used as a pharmaceutical raw material. This method was shown to be selective for lactose and was found to be quantitative. The separation was achieved due to on-capillary chelation of the lactose with borate ion. The resulting complex was detected at 195nm. An internal standard is employed to improve injection precision and detector linearity. A system peak occurred in the separation and was systematically investigated to show that it was not sample related. The method was validated and successfully submitted to regulatory authorities and is now in routine use in a number of our quality control laboratories.     

Pesticides analysis by liquid chromatography and capillary electrophoresis

Nowadays, a wide range of pesticides are used in agricultural production, and their monitoring in samples of environmental and alimentary interest is of extreme importance to ensure, among others, the safety of consumption of foods. The aim of this work is to provide updated information about the major developments in CE and HPLC in pesticide analysis, covering relevant publications between 2004 and early 2006. The use of different sample pretreatment steps to provide a suitable extraction of these compounds from the different matrices as well as to increase the sensitivity of the determination is also discussed.     

Chemical analysis of raw, dry-roasted, and honey-roasted licorice by capillary electrophoresis

In herbal medicine, licorice is usually processed using a roasting procedure which might modify the chemical compositions in licorice. To test this hypothesis, licorice root samples were roasted under various conditions (with or without honey) and subsequently extracted by refluxing with 95% ethanol. The analysis of chemical compositions of licorice root extracts was achieved by capillary electrophoresis. The running buffer has been optimized to be 50 mM sodium tetraborate (pH 9.01) containing 5 mM beta-cyclodextrin. Thermal decomposition of glycyrrhizin, which was a major ingredient in licorice, was first studied in detail, indicating the conversion of glycyrrhizin to glycyrrhetinic acid. The licorice extracts were then analyzed to indicate the above thermal conversion did occur in the licorice samples. This finding may shed some light on understanding the differences in the therapeutic values of raw versus roasted licorice in herbal medicine.     

毛细管区带电泳法测定消毒剂中三氯新

建立了消毒剂中三氯新的毛细管电泳分析方法。探讨了缓冲介质和电泳参数对三氯新测定的影响。以15mmol/LNa2HPO4(pH6.0)-乙腈(V(Na2HPO4)∶V(乙腈)=50∶50)为电泳缓冲液,三氯新在12kV电压下电泳,于254nm检测波长处测定,6min可以完成分析。本方法的检出限为0.04mg/L,线性范围0.04～2.00mg/mL(r=0.997),加标回收率在90.9%～108.2%范围内,测定值的相对标准偏差分别为峰高7.7%,迁移时间5.5%。将本法与高效液相色谱法进行比较,样品测定结果的相对误差小于10%。将所建立的方法已用于消毒剂样品中三氯新的测定。     

Determination of synthetic pharmaceuticals in phytotherapeutics by capillary zone electrophoresis with contactless conductivity detection (CZE-CD)

In this work, a method for simultaneous determination of amfepramone, fenproporex, sibutramine and fluoxetine was developed by capillary zone electrophoresis with capacitively coupled contactless conductivity detection (C⁴D) using a homemade capillary electrophoretic system. The optimized conditions for the separation of the pharmaceuticals by CZE were as follows: 50 mmol L^− 1 phosphate buffer (pH 5.0) in 50/50 (v/v) mixture of water/acetonitrile as the working electrolyte, 15 kV separation voltage, 25 °C separation temperature, hydrodynamic injection by gravity using 20 cm injection height and 60 s injection time. The detection by C⁴D was carried out by using a homemade detector, which employs a sinusoidal wave generator operating at 600 kHz frequency and 2 V_pp wave amplitude. The optimized and validated CZE-C⁴D method was applied for the determination of the studied pharmaceuticals as adulterants in phytotherapeutic formulations commercialized in Brazil for slimming purposes.     

Rapid analysis of spent fixing solutions by capillary electrophoresis

Summary A capillary electrophoretic method for the determination of thiosulphate, bromide, Fe(III)-EDTA chelate and free EDTA in spent fixing solutions has been developed. Free EDTA was complexed with Ni(II) ions prior to analysis. The optimised separations were carried out in a fused silica capillary (57 cm×75 μm I.D.) filled with a borate buffer (100mmol L⁻¹ borate, 0.2 mmol L⁻¹ tetradecyltrimethylammonium hydroxide, pH 8.5; applied voltage, −30kV) using direct UV detection at 214 nm. All four anions were well separated in less than 4 min. The method was applied to the rapid monitoring of spent fixing solutions.     

Optimization of phenolic compounds analysis by capillary electrophoresis

Operational parameters like migration time, temperature, voltage, composition of background electrolyte and content of organic modifier were optimized in CZE for the determination of lignin-like phenolic compounds.The applied background electrolyte buffer consisted of a Na₂B₄O₇, KH₂PO₄ aqueous solution, pH 9.15 using acetonitrile as organic modifier with UV-detection. Compounds, such as acetosyringone, acetovanillone, syringealdehyde, p-hydroxyacetophenone, vanillin, syringic acid, ferulic acid, p-hydroxybenzaldehyde, p-coumaric acid, vanillic acid and p-hydroxybenzoic acid were applied as reference compounds.The quality and quantity of different phenolic compounds obtained upon alkaline CuO oxidation of a commercial humic acid were determined with CZE using ethylvanillin as internal standard.The optimized CZE revealed has being an appropriate method since it is quick, sensitive and quantitative and does not require a time-consuming sample preparation.