Rapid determination of globin chains in red blood cells by capillary electrophoresis using INSTCoated fused‐silica capillary

A laboratory‐made INSTCoated fused‐silica capillary has been newly used for CE separation of four mixtures of proteins in sodium phosphate BGEs at pH 3.0 and 2.5, respectively. The obtained separation efficiencies range from 145 000 theoretical plates per meter for myoglobin to 1 216 000 m^?1 for lysozyme. A total of 49–89% of the number of theoretical plates was obtained in a commercial polyvinyl alcohol coated capillary compared to the INSTCoated capillary under the same experimental conditions, 0–86% was obtained in a laboratory polyacrylamide‐coated capillary, and only 0–6% was obtained in an uncoated fused‐silica capillary. The RSD values for the intraday repeatability for an INSTCoated capillary were 0.1–1.0% (migration time) and 0.3–2.4% (peak area); RSD values for the interday repeatability in the same capillary are 0.6–1.4% (migration time) and 2.4–5.5% (peak area); RSD values for interday repeatability between different capillaries equaled 1.7–2.1% (migration time) and 2.8–10.9% (peak area). The INSTCoated capillary has been further used for rapid determination of globin chains isolated from red blood cells. A separation of α and β chains prepared from adult blood has been completed in 3 min with a peak resolution of 1.3, and the separation of α and ^Gγ chains prepared from newborn blood took 3 min with a peak resolution of 3.6.     

基于毛细管电泳的黄芪抗氧化生物指纹图谱研究

根据评价抗氧化剂清除自由基能力的间接化学发光法,结合具有高效分离能力的毛细管电泳技术,建立了能表征中药抗氧化活性的生物指纹图谱。选取中药黄芪为研究对象,以黄芪甲苷为参照物峰,确定了6个共有峰,并首次测得黄芪甲苷对羟自由基的清除率。各共有峰相对迁移时间的RSD<1.5%,相对自由基清除率的RSD<8.4%,表明方法的精密度和重复性较好。7个产地的黄芪药材与道地药材具有良好的抗氧化活性相似度,相似度>96.5%。抗氧化生物指纹图谱对天然产物的质量控制与临床研究具有指导意义。     

Multi‐site N‐Glycan mapping study 2: UHPLC

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N‐glycosylation analysis using capillary electrophoresis of APTS‐labeled N‐glycans were presented. The corresponding results from ultra‐high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725‐2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC‐based N‐glycan analysis platforms are appropriate for general use.     

Development of fully automated quantitative capillary electrophoresis with high accuracy and repeatability

A quantitative capillary electrophoresis (qCE) was developed by utilizing a rotary type of nano‐volume injector, an autosampler, and a thermostat with cooling capacity. The accuracy and precision were greatly improved compared with conventional capillary electrophoresis. The 10 nL volume accuracy was guaranteed by the carefully designed nano‐injector with an accurate internal loop. The system repeatability (precision) in terms of RSD <0.5% for migration time and 1% for peak area were achieved by using DMSO as a test sample. We believe that this fully automated qCE system has the potential to be employed broadly in quality control and quality assurance in the pharmaceutical industry. Copyright © 2015 John Wiley & Sons, Ltd.     

Improving precision of manual hydrodynamic injection in capillary electrophoresis with contactless conductivity detection

Reproducible injection in capillary electrophoresis has been difficult to achieve with manual injection techniques using simple injection devices, such as gravity injection (siphoning) or hydrodynamic sample splitting. We demonstrate that the injection reproducibility can be improved using very simple means. With hydrodynamic sample splitter, a passive micro-metering valve can be inserted in-line to regulate the sample flow rate through the splitter interface. A significant improvement of both reproducibility and repeatability was achieved. The reproducibility of RSD of the peak areas improved from 25.4% to 4.4%, while the repeatability was below 4.1% when micro-metering valve was used. Additional simple correction that can be used to further improve the variability of injected sample volumes in any hydrodynamic injection mode in CE with conductivity detection was proposed and verified. The measured EOF peak can serve as a simple indicator of the injected volume and can be effectively used for additional correction. By a linear function between the injection volume and the peak area of the EOF, the RSD values of peak areas for both manual gravity injection and hydrodynamic sample splitter were further improved below 2% RSD. The linearity of the calibration curve was also significantly improved. The proposed correction works even with slight differences in matrix composition, as demonstrated on the analysis aqueous soil extract of model mixture of five nerve agent degradation products.     

Solid acids assisted matrix solid‐phase dispersion microextraction of alkaloids by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry

The quantification of three alkaloids is important because quantitative study is a means of assessing the reliability of the experimental method, and three alkaloids of peimine, peiminine, and peimisine are main active ingredients in Chinese Pharmacopoeia 2015. An effective method based on the matrix solid‐phase dispersion microextraction was developed for the extraction of alkaloid compounds in Fritillariae Thunbergii Bulbus. Target analytes were analyzed by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry. The optimized experimental condition was that 50 mg Fritillariae Thunbergii Bulbus was blended homogeneously with 10 mg citric acid for 5 min. Two hundred microliters of water acidized by 1 mol/L hydrochloric acid (pH = 4.5) was selected to elute tested alkaloids. The results demonstrated that the investigated method had low limits of detection (1.32–1.59 ng/mL), good recoveries (86.63–98.12%), and reproducibility (relative standard deviations of peak areas < 0.87%). The proposed matrix solid‐phase dispersion microextraction coupled with capillary electrophoresis combined with quadrupole time‐of‐flight mass spectrometry was successfully applied for the extraction of alkaloids in plants.     

Simultaneous determination of six toxic alkaloids in human plasma and urine using capillary zone electrophoresis coupled to time‐of‐flight mass spectrometry

A novel capillary zone electrophoresis separation coupled to electro spray ionization time‐of‐flight mass spectrometry method was developed for the simultaneous analysis of six toxic alkaloids: brucine, strychnine, atropine sulfate, anisodamine hydrobromide, scopolamine hydrobromide and anisodine hydrobromide in human plasma and urine. To obtain optimal sensitivity, a solid‐phase extraction method using Oasis MCX cartridges (1 mL, 30 mg; Waters, USA) for the pretreatment of samples was used. All compounds were separated by capillary zone electrophoresis at 25 kV within 12 min in an uncoated fused‐silica capillary of 75 μm id × 100 cm and were detected by time‐of‐flight mass spectrometry. This method was validated with regard to precision, accuracy, sensitivity, linear range, limit of detection (LOD), and limit of quantification (LOQ). In the plasma and urine samples, the linear calibration curves were obtained over the range of 0.50–100 ng/mL. The LOD and LOQ were 0.2–0.5 ng/mL and 0.5–1.0 ng/mL, respectively. The intra‐ and interday precision was better than 12% and 13%, respectively. Electrophoretic peaks could be identified by mass analysis.     

CE coupled to MALDI with novel covalently coated capillaries

CE offers the advantage of flexibility and method development options. It excels in the area of separation of ions, chiral, polar and biological compounds (especially proteins and peptides). Masking the active sites on the inner surface of a bare fused silica capillary wall is often necessary for CE separations of basic compounds, proteins and peptides. The use of capillary surface coating is one of the approaches to prevent the adsorption phenomena and improve the repeatability of migration times and peak areas of these analytes. In this study, new capillary coatings consisting of (i) derivatized polystyrene nanoparticles and (ii) derivatized fullerenes were investigated for the analysis of peptides and protein digest by CE. The coated capillaries showed excellent run‐to‐run and batch‐to‐batch reproducibility (RSD of migration time ≤0.5% for run‐to‐run and ≤9.5% for batch‐to‐batch experiments). Furthermore, the capillaries offer high stability from pH 2.0 to 10.0. The actual potential of the coated capillaries was tested by combining CE with MALDI‐MS for analysing complex samples, such as peptides, whereas the overall performance of the CE‐MALDI‐MS system was investigated by analysing a five‐protein digest mixture. Subsequently, the peak list (peptide mass fingerprint) generated from the mass spectra of each fraction was entered into the Swiss‐Prot database in order to search for matching tryptic fragments using the MASCOT software. The sequence coverage of analysed proteins was between 36 and 68%. The established technology benefits from the synergism of high separation efficiency and the structure selective identification via MS.     

Enhancing separation in short‐capillary electrophoresis via pressure‐driven backflow

We present a novel easy‐to‐operate and efficient method to improve the separation efficiency in short‐capillary electrophoresis by introducing steady backflow to counterbalance electro‐osmotic flow without the use of any external pressure. The backflow was easily generated by tapering the capillary end, which was achieved by heating a straight capillary and stretching it with a constant force. We investigated the net fluidic transport rate under different tip lengths and separation voltages. Good run‐to‐run repeatability and capillary‐to‐capillary reproducibility of the present method were obtained with RSD less than 1.5%, indicating the stability of the fluid transport rate in the tapered capillary, which ensures the quantification and repeatability of capillary zone electrophoresis (CZE) analysis. Enhanced separation of the tapered short capillary electrophoresis was demonstrated by CZE analyzing amino acids and positional isomers. Baseline separations were achieved in less than 60 s using a tapered capillary with the effective length of 5 cm, while no separation was achieved using a normal capillary without a tapered tip. The present study provides a promising method to use pressure‐driven backflow to enhance separation efficiency in short‐capillary electrophoresis, which would be of potential value in a wide application for fast analysis of complex samples.     

Cross-validation and evaluation of the performance of methods for the elemental analysis of forensic glass by μ-XRF, ICP-MS, and LA-ICP-MS

Elemental analysis of glass was conducted by 16 forensic science laboratories, providing a direct comparison between three analytical methods [micro-x-ray fluorescence spectroscopy (μ-XRF), solution analysis using inductively coupled plasma mass spectrometry (ICP-MS), and laser ablation inductively coupled plasma mass spectrometry]. Interlaboratory studies using glass standard reference materials and other glass samples were designed to (a) evaluate the analytical performance between different laboratories using the same method, (b) evaluate the analytical performance of the different methods, (c) evaluate the capabilities of the methods to correctly associate glass that originated from the same source and to correctly discriminate glass samples that do not share the same source, and (d) standardize the methods of analysis and interpretation of results. Reference materials NIST 612, NIST 1831, FGS 1, and FGS 2 were employed to cross-validate these sensitive techniques and to optimize and standardize the analytical protocols. The resulting figures of merit for the ICP-MS methods include repeatability better than 5 % RSD, reproducibility between laboratories better than 10 % RSD, bias better than 10 %, and limits of detection between 0.03 and 9 μg g^?1 for the majority of the elements monitored. The figures of merit for the μ-XRF methods include repeatability better than 11 % RSD, reproducibility between laboratories after normalization of the data better than 16 % RSD, and limits of detection between 5.8 and 7,400 μg g^?1. The results from this study also compare the analytical performance of different forensic science laboratories conducting elemental analysis of glass evidence fragments using the three analytical methods.

Determination of levamisole in feeds by liquid chromatography coupled to electrospray mass spectrometry on an ion trap

Test methods have to be developed by laboratories for official control to monitor possible misuse of veterinary drugs in animal productions, also through feeding stuff. A novel method for identification and quantification of levamisole in feeds by liquid chromatography coupled to electrospray mass spectrometry in an ion trap (LC/ESI‐MS/MS) is herein described; after a single‐step cleanup by liquid‐liquid extraction from the feed and separation by reversed‐phase liquid chromatography, levamisole was determined and unambiguously confirmed by tandem mass spectrometry, on the basis of two product ions. The method was in‐house validated, according to the Regulation 882/2004/EC, evaluating trueness, repeatability, within‐laboratory reproducibility, ruggedness, specificity, and the limit of quantification (LOQ). The method is reliable and specific for complete and complementary feeds for pigs, cattle, rabbits and poultry; very good mean recoveries (higher than 92 %) and precision (RSD values < 15.2%) were attained. The LOQ at 2.0 mg/kg was verified. Moreover, we describe how the method was developed to support Italian Police investigations regarding illegal treatments of pigs; in this case, since the drug(s) added to the feed were unknown, a preliminary untargeted analysis was performed by full scan mass spectrometry on an ion trap, from 50 up to 2000 m/z; the presence of levamisole was hypothesised, on the basis of the most abundant ion and its fragmentation pattern. Then, levamisole was unambiguously confirmed by the ion trap LC/ESI‐MS/MS method. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of two glutaraldehyde immobilization techniques for solid-phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometry

Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively.     

Parameters affecting reproducibility in capillary electrophoresis

It is well known that poor quantitative reproducibility substantially limits the practical implementation of capillary electrophoresis (CE) separations in chemical analysis. The principal sources of variance in observed peak areas are irreproducible flow rate, which influences on-column detector response, and inconsistent injection volume or amount. An overview of studies by researchers to address the reproducibility issue will be presented. In addition, current efforts in our laboratory to assess sources of quantitative variance for separations of dansylated amino acids using an automated CE system are presented and related when appropriate to the body of existing knowledge on this important topic. A comparison of different injection methods (hydrostatic vs. electrokinetic) and approaches (e.g., high vs. low pressure), the effect of random changes in electroosmotic flow (EOF) due to air bubbles in the CE capillary, and choice of certain peak integration parameters in terms of peak area reproducibility are presented. Under optimum conditions relative standard deviation (RSD) values in raw peak area are typically 2.0%. With nonoptimum conditions (e.g., with air bubbles in capillary), RSD values can substantially degrade. However, normalizing with retention times, internal standards, or observed electrophoretic current produces RSD values in a range of 1.4-2.3%.     

连翘的毛细管电泳指纹图谱研究

采用毛细管区带电泳法,以75 mmol/L硼砂溶液(用0.1 mmol/L氢氧化钠溶液调pH 9.7)为背景电解质,运行电压14 kV,检测波长228 nm,重力进样15 s(高度9 cm),建立了连翘药材的毛细管电泳指纹图谱(CEFP)。将10个不同产地的连翘药材进行比较,按各电泳峰共有率fi≥70%作为选择电泳指纹峰的依据,确定连翘的毛细管电泳指纹峰为29个。在方法的精密度和重复性试验中,各指纹峰的相对迁移时间的相对标准偏差(RSD)不大于5%,相对峰面积的RSD约为2%~15%。10个产地的连翘药材的CEFP与其对照CEFP的相似度均不低于0.94。同时,采用指纹图谱信息量指数I和相对信息量指数Ir评价了10个产地连翘药材的质量差异。     

On-line pervaporation-capillary electrophoresis for the determination of volatile acidity and free sulfur dioxide in wines

Pervaporation has been coupled on-line to capillary electrophoresis (CE) by a simple interface consisting of a modified CE vial. The approach allows volatile analytes to be removed and injected into the capillary meanwhile the sample matrix remains in the pervaporator. By this approach volatile acidity and free sulfur dioxide have been simultaneously determined in wines. The detection limits (LODs) are 1.25 and 5.00 microg/mL, the quantification limits 4.12 and 16.50 microg/mL, and the linear dynamic ranges between LOD and 50 microg/mL and between 0.1 and 0.9 g/L for free sulfur dioxide and volatile acidity, respectively. The repeatability and within laboratory reproducibility, expressed as relative standard deviation (RSD), are 1.61% and 3.00% for free sulfur, and 3.35% and 4.58% for volatile acidity, respectively. The optimal pervaporation time and the time necessary for the individual separation-detection of the target analytes are 6 and 5 min, respectively. The analysis frequency is 7 h(-1) and the sample amount necessary is less than 7 mL. The proposed method and official methods for the analytes were applied to 32 wine samples. A two-tailed t-test was used to compare the methods, which yielded similar results. The errors, expressed as RSD for the two parameters, ranged between 1.3 and 4.1%.     

Capillary electrophoresis using core‐based hyperbranched polyethyleneimine (CHPEI) static‐coated capillaries

With unique 3‐D architecture, the application of core‐based hyperbranched polyethyleneimine (CHPEI), as a capillary coating in capillary electrophoresis, is demonstrated by manipulation of the electroosmotic mobility (EOF). CHPEI coatings (CHPEI5, M_w ≈? 5000 and CHPEI25, M_w ≈? 25 000) were physically adsorbed onto the inner surface of bare fused‐silica capillary (BFS) via electrostatic interaction of the oppositely charged molecules by rinsing the capillaries with different CHPEI aqueous solutions. The EOF values of the coated capillaries were measured over the pH range of 4.0–9.0. At higher pH (pH >6) the coated capillary surface possesses excess negative charges, which causes the reversal of the EOF. The magnitudes of the EOF obtained from the coated capillaries were three‐fold lower than that of BFS capillary. Desirable reproducibility of the EOF with % RSD (n = 5) ? 2 was obtained. Effect of ionic strength, stability of the coating (% RSD = 0.3) and the dependence of the EOF on pH (% RSD = 0.5) were also investigated. The CHPEI‐coated capillaries were successfully utilized to separate phenolic compounds, B vitamins, as well as basic drugs and related compounds with reasonable analysis time (<20 min) and acceptable migration‐time repeatability (<0.7% RSD for intra‐capillary and <2% RSD for inter‐capillary).     

Coupling of CE‐MS for protein and peptide analysis

The review is focused on the latest developments in the analysis of proteins and peptides by capillary electrophoresis techniques coupled to mass spectrometry. First, the methodology and instrumentation are overviewed. In this section, recent progress in capillary electrophoresis with mass spectrometry interfaces and capillary electrophoresis with matrix‐assisted laser desorption/ionization is mentioned, as well as separation tasks. The second part is devoted to applications—mainly bottom‐up and top‐down proteomics. It is obvious that capillary electrophoresis with mass spectrometry methods are well suited for peptide and protein analysis (proteomic research) and it is described how these techniques are complementary and not competitive with the often used liquid chromatography with mass spectrometry methods.     

Matrix‐free laser desorption/ionization mass spectrometry using silicon glancing angle deposition (GLAD) films

Glancing angle deposition (GLAD) was used to fabricate nanostructured silicon (Si) thin films with highly controlled morphology for use in laser desorption/ionization mass spectrometry (DIOS‐MS). Peptides, drugs and metabolites in the mass range of 150–2500 Da were readily analyzed. The best performance was obtained with 500 nm thick films deposited at a deposition angle of 85°. Low background mass spectra and attomole detection limits were observed with DIOS‐MS for various peptides. Films used after three months of dry storage in ambient conditions produced mass spectra with negligible low‐mass noise following a 15 min UV‐ozone treatment. The performance of the Si GLAD films was as good as or better than that reported for electrochemically etched porous silicon and related materials, and was superior to matrix‐assisted laser desorption/ionization (MALDI)‐MS for analysis of mixtures of small molecules between 150–2500 Da in terms of background chemical noise, detection limits and spot‐to‐spot reproducibility. The spot‐to‐spot reproducibility of signal intensities (100 shots/spectrum) from 21 different Si GLAD film targets was ±13% relative standard deviation (RSD). The single shot‐to‐shot reproducibility of signals on a single target was ±19% RSD (n = 7), with no indication of sweet spots or mute spots. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of fentanyl derivatives at trace levels with nonaqueous capillary electrophoresis-electrospray-tandem mass spectrometry (MS(n), n = 2, 3): analytical method and forensic applications

The identification of fentanyl derivatives at trace levels employing capillary electrophoresis coupled to electrospray ionization tandem mass spectrometry (CE-ESI-MS(n) , n = 2, 3) is presented. The studied synthetic opioid fentanyl and its derivatives have an exceeding analgesic potency which can be up to 8000 times higher that of morphine. Apart from their therapeutical applications, there is an abuse of them in the drug scene as a heroin substitute. The identification of these opioids at trace levels is of further significant forensic interest with respect to recent seizures of clandestine fentanyl laboratories in Germany. In this work, a nonaqueous capillary electrophoresis (NACE)-ESI-MS(n) procedure was developed for the separation and identification of six fentanyl derivatives including fentanyl, cis- and trans-methylfentanyl, sufentanil, alfentanil, and carfentanil. Their fragmentation pattern in MS(n) experiments were investigated as well as the influence of the sheath-liquid mixture and the influence of the inside diameter of the fused silica capillary on the peak shape and the signal to noise ratio. Method validation included determination of the detection limits (about 1-2 nmol/L) and the repeatability of migration time (at most 0.07% relative standard deviation). The NACE-MS procedure was successfully applied for the analysis of real samples from seizures in illegal fentanyl laboratories.     

Development and validation of a capillary electrophoresis method for the characterization of sulfoethyl cellulose

To characterize sulfoethyl cellulose el samples, a capillary electrophoresis method was developed and validated sulfoethyl cellulose el was hydrolyzed, and the resulting d ‐glucose derivatives were analyzed after reductive amination with 4‐aminobenzoic acid using 150 mM boric acid, pH 9.5, as background electrolyte at 20°C and a voltage of 28 kV. Peak identification was derived from capillary electrophoresis with mass spectrometry using 25 mM ammonia adjusted to pH 6.2 by acetic acid as electrolyte. Besides mono‐, di‐, and trisulfoethyl d ‐glucose small amounts of disaccharides could be identified resulting from incomplete hydrolysis. The linearity of the borate buffer‐based capillary electrophoresis method was evaluated using d ‐glucose in the concentration range of 3.9–97.5 μg/mL, while limits of detection and quantification derived from the signal‐to‐noise ratio of 3 and 10 were 0.4 ± 0.1 and 1.2 ± 0.3 μg/mL, respectively. Reproducibility and intermediate precision were determined using a hydrolyzed sulfoethyl cellulose el sample and ranged between 0.2 and 8.8% for migration times and between 0.3 and 10.4% for peak area. The method was applied to the analysis of the degree of substitution of synthetic sulfoethyl cellulose el samples obtained by variation of the synthetic process and compared to data obtained by elemental analysis.