CE frontal analysis based on simultaneous UV and contactless conductivity detection: a general setup for studying noncovalent interactions

CE frontal analysis (CE-FA) has been established as a powerful tool to study noncovalent interactions between macromolecules and small molecules such as drug substances or pharmaceutical excipients. However, when using traditional commercial CE instrumentation, a serious drawback is related to the fact that only UV-active compounds can be studied. In recent years, contactless conductivity detection has become an attractive alternative to UV detection in CE due to its high versatility. In this study, we combine contactless conductivity detection and UV detection in a highly versatile setup for profiling noncovalent interactions between low-molecular-weight molecules and macromolecules. In the case of molecules having a chromophore the setup allows determination of binding constants using two independent detectors. The new contactless conductivity detection cell is compatible with commercial CE instrumentation and is therefore easily implemented in any analysis laboratory with CE expertise.     

Recent advances in the application of capillary electrophoresis for food analysis

This article reviews recent developments in the application of capillary electrophoresis (CE) for the analysis of foods and food components. CE has been applied to a number of important areas of food analysis and is fast becoming an established technique within food analytical and research laboratories. Papers are reviewed that were published during the two years to date following the previous review (Electrophoresis 2001, 22, 4197-4206).     

Capillary electrophoresis/tandem mass spectrometry for analysis of proteins from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis

Capillary electrophoresis/time-of-flight mass spectrometry(CE/TOFMS) has been used for analysis of in-gel digests of protein spots excised from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). An off-line purification and preconcentration procedure with a Zip Tip is used before CE/TOFMS analysis which allows for detection of protein spots with <1 picomole of material from 2-D gels. The off-line procedure provides sufficient purification for analysis while maintaining the quality of the CE separation. Using this procedure, several proteins from Coomassie Blue and zinc negatively stained gels are identified by the peptide maps generated and database searching. CE/TOF tandem mass spectrometry is used for the confirmation of database searching results and structural analysis of peptides that do not match the expected peptide maps obtained from the database in order to identify structural modifications. Several modifications were pinpointed and identified by this method.     

Generic capillary electrophoresis conditions for chiral assay in early pharmaceutical development

Generic capillary electrophoresis (CE) conditions have been implemented for chiral separations in early pharmaceutical development. The chiral CE separations of several pharmaceutical samples at different stages of development, i.e., discovery, process chemistry, and investigative new drug application, have been obtained using sulfated beta-cyclodextrin (CD). Several sulfated beta-CDs have been screened to select an appropriate enantioselective agent. The use of a generic CE method allows for a convenient and rapid chiral recognition of different weak bases, with minimal or no method development. CE using sulfated beta-CD for the chiral separation of N-benzoyl methyl piperazine has been validated for linearity, precision, accuracy, limits of detection and quantitation (LOD, LOQ). Although less sensitive than a specific liquid chromatography method using a Chiralpak AD column, the overall performance of the chiral CE method was found comparable. Validation data demonstrate that a LOD of 0.1%, sufficient to fulfill regulatory requirements, is achievable by chiral CE.     

Applications of CE SDS gel in development of biopharmaceutical antibody-based products

CE SDS gel technique offers many advantages over the traditional labor-intensive SDS PAGE slab gel technology. The CE-based method has increasingly been applied to many protein analysis applications. Specific examples are provided for monoclonal antibody (mAb), though the technique can be adapted to many other therapeutic protein products. Applications of CE SDS gel method using Beckman PA800 with UV detection are presented and discussed with respect to mAb analysis, such as purity, quantitation of non-glycosylated heavy chain (NGHC) peak, identity, and stability. The stability of mAb is evaluated with respect to formulation buffer, accelerated temperature stress, UV light-exposure, and high pH conditions. Both reducing and non-reducing CE SDS gel conditions were applied and optimized to characterize mAb products. The data presented provides a "taste" of what CE SDS gel method can do to support the development of mAb products from early clone screening for product quality to the final product characterization. Since the CE SDS gel method is automatable, quantitative, robust, and allows for relatively high throughput, it provides both great analytical capacity and product coverage for a wide spectrum of protein product development in biopharmaceutical industry.     

Determination of Proteinogenic Amino Acids in Human Plasma by Capillary Electrophoresis with Contactless Conductivity Detection

Capillary electrophoresis (CE) with contactless conductivity detection (CCD) represents a viable alternative to liquid chromatography in the analysis of amino acids (AA) in human plasma. After optimizing the composition of the background electrolyte (BGE), and introducing simple plasma pretreatment to remove spurious protein components, the CE/CCD methods allows determination of 18 from 20 proteinogenic AAs, three nonproteinogenic AAs and creatinine in 60 minutes. AAs are separated in their cationic forms in BGE containing 1.7 M acetic acid and 0.1% hydroxyethylcellulose, pH 2.2. LODs for individual AAs vary in an acceptable range with minimum of 4.3 μM for Arg and maximum of 42.9 μM for Cys. Mean concentrations and concentrations ranges for AAs in human plasma samples from 9 healthy individuals are found to agree well with those determined by an LC method in other two laboratories.     

CE frontal analysis employing contactless conductivity detection for determination of CMCs of non-UV absorbing charged surfactants

Micellar systems composed of surfactants are used extensively in academia and industry for many different applications. In this work a highly versatile CE method for determination of CMCs of charged surfactants has been developed. In the case of positively charged surfactants a coating procedure of the fused-silica capillary was used, whereas negatively charged surfactants were analyzed using uncoated capillaries. The CE method is based on frontal analysis (FA) employing use of contactless conductivity and UV detection. The main advantages of the method are that it can be used for non-UV absorbing surfactants without introducing marker compounds which previously has been found to affect CMCs, requires very limited sample volume and is easily implemented and automated using standard CE equipment. The fact that counterions and different aggregated states are separated allows a detailed characterization of the micelle systems using the developed method. In the case of UV absorbing surfactants similar results were obtained employing contactless conductivity and UV detection. Finally, CMCs obtained using conductometry gave similar results as compared to the developed CE-FA procedure.     

Analysis of serotonin in brain microdialysates using capillary electrophoresis and native laser-induced fluorescence detection

Serotonin or 5-hydroxytryptamine (5-HT) is a major neurotransmitter in the central nervous system. In this work, a method for analyzing 5-HT in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. A pH-mediated in-capillary preconcentration of samples was performed, and after separation by capillary zone electrophoresis, native fluorescence of 5-HT was detected by a 266 nm solid-state laser. The separation conditions for the analysis of 5-HT in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical bases. Separation of 5-HT was performed using a 80 mmol/L citrate buffer, pH 2.5, containing 20 mmol/L hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and +30 kV voltage. The detection limit was 2.5 x 10(-10) mol/L. This method allows the in vivo brain monitoring of 5-HT using a simple, accurate CE measurement in underivatized microdialysis samples.     

Selective digestion and novel cleanup techniques for detection of benzo[a]pyrene diol epoxide-DNA adducts by capillary electrophoresis/mass spectrometry

Benzo[a]pyrene (BP) is a ubiquitous environmental polycyclic aromatic hydrocarbon (PAH) which, upon metabolic conversion to reactive benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), has been found to attach covalently to DNA. Given the low level of DNA adducts typically present in vivo or in vitro, an essential first step prior to capillary electrophoresis/mass spectrometry (CE/MS) (or liquid chromatography/mass spectrometry (LC/MS)) analysis of the DNA digests is the removal of the bulk non-adducted nucleotides, enzymes or salts, and isolation of enriched adducts. This report focuses on the development of novel sample handling methods aimed at facilitating the analysis of BPDE-DNA adducts by CE/MS. This approach involves a simple variation on the digestion procedure, in combination with the use of metal affinity ZipTips for the more efficient cleanup of BPDE-DNA adducts formed in vitro for subsequent CE/MS analysis. The previously described digestion procedure, consisting of micrococcal nuclease, spleen phosphodiesterase and nuclease P1, allows for selective dephosphorylation of normal nucleotides, while leaving adducted nucleotides intact. Metal affinity ZipTips, typically used for selective extraction of phosphopeptides, were used here for extraction of adducted nucleotides. The utility of metal affinity SPE was tested on mixtures of dG and dGp, wherein nucleotide extracts contained no detectable nucleosides by CE/UV analysis. An in vitro BPDE-DNA incubation was then digested using the above procedure. Metal affinity solid-phase extraction (SPE) was subsequently used for the selective isolation of phosphorylated components, i.e., adducted nucleotides, from the mixture of enzymes and non-adducted nucleosides. SPE extracts were enriched in nucleotide adducts and analyzed using sample stacking and CE/MS. This method has several advantages over previously described cleanup procedures for dGp-BPDE adducts: fast, simple, uses commercially available materials, no need for excessive dilution (small scale), the suitability for use with automation, and possible applicability to other bulky hydrophobic adducts.     

Evaluation of Liquid Chromatography and Capillary Electrophoresis for the Elucidation of the Artificial Colorants Brilliant Blue and Azorubine in Red Wines

Summary Liquid chromatography (LC) and capillary electrophoresis (CE) have been compared for the analysis of the dyes brilliant blue and azorubine in red wines. A liquid-liquid extraction procedure followed by an ion-pair LC method was developed to separate the dyes from the wine polyphenols allowing reliable UV-spectral identification of the target dyes with limits of detection of 10 and 20 ppb for azorubine and brilliant blue, respectively. Because adulteration of wine with dyes is usually in the ppm level, CE proved to be a good alternative for the LC method. CE could be applied after a simple sample clean-up step by SPE eliminating interference from the bulk of the polyphenols. Although LC proved to be more sensitive compared to CE, the latter is more effective in reducing interferences from other wine components and showed the typical advantages of CE such as low solvent consumption and speed of analysis.     

Immunoaffinity chromatographic isolation of prostate-specific antigen from seminal plasma for capillary electrophoresis analysis of its isoforms

Prostate-specific antigen (PSA) concentration in serum has been the biomarker employed for prostate cancer diagnosis in the last two decades. However, new more specific biomarkers allowing a better differentiation of cancer from non-malignant prostate diseases are necessary. Glycosylation of PSA gives rise to different forms of the protein which can be separated into several isoforms by analytical techniques, such as CE. Because PSA glycosylation is influenced by pathological conditions, the CE pattern of PSA isoforms could be different in prostate cancer than in non-malignant prostate diseases. To study this CE pattern of PSA, prior purification of the protein from the biological fluid is mandatory. In this study an immunoaffinity chromatography method which allows PSA purification without altering the CE pattern is developed. An in-house prepared column produced with commercial anti-PSA antibodies is employed. The use of 1 M propionic acid as elution agent provides higher than 40% recovery of high purity PSA. CE analysis of PSA immunopurified from seminal plasma of a healthy individual shows the same 8 peaks as the commercially available PSA standard. Sample preparation only requires dilution with phosphate buffered saline prior to immunoaffinity purification. High repeatability for the sample preparation step was achieved (RSD% for percentage of corrected peak area in the range 0.6–5.3 for CE analysis of three independently purified seminal plasma aliquots compared to range 0.8–4.9 for a given aliquot analyzed three times by CE). IAC of five microliters seminal plasma provided enough PSA to achieve signal/noise ratio larger than 5 for the smallest CE isoforms.     

Single-stranded DNA-binding protein facilitates gel-free analysis of polymerase chain reaction products in capillary electrophoresis

It has been recently demonstrated that single-stranded DNA-binding protein (SSB) can facilitate quantitative analyses of DNA, RNA, and proteins in gel-free capillary electrophoresis (CE). Here, we report the application of SSB-mediated gel-free CE for analyses of polymerase chain reaction (PCR) products. The unique ability of SSB to bind ssDNA but not double-stranded DNA (dsDNA) allows efficient separation of three types of DNA molecules in the PCR reaction mixture: primers, products (amplified templates), and by-products, which originate from non-specific DNA hybridization. SSB-mediated gel-free CE analysis of PCR products combines simplicity, high sensitivity, and outstanding quantitative capabilities. The ability of the method to distinguish between products and by-products makes this method an indispensable tool in preparative PCR (e.g., in the development of nucleotide aptamers).     

Sodium glycylglycine as effective electrolyte run buffer for ascorbic and uric acid separation by CZE: a comparison with two other CE assays

A new CE method for ascorbic acid (AA) and uric acid (UA) detection in human plasma has been developed. Analytes were resolved in less than 4 min by employing sodium glycylglycine (Glygly) as electrolyte run buffer at pH 8.0. Using the diode array detector ability to measure multiple wavelengths simultaneously, detection was optimized by monitoring the run at 262 nm for AA and at 288 nm for UA. Electrophoretic parameters such as resolution, migration times, efficiency, and peak areas of this new method were compared to those obtained by the two CE assays described in literature, in which the analytes separation was achieved by using sodium borate (that allows faster migration times but poor resolution) or tricine (with the highest resolution but elevated migration times) as electrolyte run buffer. Sodium Glygly allows to obtain the same good resolution given by the tricine buffer but with the faster analysis times of the sodium borate run buffer. Ascorbate and urate levels were measured in 35 healthy volunteers by the three methods and the obtained data were compared by three different statistical tests (mountain plot, Passing-Bablok regression, and Bland-Altman test) in order to verify the accuracy of our proposed method.     

A novel technique for on-capillary preconcentration of anionic compounds applied to the trace analysis of rapamycin in human blood by capillary electrophoresis

A capillary electrophoretic (CE) method with UV detection at 278 nm has been developed for analysis of the immunosuppressant rapamycin (sirolimus) in human blood at low microg.L(-1) levels. Separation has been achieved in an acidic carrier electrolyte containing sodium dodecyl sulfate and 20% v/v methanol. For sample cleanup and preconcentration, both an off-line solid-phase extraction step using a silica-based reversed-phase material and a newly developed on-capillary focusing technique have been employed. The subsequent treatment of rapamycin under alkaline conditions leads to a cleavage of the lacton bond of the molecule, generating a negatively charged carboxylic group which allows electrokinetic injection into the CE instrument. During the injection process, the negatively charged analyte migrates into an acidic carrier electrolyte, so that it becomes neutral due to protonation and is focused at the capillary inlet. Injection times of 300 s at -7.5 kV could be applied without band-broadening. Results for real samples indicated that the method is fully suited for routine applications and may be an attractive alternative to established liquid chromatographic techniques.     

高效毛细管电泳在高速逆流色谱的溶剂系统选择过程的应用

对高产毛细管电泳在高速逆流色谱溶剂系统选择过程的应用进行了研究。应用高效毛细管电泳分析高速逆流色谱溶剂系统两相中样品的组成，可以利用其高的分辨率使各组分得到良好的分离，并且容易定量，有利于迅速了解各组分在两相溶剂中的分配情况。根据各组分的相对含量变化判断溶剂系统的适用性，具有实际意义。     

Efficient recovery of electrophoretic profiles of nucleoside metabolites from urine samples by multivariate curve resolution

Chemometric techniques usually employed in purity assessment and resolution of multicomponent peaks have been applied to analytical data from complex biological samples obtained with CE‐DAD. In the assessment of the purity of the electrophoretic peaks, the orthogonal projection approach, the orthogonal projection approach with Durbin–Watson criterion, and the simple‐to‐use interactive self‐modeling mixture analysis method have been employed. Multivariate curve resolution with alternating least squares has been successfully implemented to resolve co‐migrating peaks of metabolites in CE‐DAD and to recover qualitative and quantitative information about co‐migrating components of urine extract. The main challenge consisted of developing high‐quality multivariate curve resolution with alternating least squares models of multicomponent peaks acquired during the CE analysis of nucleoside patterns in 18 urine samples. The recovered ultraviolet visible (UV–Vis) spectra have been employed to identify additional nucleosides, such as 1‐methylinosine, 2‐methylguanosine, and 1‐methylguanosine, whose presence in the metabolic profile produced by the applied CE‐DAD method has not yet been recognized. Concentration profiles of these compounds can be used in metabonomic studies.     

Application of CE in hydrodynamically closed systems for analysis of bioactive compounds in food

CE is a family of electrokinetic separation techniques that separate compounds based upon differences in electrophoretic mobilities, phase partitioning, pI, molecular size, or a combination of one or several of these properties. CE has been used in several modes to analyze and characterize a wide variety of analytes from simple inorganic ions, small organic molecules, peptides, proteins, nucleic acids to virus, microbes and particles. Food consists of a complex mixture of a variety of components, many of which are biologically active. Components classified as "nutrients" are essential for growth, maintenance, and repair of the body. Other food constituents, typically occurring in small quantities, are classified as "biologically active substances" and they have beneficial or harmful effects on human health. There are two types of biologically active substances in food - naturally occurring and food additives. The bioactive compounds of food that will be mentioned in this review are inorganic and organic acids, amino acids, vitamins, phenolic compounds, biogenic amines, antinutrients, toxins, etc. This review is focused on the application of CE with hydrodynamically closed system (suppression of EOF) for the analysis of the above-mentioned compounds. CE can be an alternative method to HPLC or other methods for analysis of bioactive compounds in food. The main advantages of CE are low running cost (at least ten times than HPLC) and consideration to environment (hundreds of microliters of diluted water based electrolyte per analysis).     

Screening of aflatoxins in feed samples using a flow system coupled to capillary electrophoresis

A method is proposed which presents a new approach to the joint use of capillary electrophoresis (CE) commercial equipment and a flow system. This flow system allows the total determination of several compounds by using a fluorimetric screening system. The individual determination for each analyte is performed by the CE proposed method. The screening procedure uses simple equipment and operations and provides a yes/no binary response that occasionally requires confirmation. A fast, simple, and reliable method has been developed in order to determine the most frequent mycotoxins in feed samples using micellar electrokinetic capillary chromatography (MECC). An extraction step followed by a purification step was carried out on the samples in order to remove interference substances before analysis. A C18 column was chosen to concentrate the mycotoxins, and the analytes were eluted from C18 using methanol. The MECC method allows the separation of six mycotoxins within 50 min with a reproducibility as RSD between 7.45 and 13.06%, and a limit of detection (LOD) between 0.02 and 0.06 mg l(-1) for all the mycotoxins. These LODs were clearly below legal limits (0.05 mg l(-1)).     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。     

Rapid determination of salbutamol in pharmaceutical preparations by chiral capillary electrophoresis

A fast and simple method of chiral capillary electrophoresis (CE) has been applied to the analysis of salbutamol in different pharmaceutical preparations. Using of a 25 mM acetate buffer (pH 5.0), containing 13.1 mg/mL carboxymethyl-beta-cyclodextrin (CM-beta-CD), an applied voltage of 20 kV and a temperature of 25 degrees C, the enantiomers of salbutamol could be separated in about 2 min. Three different pharmaceutical preparations (two syrups, one oral solution, and two kind of tablets) containing a racemate of salbutamol were injected directly in the CE system, following dilution in dimethyl sulfoxide (DMSO). Appreciable differences in the retention times were observed for salbutamol enantiomers in the different formulations studied, which were attributed to the effect of the matrix components on the electrophoretic mobility. The standard addition method was used for the calibration due to the existence of matrix interferences. Finally, the stability of the enantiomers of salbutamol in the oral solution was studied calculating the enantiomeric ratio values when the solution was injected immediately after being opened in the first case and after being opened and stored in the fridge for two months in the second case.