Development of a capillary electrophoretic method for the separation of diastereoisomers of a new human immunodeficiency virus protease inhibitor

A capillary electrophoretic (CE) method was developed for the separation of diastereoisomers of a new human immunodeficiency virus (HIV) protease inhibitor TMC114. In total 16 isomers of this drug have been synthesized (eight pairs of enantiomers). We succeeded in the separation of the eight diastereoisomers, but no enantiomers could be separated. Because of the high similarity and water-insolubility of these isomers, the separation is a real challenge. Different CE modes were tried out: capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), micellar electrokinetic capillary chromatography (MEKC), and microemulsion electrokinetic capillary chromatography (MEEKC). Only MEEKC offered resolution of these compounds.     

Method Development of Enantiomer Separations by Affinity Capillary Electrophoresis,Cyclodextrin Electrokinetic Chromatography and Capillary Electrophoresis-Mass Spectrometry

 Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection     

不同缓冲体系对毛细管电泳法分离15种核苷类化合物效果的比较

对毛细管电泳法分离15种核苷类化合物所用的不同缓冲液体系进行了系统比较,确定不同模式毛细管电泳法分析多种核苷类化合物的最适合背景缓冲液体系(BGE)。分别以四硼酸钠、磷酸氢二钠、乙酸钠、碳酸氢钠、乙酸铵和乙二胺(DEA)为背景电解质,对毛细管区带电泳(CZE)、毛细管电泳-电喷雾飞行时间质谱(CE-ESI-TOF/MS)以及胶束电动毛细管电泳(MEKC)3种模式进行比较,并对其中几种优势缓冲体系进行了优化。结果表明,CZE模式下使用四硼酸钠和磷酸氢二钠缓冲体系无法同时分离15种核苷类化合物,因此只适用于分析核苷类化合物数量较少的样品。而使用含有2%丙酮的300 mmol/L DEA能完全分开15种核苷类化合物,且分辨率和峰形良好。MEKC模式下,以25 mmol/L磷酸氢二钠(添加70 mmol/L十二烷基磺酸钠(SDS))为缓冲盐的分离结果最佳,并且此方法能成功应用于海洋生物海葵中核苷类化合物的分离。CE-ESI-TOF/MS分析中,以20 mmol/L乙酸铵(pH 10.0)为背景电解质,正离子模式检测,15种核苷类化合物的质谱信号均良好,检测灵敏度明显优于文献中报道的使用DEA缓冲体系的结果。本研究阐明了不同缓冲体系对15种核苷类化合物分离的适用性,为毛细管电泳技术在复杂基质中多种核苷类化合物的分离方法中的应用奠定了基础。     

Comparative study of capillary zone electrophoresis and micellar electrokinetic chromatography for the separation of twelve aromatic sulphonate compounds

Summary Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), were investigated for the separation of 12 aromatic sulphonate compounds. In CZE, although the voltage applied, the buffer concentration and the pH were optimized for effective separation of the compounds studied, under the best conditions four of the five amino compounds coeluted, as did naphthalene-1-sulphonic acid and naphthalene-2-sulphonic acid. In MEKC, sodium dodecyl sulphate (SDS) and Brij 35 were chosen as the anionic and nonionic surfactants and the effect of the concentration of micelles was examined. The effect of adding methanol as the organic modifier was also investigated with each of these micellar systems. All the analytes, including the isomers, were completely separated by use of MEKC with Brij 35 but when SDS was used only 11 compounds were separated because two amino compounds coeluted.     

Recent advances of capillary electrophoresis in pharmaceutical analysis

This review covers recent advances of capillary electrophoresis (CE) in pharmaceutical analysis. The principle, instrumentation, and conventional modes of CE are briefly discussed. Advances in the different CE techniques (non-aqueous CE, microemulsion electrokinetic chromatography, capillary isotachophoresis, capillary electrochromatography, and immunoaffinity CE), detection techniques (mass spectrometry, light-emitting diode, fluorescence, chemiluminescence, and contactless conductivity), on-line sample pretreatment (flow injection) and chiral separation are described. Applications of CE to assay of active pharmaceutical ingredients (APIs), drug impurity testing, chiral drug separation, and determination of APIs in biological fluids published from 2008 to 2009 are tabulated.     

Separation of isomers of dienoic acids by electromigration techniques

Different capillary electromigration techniques were employed to resolve geometrical isomers of sorbic acid, decadienoic acid, and ethyl sorbate. Since these substances differ in their polarity, shape, and size, various electromigration approaches were investigated to separate the four geometrical isomers of each compound. With capillary electrophoresis (CE) modified with a cyclodextrin (β‐CD) the four isomers of sorbic acid were separated using a buffer that consists of 60 mM tetraborate and 8 mg/mL β‐CD. The separation of decadienoic acid geometrical isomers was not possible, even at elevated tetraborate and cyclodextrin concentrations. The four isomers of decadienoic acid were successfully separated using micellar electrokinetic chromatography (MEKC) with a buffer consisting of 30 mM tetraborate and 100 mM SDS and microemulsion electrokinetic chromatography (MEEKC). Ethyl sorbate is the least polar of all the studied substances and its isomers could not be separated by MEKC or MEEKC. The resolution was improved and isomers were fully separated using capillary electrochromatography (CEC) with ODS stationary phase and a mobile phase consisting of 10 mM boric acid in 50% acetonitrile. Minor differences in the polarity and the shape of isomers and high resolving power of the applied techniques were sufficient for separation of very similar compounds. We have shown that versatile electromigration techniques can be applied for separation of geometrical isomers of dienoic acids and their esters.     

毛细管电泳在手性氨基酸拆分中的研究进展

毛细管电泳(capillary electrophoresis,CE)作为一种强有力的手性分离技术,由于操作简单、试剂消耗少及柱效高等优点,受到广泛关注,是近年来手性分离领域的研究热点.氨基酸是组成蛋白质的基本单元,且大多数氨基酸具有手性中心,手性氨基酸是生命体系的一个重要特征.具有手性中心的氨基酸,其对映体间的生物活性往往存在着较大的差异,因此,氨基酸的手性拆分对了解人体及动物生命活动起着举足轻重的作用.主要总结了近5年来毛细管电泳的3种分离模式(毛细管区带电泳、胶束电动毛细管色谱、毛细管电色谱)在氨基酸手性拆分中的发展和应用.     

Optimization of solid-phase extraction and solid-phase microextraction for the determination of alpha- and beta-endosulfan in water by gas chromatography-electron-capture detection

The electrophoretic behaviour of ionizable and neutral alkylxanthines commonly used in pharmaceutical preparations was studied. The performance of various separation modes including capillary zone electrophoresis (CZE), cyclodextrin electrokinetic chromatography, and micellar electrokinetic chromatography (MEKC) with either sodium dodecyl sulfate (SDS) or bile salts as surfactants, was assessed. CZE in an alkaline medium successfully separates ionizable xanthines and dyphylline. The addition of carboxymethyl-β-cyclodextrin to the background electrolyte allows only partial resolution of neutral xanthines. Based on MEKC results, bile salts exhibit more discrimination ability than SDS to separate similar xanthines. The best results are provided by taurodeoxycholic acid, which ensures baseline separation of xanthines.     

Enantiomer separation by capillary electrophoresis using DEAE-dextran and aminoglycosidic antibiotics

Summary Diethylaminoethyl (DEAE) dextran hydrochloride and three kinds of aminoglycosidic antibiotics; fradiomycin sulfate, kanamycin sulfate and streptomycin sulfate, were employed as chiral selectors in capillary electrophoresis, enantiomer separation. These selectors are cationic or basic because of amino functionality and therefore used for enantiomer separation of acidic compounds. To avoid adsorption of the basic or cationic selectors on the capillary inner surface, a coated capillary was employed. Among those tested, enantiomers of binaphthyl compounds and synthetic intermediates of diltiazem analogues were separated. Methanol addition was effective for the improvement of peak shape and resolution.     

Enantioseparation of benzoins and enantiomer migration reversal of hydrobenzoin in capillary zone electrophoresis with dual cyclodextrin systems and borate complexation

Rapid, precise, accurate, and reproducible methodology using capillary electrophoresis (CE) with dynamically coated capillaries for the analysis of heroin and its basic impurities and adulterants is presented. Highly selective determination of the above solutes is obtained by analyzing the same sample preparation by two CE methods. For the determination of heroin, its basic impurities and basic adulterants, dynamic coating of the capillary surface is accomplished using a commercially available reagent kit with an added cyclodextrin ((CD) polycation coating followed by polyanion coating with dimethyl-beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin). The addition of a cyclodextrin to the run buffer significantly improves the separation of these solutes. Neutral, acidic, and weakly basic adulterants which migrate near or after t0 do not interfere with the more mobile basic solutes. The determination of neutral, acidic, and weakly basic adulterants in heroin is accomplished using a modification of the above commercially available reagent kit. After first coating with a polycation, a negative coating is obtained using a surfactant sodium dodecyl sulfate. Micellar electrokinetic chromatography (MEKC) with dynamically coated capillaries gives an excellent separation of the neutral, acidic, and weakly basic solutes, with considerably shorter run times compared to conventional MEKC. In addition for this system, most basic solutes in heroin have longer migration times than the uncharged and acidic compounds.     

Comparative study on the enantiomer separation of 1,1'-binaphthyl-2,2'diyl hydrogenphosphate and 1,1'-bi-2-naphthol by liquid chromatography and capillary electrophoresis using single and combined chiral selector systems

The chiral recognition ability of single and dual selectors, that were used as additives, have been investigated by HPLC and CE. Native beta- and gamma-cyclodextrins, permethylated beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, cholic acid and taurodeoxycholic acid sodium salts were applied as chiral selectors, whereas the atropisomers of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate, and 1,1'-bi-2-naphthol served as model compounds. It was found that all investigated selectors, except for gamma-cyclodextrin, display the same affinity pattern for binaphthyl enantiomers, i.e., binding the S more strongly than the R enantiomer. However, the differences in the phase distribution of chiral selectors led to the opposit elution order of enantiomers: with cyclodextrins, the first eluted is S enantiomer, while R is the first eluted for bile salts. Under the conditions studied, cyclodextrins (except gamma-cyclodextrin), as well as cholic acid sodium salts acting singly, enable the separation of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate enantiomers both by HPLC and CE methods, while 1,1'-bi-2-naphthol enantiomers were resolved only under CE conditions with permethylated cyclodextrin or bile salts. In both techniques the application of dual systems could improve resolution or make it worse (oreven cancel), depending on the sign of enantioselectivity of particular selectors, their concentrations and localization: mobile or stationary phase. It has been found that the mechanism of separation as well as interactions occurring between two selectors may be followed by using combined HPLC and CE methods. The obtained results proved that, as well as beta-CD, TM-beta-D and gamma-CD also form inclusion complexes with cholic acid sodium salts. The reversal of elution order may be realized by two procedures: changing a single selector, i.e., cyclodextrin on cholic acid sodium salt or vice versa, and by changing the proportion of selectors in the combined bile salt-cyclodextrin system.     

Enantioseparation of thalidomide and its hydroxylated metabolites using capillary electrophoresis with various cyclodextrins and their combinations as chiral buffer additives.

The separation of thalidomide (TD) and its hydroxylated metabolites including their simultaneous enantioseparation was studied in capillary electrophoresis (CE) using four different randomly substituted charged cyclodextrin (CD) derivatives, the combinations of some of them with each other, and beta-CD. TD, as well as two metabolites recently found in incubations of human liver microsomes and human blood, 5-hydroxythalidomide (5-OH-TD) and one of the diastereomeric 5'-hydroxythalidomides (5'-OH-TD), are neutral compounds. Therefore, they were resolved using charged chiral selectors in CE. Two different separation modes (normal polarity and carrier mode) and two different capillaries (fused-silica and polyacrylamide-coated) were tested. Based on the behavior of the individual CDs, their designed combinations were selected in order to improve the separation selectivity and enantioselectivity. Under optimized conditions all three chiral compounds and their enantiomers were resolved simultaneously.     

Dual opposite injection electrokinetic chromatography: nonionic microemulsion pseudostationary phase and novel approach to electrokinetic sampling bias

Dual opposite injection capillary electrophoresis (DOI-CE) is a family of CE techniques in which the sample is introduced into both ends of the capillary. For the analysis of compounds with widely varying pKa values using a voltage-driven separation scheme, DOI-CE is superior to conventional CE with sample introduction at only one end of the capillary due to DOI-CE's broader elution window. To enhance the DOI-CE separation, a running buffer with a microemulsion system was developed. Since DOI-CE works best under conditions of low electroosmotic flow (EOF), the suppression of EOF via the addition of a multiply charged cation (e.g., Zn2+) to the buffer was investigated, and was found to suppress the EOF effectively at moderate concentrations (2.5-10 mM). Three different dual opposite injection modes were studied: simultaneous electrokinetic injection, sequential electrokinetic injection, and sequential hydrodynamic injection. The injection bias in the first two electrokinetic injection modes was compared with the sequential hydrodynamic injection. Corrections in the bias of the electrokinetic injections were discussed, and an improved approach was suggested. Finally, the effect of the relative concentration of the multiply charged cation in the sample plug and running buffer on the peak shape of co-electroosmotic and counter-electroosmotic ions was examined, and found to be much more influential on the latter.     

Enantioselective HRGC Separation of Toxaphene Congeners by Ethylated γ‐Cyclodextrins

The enantioselectivity of three batches of octakis‐(2,3,6‐tri‐O‐ethyl)‐γ‐cyclodextrin (TEG‐CD), which differed significantly in their degree of ethylation, is reported for toxaphene congeners. The cyclodextrin composition was determined by high performance liquid chromatography‐mass spectrometry. Columns prepared with almost fully ethylated cyclodextrin showed no enantiomer separation. Increasing the average number of free OH groups up to an optimum of 3.8 allowed to resolve the following toxaphene congeners into enantiomers: Parlar no. 11, 12, 15, 21, 25, 32, 38, 42.1, 42.2, 50, 51, 56, 58, 59, 62, 69 as well as B8‐1412. More free OH groups did not improve the enantiomer resolution. The structure of the polysiloxane used for dilution of TEG‐CD also had an influence on the enantiomer separations achieved. Compared to OV 1701‐OH, the use of PS 086 significantly improved the enantiomer resolutions of the separated congeners. However, neither increasing the column length nor the cyclodextrin amount in the stationary phase led to better separation results.     

Orthogonal separations of nicotine and nicotine-related alkaloids by various capillary electrophoretic modes

The migration behaviour of nicotine and related tobacco alkaloids was investigated using three different capillary electrophoretic (CE) modes. Novel separations were achieved both using microemulsion electrokinetic chromatography (MEEKC) and nonaqueous CE (NACE). Improved resolution compared to previous studies was obtained using free-solution CE (FSCE). Each technique resulted in different, orthogonal separation selectivity. The suitability of each method for application to the analysis of nicotine lozenges is discussed. The FSCE method was applied to the analysis of nicotine lozenges due to its compatibility with an established lozenge extraction solvent. The method used gave good injection precision and linearity. Good agreement of CE and high-performance liquid chromatography (HPLC) results was obtained. The CE method is therefore considered suitable for the quantitative determination of nicotine in nicotine lozenges.     

Combination of capillary electrophoresis and molecular modeling to study the enantiomer affinity pattern between β‐blockers and anionic cyclodextrin derivatives in a methanolic and water background electrolyte

In order to have deep insights into the mechanisms of enantiomer affinity pattern in both aqueous and non‐aqueous systems, an approach combining capillary electrophoresis and molecular modeling was undertaken. A chiral β‐blocker; acebutolol, was enantioseparated in aqueous capillary electrophoresis and non‐aqueous capillary electrophoresis using two anionic β‐cyclodextrin derivatives. The enantiomer affinity pattern of acebutolol was found to be opposite when an aqueous background electrolyte was replaced with non‐aqueous background electrolyte in the presence of heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin but remained the same in the presence of heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin. Molecular docking of acebutolol into two β‐cyclodextrin derivatives indicated two distinct binding modes called ‘up’ and ‘down’ conformations. After structure optimization by molecular dynamics and energy minimization, both enantiomers of acebutolol were preferred to the ‘up’ conformation with heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin while ‘down’ conformation with heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin. The further calculation of the complex energy with solvent effect indicated that heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin had higher affinity to S‐acebutolol than R‐acebutolol in non‐aqueous capillary electrophoresis while it showed better binding to R‐acebutolol in aqueous capillary electrophoresis. However, the heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin bound better to R‐acebutolol in both aqueous and non‐aqueous capillary electrophoresis, implying that the binding mode played more important role in chiral separation of heptakis(2,3‐di‐O‐methyl‐6‐sulfo)‐β‐cyclodextrin while the solvent effect had prevailing impact on heptakis(2,3‐di‐O‐acetyl‐6‐sulfo)‐β‐cyclodextrin.     

A decade of capillary electrophoresis

Since the introduction of the first commercial capillary electrophoresis (CE) instrument a decade ago, CE applications have become widespread. Today, CE is a versatile analytical technique which is successfully used for the separation of small ions, neutral molecules, and large biomolecules and for the study of physicochemical parameters. It is being utilized in widely different fields, such as analytical chemistry, forensic chemistry, clinical chemistry, organic chemistry, natural products, pharmaceutical industry, chiral separations, molecular biology, and others. It is not only used as a separation technique but to answer physicochemical questions. In this review, we will discuss different modes of CE such as capillary zone electrophoresis, micellar electrokinetic chromatography, capillary gel electrophoresis, capillary isoelectric focusing, and capillary electrochromatography, and will comment on the future direction of CE, including array capillary electrophoresis and array microchip separations.     

毛细管电泳分析中手性化合物的定性检测

毛细管电泳（CE）作为一种新型分离分析技术,具有分离效率高、分析速度快、样品用量少、分离模式灵活多样等众多优势,在手性物质分离等领域应用广泛。在以往的工作中,手性化合物的CE分离模式、手性拆分剂选择及提高分离度等研究已作了详尽报道,而成功分离后的手性物质定性、对映体出峰顺序确认等问题也至关重要。该文以CE手性化合物分离分析中是否依赖标准品分类,及其定性检测方法进行了总结。     

Determination of Low-Molecular-Mass Phenolic and Non-Phenolic Lignin Degradation Compounds in Wood Digestion Solutions by Capillary Electrophoresis

 Different capillary electrophoretic techniques were investigated and compared for the separation of low-molecular-mass phenolic and neutral lignin degradation compounds. Simple capillary zone electrophoresis (CZE) was not suited for this problem. Switching to micellar electrokinetic capillary chromatography (MEKC) by adding micelle-forming reagents to the carrier electrolyte enhanced the separation performance considerably. Alternatively, microemulsion electrokinetic chromatography (MEEKC) was investigated. This is a CE technique in which analytes interact with moving oil droplets present in a microemulsion buffer. Using the optimized carrier electrolyte systems and a 60 cm×50 μm I.D. bubble cell capillary it was possible to analyse solutions of different wood digestion procedures and to investigate differences in lignin degradation products during use of different kinds of wood (eucalyptus, beech, scotch pine and acacia). Received August 25, 1999. Revision April 13, 2000