Recent advances in enantiomer separations by affinity capillary electrophoresis using proteins and peptides

Enantiomer separations by capillary electrophoresis (CE), using proteins as chiral selectors--affinity capillary electrophoresis (ACE) with free solutions and capillary electrochromatography (CEC)--with protein immobilized capillaries, are reviewed. The separation principle, recent advances in this field and some interesting topics are presented. In ACE, various enantiomer separations have been already reported using either plasma proteins or egg white ones. Miscellaneous proteins were also explored in the last few years. On the contrary, only a limited number of enantiomer separations have been successfully achieved in CEC. CEC is not yet mature enough to date, and further investigations, such as efficiency, durability and reproducibility of capillaries, will be necessary for the use of routine analyses. The study of enantioselective drug-protein binding is important in pharmaceutical developments. Some applications including high-performance CE/frontal analysis (HPCE/FA) are introduced in this paper.     

Semi-preparative chromatographic purification of the enantiomers S-(−)-amlodipine and R-(+)-amlodipine

Pharmacokinetic studies of optically pure compounds after single enantiomer administration are becoming increasingly important. The process of racemization in vivo can diminish all expected advantages of single enantiomer treatment. Amlodipine, one of the calcium channel blockers, currently used in therapy as a racemate, is one of such drugs under study. In order to administer single enantiomers of amlodipine to healthy volunteers both were chromatographically purified and characterised. The two optical isomers of amlodipine, active S-(−)- and non-active R-(+)-amlodipine, were purified using chromatographic procedure adopted from the analytical separation. Enantiomers were successfully converted to benzenesulphonic salt without any racemization. All semi-preparative purifications were monitored with complementary analytical methods, HPLC and CE, along with the determination of optical activity so that the final product was sufficiently defined for further in vivo studies. The analytical method developed for the determination of plasma concentrations of each enantiomer of amlodipine in these studies is also briefly described.     

Semi-preparative chromatographic purification of the enantiomers S-(−)-amlodipine and R-(+)-amlodipine

Pharmacokinetic studies of optically pure compounds after single enantiomer administration are becoming increasingly important. The process of racemization in vivo can diminish all expected advantages of single enantiomer treatment. Amlodipine, one of the calcium channel blockers, currently used in therapy as a racemate, is one of such drugs under study. In order to administer single enantiomers of amlodipine to healthy volunteers both were chromatographically purified and characterised. The two optical isomers of amlodipine, active S-(−)- and non-active R-(+)-amlodipine, were purified using chromatographic procedure adopted from the analytical separation. Enantiomers were successfully converted to benzenesulphonic salt without any racemization. All semi-preparative purifications were monitored with complementary analytical methods, HPLC and CE, along with the determination of optical activity so that the final product was sufficiently defined for further in vivo studies. The analytical method developed for the determination of plasma concentrations of each enantiomer of amlodipine in these studies is also briefly described.     

Chiral discrimination by HPLC and CE and antifungal activity of racemic fenticonazole and its enantiomers

Fenticonazole is a chiral antifungal agent, used in therapy as the racemic mixture. The investigation on the chirality of fenticonazole is reported in this study. rac-Fenticonazole was resolved by HPLC and by capillary electrophoresis (CE). The chiral stationary phase (CSP), used in HPLC, was Daicel OD-H, a commercial phase, which allowed the separate collection of the two enantiomers. The chiral selectors used for CE were some cyclodextrin derivatives. The analysis time required from CE was about the half the HPLC enantioseparation time. The biological activity of the rac-mixture and each individual enantiomer was tested against Cryptococcus neoformans and two Aspergillus nidulans strains. The minimum inhibitory concentration (MIC) evaluation showed that the eutomer was the enantiomer chromatographically more retained and had a longer migration time in the electrophoretic enantioseparation. The CD spectrum of the eutomer showed a positive Cotton effect.     

Simultaneous enantioselective separation of azelastine and three of its metabolites for the investigation of the enantiomeric metabolism in rats. I. Liquid chromatography-ionspray tandem mass spectrometry and electrokinetic capillary chromatography

Enantioselective separation methods and the enantioselective determination of the anti-allergic drug azelastine and of three of its main phase I metabolites in a biological matrix underwent chromatographic and electrophoretic investigations. An enantioselective assay of a coupling of HPLC using a beta-cyclodextrin chiral stationary phase to ionspray tandem mass spectrometry is presented. Additionally, this assay is compared to another enantioselective assay using electrokinetic capillary chromatography with beta-cyclodextrin and carboxymethyl-beta-cyclodextrin in polyacrylamide-coated capillaries. For capillary electrophoresis (CE) the importance of polyacrylamide coating for the validation of this separation method is highlighted. Extracted rat plasma samples of enantioselective metabolism studies were measured by both validated assays. Differences in the pharmacokinetics and pharmacodynamics were evaluated for the main substance azelastine and its main metabolite demethylazelastine. So, a first hint about the enantioselectivity of biotransformation of azelastine in rats was seen after oral application of either enantiomer or the racemate to rats.     

Role of chemical structure in molecular recognition by transferrin

Studies of molecular recognition of chiral compounds by proteins are of importance from many points of view. The biological role of proteins in their interaction with small molecules is of fundamental interest and can be used in many different fields, for instance for in vitro analysis of optically active compounds. Studies in these areas need a detailed study of the interaction sites on the protein surface and the relationship between chemical structure and the complex formation ability of small molecules, such as drugs. The electrophoretic migration of charged compounds through a protein zone may provide information about the surface properties of the macromolecule in the interaction site. The interaction of human serum transferrin with tryptophan-methyl- (TME), ethyl- (TEE) and butyl-esters (TBE) has been investigated by capillary electrophoresis (CE) and model calculations. Differences in the separation of tryptophan derivatives were obtained by varying experimental parameters such as, pH, ionic strength of background electrolyte and the length of transferrin zone. Limited separation of the enantiomer pairs were observed at pH 5 and 7 with a maximum resolution at pH 6. The size of the ligands coupled to the chiral centre has importance in stereoselective recognition; however, a direct comparison of resolution different in same runs may lead to false conclusion if the experimental conditions are not comparable. With a careful evaluation of the data we obtained significant differences between the resolution of the smallest enantiomer pair compared to those of tryptophan derivatives with longer alkyl chains.     

A convenient and validated enantiomer separation of chiral aliphatic amines as nitrobenzoxadiazole derivatives on polysaccharide‐derived chiral stationary phases under simultaneous ultraviolet and fluorescence detection

A convenient method using a fluorogenic agent, 4‐chloro‐7‐nitro‐1,2,3‐benzoxadiazole (NBD‐Cl), was developed for enantiomer separation of chiral aliphatic amines including amino alcohols by normal high‐performance liquid chromatography. The enantiomer separation of chiral aliphatic amines as NBD derivatives was performed on six covalently bonded and four coated‐type polysaccharide‐derived chiral stationary phases (CSPs) under simultaneous ultraviolet (UV) and fluorescence detection (FLD). Among the covalently bonded CSPs, Chiralpak IE showed the best enantiomer separation for most analytes. The other CSPs also showed good enantioselectivity except for Chiralpak IB. On the other hand, Chiralpak AD‐H and Amylose‐1 generally exhibited better enantiomer separation of NBD derivatized chiral amines among the coated CSPs. The developed analytical technique was also applied to determine the optical purity of commercially available (R)‐ and (S)‐leucinol; the impurity was found to be 0.06%. The developed method was validated and proved to be an accurate, precise, sensitive, and selective method suitable for separation of chiral aliphatic amines as NBD derivatives under simultaneous UV and FLD.     

Separation methods for pharmacologically active xanthones

Xanthones, as a kind of polyphenolic natural products with many strong bioactivities, are attractive for separation scientists due to the similarity and diversity of their structures resulting in difficult separation by chromatographic methods. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are traditional methods to separate xanthones. Recently, capillary electrophoresis (CE), as a micro-column technique driven by electroosmotic flow (EOF), with its high efficiency and high-speed separation, has been employed to separate xanthones and determine their physicochemical properties such as binding constants with cyclodextrin (CD) and ionization constants. Since xanthones have been used in clinic treatment, the development of chromatographic and CE methods for the separation and determination of xanthones plays an essential role in the quality control of some herbal medicines containing xanthones. This article reviewed the separation of xanthones by HPLC, TLC and CE, citing 72 literatures. This review focused on the CE separation for xanthones due to its unique advantages compared to chromatographic methods. The comparison of separation selectivity of different CE modes including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC) and capillary electrochromatography (CEC) was discussed. Compared with traditional chromatographic methods such as HPLC and TLC, CE has higher separation efficiency, faster separation, lower cost and more flexible modes. However, because of low sensitivity of UV detector and low contents of xanthones in herbal medicines, CE methods have seldom been applied to the analysis of real samples although CE showed great potential for xanthone separation. The determination of xanthones in herbal medicines has been often achieved by HPLC. Hence, how to enhance CE detection sensitivity for real sample analysis, e.g. by on-line preconcentration and CE-MS, would be a key to achieve the quantitation of xanthones.     

LC/CE-MS tools for the analysis of complex arabino-oligosaccharides

Recently, various branched arabino-oligosaccharides as present in a sugar beet arabinan digest were characterized using NMR. Although HPAEC often has been the method of choice to monitor the enzymatic degradation reactions of polysaccharides, it was shown that HPAEC was incapable to separate all known linear and branched arabino-oligosaccharides present. As this lack of resolution might result in an incorrect interpretation of the results, other separation techniques were explored for the separation of linear and branched arabino-oligosaccharides. The use of porous-graphitized carbon liquid chromatography with evaporative light scattering and mass detection as well as capillary electrophoresis with laser-induced fluorescence and mass detection demonstrated the superiority of both the techniques toward HPAEC by enabling the separation and unambiguous identification of almost all the linear and branched arabino-oligosaccharides available. The elution behavior of all arabino-oligosaccharides for the three tested separation techniques was correlated with their chemical structures and conclusions were drawn for the retention mechanisms of the arabino-oligosaccharides on the different chromatographic and electrophoretic systems. The combination of the elution/migration behavior on LC/CE and the MS fragmentation patterns of the arabino-oligosaccharides led to the prediction of structures for new DP6 arabino-oligosaccharides in complex enzyme digests.     

Enantiomer self-disproportionation and chiral stationary phase based selective chiral separation of organic compounds

In modern chromatography, chiral stationary phase (CSP) and enantiomer self-disproportionation (ESD) are new inventions of packing material offer a guarantee for a successful enantiomeric separation. All CSPs were synthesized by chemical bonding of the relevant organic moieties onto a porous parent silica material for the separation of various racemic mixtures whereas achiral silica matrix was used for separation of non-racemic mixtures in ESD. Our present study provides to establish an understanding on the entire enantio-selective profile of amino alcohol based CSP as well as ESD and their precise utilization for high success rates for selective enantiomer separation with its appropriateness.     

Analysis of sugars in traditional Chinese drugs

This review is presented of chromatography and electromigration methods currently in use to determine sugars in traditional Chinese drugs: gas chromatography (GC), high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel column chromatography (GCC), paper chromatography (PC) and thin layer chromatography (TLC), capillary electrophoresis (CE) and gel electrophoresis (GEP). The detection methods combined with above separation methods including ultra-violet, mass spectra, fluorescent light, refractive index (RI), electrochemical detection are also described. For the complicacy of structural analysis of polysaccharides in traditional Chinese drugs, the hyphenation procedures concerned with this analysis are introduced in this article too.     

Chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether for enantiomer separation of amino compounds using a normal mobile phase

In order to apply the excellent chiral recognition ability of chiral pseudo-18-crown-6 ethers that we developed to chiral separation, we prepared a chiral stationary phase (CSP) by immobilizing a chiral pseudo-18-crown-6-type host on 3-aminopropyl silica gel. A chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. A liquid chromatography system using this CSP combined with the detection by mass spectrometry was used for enantiomer separation of amino compounds. A normal mobile phase can be used on this CSP as opposed to conventional dynamic coating-type CSPs. Enantiomers of 18 common natural amino acids were efficiently separated. The chiral separation observed for amino acid methyl esters, amino alcohols, and lipophilic amines was fair using this HPLC system. In view of the correlation between the enantiomer selectivity observed in chromatography and the complexion in solution, the chiral recognition in host-guest interactions might contribute to this enantiomer separation.     

Microbial carbohydrate esterases deacetylating plant polysaccharides

Several plant polysaccharides are partially esterified with acetic acid. One of the roles of this modification is protection of plant cell walls against invading microorganisms. Acetylation of glycosyl residues of polysaccharides prevents hydrolysis of their glycosidic linkages by the corresponding glycoside hydrolases. In this way the acetylation also represents an obstacle of enzymatic saccharification of plant hemicelluloses to fermentable sugars which appears to be a hot topic of current research. We can eliminate this obstacle by alkaline extraction or pretreatment leading to saponification of ester linkages. However, this task has been accomplished in a different way in the nature. The acetyl groups became targets of microbial carbohydrate esterases that evolved to overcome the complexity of the plant cell walls and that cooperate with glycoside hydrolases in plant polysaccharide degradation. This article concentrates on enzymes deacetylating plant hemicelluloses excluding pectin. They are currently grouped in at least 8 families, specifically in CE families 1–7 and 16, originally assigned as acetylxylan esterases, the enzymes acting on hardwood acetyl glucuronoxylan and its fragments generated by endo-β-1,4-xylanases. There are esterases deacetylating softwood galactoglucomannan, but they have not been classified yet. The enzymes present in CE families 1–7 differ in structure and substrate and positional specificity. There are families behaving as endo-type and exo-type deacetylates, i.e. esterases deacetylating internal sugar residues of partially acetylated polysaccharides and also esterases deacetylating non-reducing end sugar residues in oligosaccharides. With one exception, the enzymes of all mentioned CE families belong to serine type esterases. CE family 4 harbors enzymes that are metal-dependent aspartic esterases. Three-dimensional structures have been solved for members of the first seven CE families, however, there is still insufficient knowledge about their substrate specificity and real physiological role. Current knowledge on catalytic properties of the selected families of CEs is summarized in this review. Some of the families are emerging also as new biocatalysts for regioselective acylation and deacylation of carbohydrates.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

无胶筛分毛细管电泳分离DNA片段的现状与展望

毛细管电泳已DNA片段分离分析的重要手段。本简述了毛细管电泳中采用无胶筛分介质分离DNA片段的机理研究,介绍了筛分介质近年的研究发展状况,依据分离介质的化学组成,分单聚物、共聚物和混聚物等3个部分进行了评述,并对其发展前景进行了展望。     

Enantiomer self-disproportionation of chiral compounds on achiral ordered mesoporous silica M41S and regular silica gel as a stationary phase

Chromatographic behavior of nonracemic mixtures, viz., mandelic acid and stilbene oxide as analytes has been studied in detailed by enantiomer self-disproportionation on achiral ordered mesoporous material M41S and regular silica gel as stationary phases. Enantiomer self-disproportionation gave enhanced separation of analytes. The extent and magnitude of enantiomer self-disproportionation is dependent on the optical purity of the starting non-racemic molecules, presence of intermolecular hydrogen bonding/pi-pi interactions and the nature of eluents used. The present study and previous literature data suggest that percentage ee of a nonracemic mixture needs to be determined before any chromatographic purification is taken up as enantiomer self-disproportionation phenomenon could occur during purification. The data show that enantiomer self-disproportionation of nonracemic mixtures can be harnessed for its enantioenrichment on inexpensive achiral stationary phases.     

Enantioseparation of citalopram enantiomers by capillary electrophoresis: Method development through experimental design and computational modeling

Citalopram (CIT) is a frequently used modern antidepressant that inhibits selectively serotonin reuptake in the brain. It has a chiral center in its structure and is used in therapy as both racemic mixture and pure enantiomer as its pharmacological effect is almost entirely associated with S-CIT. The aim of this study was the development of a simple and rapid capillary electrophoresis (CE) method for the separation and quantification of CIT enantiomers. To establish the optimum chiral selector, several native and derivatized, neutral, and ionized cyclodextrins (CDs) were examined at different pH levels. An experimental design strategy was adopted for method optimization; a fractional factorial design was applied for screening purposes to identify significant experimental factors followed by a face-centered central composite design used for optimization purposes. Computational modeling was used to obtain information on the interaction energy and the geometry of the complexes to aid in the understanding of chiral separation mechanism. The best results were obtained when using a 25-mM phosphate buffer at pH 7.0, 3-mM CM-β-CD as chiral selector, 17.5°C temperature, 15-kV voltage, and 50 mbar/s hydrodynamic injection. The separation time was fast, below 3 min, and the migration order was S-CIT followed by R-CIT. The analytical performance of the method was verified in terms of precision, linearity, accuracy, sensibility, and robustness, and the method was applied for the determination of CIT enantiomers from pharmaceutical preparations.     

Enantiomeric separation of N‐protected amino acids by non‐aqueous capillary electrophoresis using quinine or Tert‐butyl carbamoylated quinine as chiral additive

A capillary electrophoretic (CE) method for the enantioseparation of N‐protected chiral amino acids was developed using quinine and tert‐butyl carbamoylated quinine as chiral selectors added to nonaqueous electrolyte solutions (NACE). A series of various N‐derivatized amino acids were tested as chiral selectands, and in order to optimize the CE enantioseparation of these compounds, different parameters were investigated: the nature of the organic solvent, the combination of different solvents, the nature and the concentration of the background electrolyte, the selector concentration, the capillary temperature, and the applied voltage. The influence of these factors on the separation of the analyte enantiomers and the electroosmotic flow was studied. Generally, with tert‐butyl carbamoylated quinine as chiral selector, better enantioseparations were achieved than with unmodified quinine. Optimum experimental conditions were found with a buffer made of 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM tert‐butyl carbamoylated quinine in an ethanol–methanol mixture (60:40 v/v). Under these conditions, DNB‐Leu enantiomers could be separated with a selectivity factor (α) of 1.572 and a resolution (Rs) of 64.3; a plate number (N) of 127,000 and an asymmetry factor (As) of 0.93 were obtained for the first migrating enantiomer. Chirality 11:622–630, 1999. © 1999 Wiley‐Liss, Inc.     

Clinical applications of capillary electrophoresis

Capillary electrophoresis (CE) is an extremely sensitive technique, which has been used in the clinical laboratory for almost 10 yr. The components of CE instrumentation are described, as are injection modes, buffers, and effects of electroosmotic flow. The modes of separation used in CE, namely, capillary zone electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic capillary chromatography, are explained. References for 26 different clinical applications of CE are included, among them assays that are used routinely as well as niche assays for specialized applications of CE. Verification of CE assays, current instrumentation, and future development of CE in the clinical laboratory are addressed.