Chiral Separation In Capillary Electrophoresis Research Articles

Simultaneous Separation of Different Types of Amphetamine and Piperazine Designer Drugs by Capillary Electrophoresis with a Chiral Selector

The recent emergence of a new class of piperazine-type compounds has brought about the need for laboratory screening methods for both seized drugs and toxicological samples. These piperazine compounds, which include 1-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), exhibit comparable physiological effects and can be substituted for the classic amphetamine-type drugs. We have optimized a chiral capillary electrophoresis (CE) separation that detects a set of 6 piperazine and 4 chiral amphetamine compounds in under 23 min using a 200 mM phosphate buffer at a pH = 2.8 with 20 mM hydroxypropyl- beta-cyclodextrin (HPbeta3CD). In addition to the above compounds, a series of "clandestine" BZP diHCl samples were also analyzed using this method to assess the ruggedness of the procedure. The novel CE separation was tailored to simultaneously detect these piperzine compounds in addition to amphetamine-type drugs. Distinct migration time and UV-spectral data were obtained for all compounds of interest.

Phospholipid-lysozyme coating for chiral separation in capillary electrophoresis.

A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was developed for the chiral capillary electrophoretic (CE) separation of D- and L-tryptophan. As a kind of carriers, coated as phospholipid membranes onto the inner wall of a fused-silica capillary, liposomes are able to interact with basic proteins such as lysozyme, which may reside on the surface of the phospholipid membrane or permeate into the middle of the membrane. The interaction results in strong immobilization of lysozyme in the capillary. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the electroosmotic flow (EOF) was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. The liposome composition, the running buffer, and the capillary inner diameter all affected the chiral separation of D- and L-tryptophan. Coating with 4 mM M1C4 and then 1 mM phosphatidylcholine (PC)/phosphatidylserine (PS) (80:20 mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution. Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D- and L-tryptophan was achieved without lysozyme in the running buffer. The effects of different coating procedures and separation conditions on separation were evaluated, and the M1C4-liposome and liposome-lysozyme interactions were elucidated. The usefulness of protein immobilized into phospholipid membranes as a chiral selector in CE is demonstrated for the first time.

Influence of peak measurement parameters on the quality of chiral electrophoretic separations.

The effect of nine peak measurement/analysis parameters on chiral capillary electrophoresis (CE), followed by UV detection, was studied. The parameters pertained UV-detection (detection wavelength, reference wavelength, and wavelength bandwidths), signal processing (data acquisition rate, type and amount of filtering) and peak detection (detection threshold and peak width). The influence of these factors on the chiral separation of dimethindene enantiomers was studied at two different concentrations (i.e., at high and low signal-to-noise (S/N) ratio) by the means of experimental designs. The electropherogram characteristics considered were the resolution between the two enantiomers, the peak areas, and the S/N ratio. A D-optimal design was first used as screening design to identify the most critical parameters. Afterwards, a modelling of the different responses as a function of these critical parameters was performed based on the results of a face-centered central composite design. The results showed that the signal-processing parameters should be carefully selected when developing a CE separation since very important variations in the separation, the S/N ratio and the peak area of the substances can occur by setting these parameters at different levels. The detection wavelength should also be carefully chosen for optimal peak area measurement. The role of these parameters becomes more important with decreasing concentration of the analytes (i.e., low S/N ratio). This study showed that the peak measurement/analysis parameters should be optimized as the chemical and physical parameters of a method. They also should always be well specified in order to allow a good transfer of a method from one instrument to another.

Robustness testing of chiral separations by capillary electrophoresis using highly-sulfated cyclodextrins

The robustness of a generic method for chiral separation in capillary electrophoresis using highly-sulfated cyclodextrins in a low pH phosphate buffer and the “short-end injection technique” was studied. In this study, we focused on the robustness of the separations and not of the quantitative analysis of the enantiomers. The robustness was evaluated for the enantiomeric separation of a basic (propranolol), a neutral (praziquantel) and an acidic (warfarin) compound. The influence of eight factors which were believed to affect significantly the separations was studied using a 11-factor, 12-experiment Plackett–Burman design. Statistical interpretation of the factor effects on different analytical responses (selectivity and resolution) was performed. The separations of the three compounds could be considered as rather robust as the factor effects were generally not significant ( α=0.05) and small.

Fast enantioseparation of arylglycine amides by capillary electrophoresis with highly sulfated-β-cyclodextrin as a chiral selector

Nine racemic arylglycine amides were synthesized and successfully enantioseparated by capillary electrophoresis (CE) using highly sulfated β-cyclodextrin (HS-β-CD) as a chiral selector. Baseline enantioseparation of the analytes was obtained around neutral pH but not in the acidic conditions that are commonly used. HS-β-CD content, buffer pH, type and concentration, and organic modifier concentration were studied and optimized for fast and efficient separation. A chiral CE separation system composed of 1.5% (w/v) HS-β-CD, 0 to 10% (v/v) methanol and 20 m M 3-( N-morpholino)propanesulfonic acid at pH 6.5 was shown suitable for baseline enantioseparation of the mentioned amides within 6 min, including simultaneous enantioseparation of three positional isomer series (methyl-, methoxyl or chloro-substituted). By using this system, d-enantiomers migrated ahead of the l-enantiomers and the enantiomeric resolution order of arylglycine amides was more or less parallel to the p K a order of the analytes.

Enantiomer migration order in chiral capillary electrophoresis.

Enantiomer migration order (EMO) in chiral capillary electrophoresis (CE) represents a challenging issue, referred to in less than 20% of the articles on CE enantioseparations. This review article will (i) illustrate the actuality of the topic, (ii) discuss some technical problems related to EMO in CE enantioseparations, (iii) examine the principal differences between CE and other separation techniques from the viewpoint of enantiomer elution order, (iv) demonstrate the potential for a designed reversal of EMO in CE, and (v) emphasize the importance of studying EMO for better understanding of chiral CE as well as its more effective application. Along with CE, the results obtained by other instrumental techniques such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), X-ray crystallography, as well as molecular modeling calculations will be shortly discussed. Rather than referring to all published examples of the opposite migration order of enantiomers in CE, the emphasis will be on general aspects. Recently, the reversal of the EMO was described in detail in a book chapter (Chankvetadze, B., Capillary Electrophoresis in Chiral Analysis, Wiley & Sons, Chichester, UK 1997, Chapter 12) as well as in three review articles.

Chiral analysis of aliphatic short chain organic acids by capillary electrophoresis

Short chain organic acids play an important role in different areas such as biochemistry, clinical chemistry, or the food industry. The enantiomeric ratio of chiral metabolites is an important parameter for the understanding of metabolic processes and in many cases it can serve diagnostic purposes. On the other hand, the presence of racemates in food products could indicate the use of organic acids as additives; this is not always permitted and needs to be controlled. The short chain of these acids makes difficult the three point interaction generally accepted as necessary for chiral recognition. Relatively recent publications have demonstrated the feasibility of their direct chiral separation in capillary electrophoresis by various techniques utilizing exchange capillary electrophoresis, macrocyclic antibiotics, cyclodextrins, ion-pair method, and transition metal complexes. The present article describes existing methods and strategies proposed to advance these areas.

Recent developments in chiral capillary electrophoresis and applications of this technique to pharmaceutical and biomedical analysis.

This paper provides an overview of the current status of chiral capillary electrophoresis (CE). The emphasis is placed on the application of CE in chiral separation of various racemic compounds. During the last two years about 280 papers, several review articles, and two entire issues, edited by S. Fanali (Electrophoresis 1999, 20, 2577-2798, and H. Nishi and S. Terabe (J. Chromatogr. A 2000, 879, 1-471.) have been devoted to chiral CE. Enantiomeric separations of various compounds, e.g., pharmaceuticals, drug candidates, drugs and related metabolites in biological fluids, amino acids, di- and tri peptides, pesticides and fungicides, have been performed using different chiral selectors. Native and derivatized cyclodextrins continue to be the most widely used chiral selectors. Other chiral selectors such as natural and synthetic chiral micelles, crown ethers, chiral ligands, proteins, oligo- and polysaccharides, and macrocyclic antibiotics have also been applied to chiral CE separations.

Modeling and optimization of the chiral selectivity of basic analytes in chiral capillary electrophoresis with negatively charged cyclodextrins using electrochemical detection.

A mathematical model concerning the separation selectivity of basic analytes in chiral capillary electrophoresis (CE) modified with negatively charged cyclodextrins (CDs) has been presented to describe the dependence of chiral selectivity on the buffer pH and the chiral selector concentration. The electrophoretic method to determine the parameters of the model has also been developed. The model has been tested with racemic epinephrine and isoproterenol as target analytes and sulfonated beta-CD as chiral selector. The agreements have been found between the calculated and the measured values. Some significant conclusions to optimize chiral CE separation have been derived from the model and proven by the experiments. Electrochemical detection was used to meet the requirement of the low introduced concentration of analytes.

Chiral Separations in Capillary Electrophoresis

The marked increase in the number of communications on the utilization of electrophoresis for practical chiral separations within the last three years is the most evident, and the most important fact. It reveals that the basic period of intensive research in the field is finished. The search for chiral selectors discriminating racemates in a reasonably analytical manner and the study of both the mechanism and physicochemical aspects of the chiral discrimination process were the main features of that period. Here, we review the state of the art in the field and state the references of the related literature up to the end of 1998.

Recent innovations in the use of charged cyclodextrins in capillary electrophoresis for chiral separations in pharmaceutical analysis.

A review is presented on the use of charged cyclodextrins (CDs) as chiral selectors in capillary electrophoresis (CE) for the separation of analytes in pharmaceutical analysis. An overview is given of theoretical models that have been developed for a better prediction of the enantiomeric resolution and for a better understanding of the separation mechanism. Several types of charged CDs have been used in chiral capillary electrophoretic separation (anionic, cationic, and amphoteric CDs). Especially the anionic CDs seem to be valuable due to the fact that many pharmaceutically interesting compounds can easily be protonated (e.g., amine groups). For that reason several anionic CDs are now commercially available. Cationic and amphoteric CDs are less common in chiral analysis and only a few are commercially available. Attention is paid to the most common synthesis routes and the characterization of the CDs used in chiral capillary electrophoretic separations. The degree of substitution in the synthesized CDs may vary from one manufacturer to another or even from batch to batch, which may have a detrimental effect on the reproducibility and ruggedness of the separation system. In Sections 4, 5, and 6 the applications of anionic, cationic, and amphoteric CDs for the chiral separation in CE are described. Many interesting examples are shown and the influence of important parameters on the enantioselectivity is discussed.

When a truck becomes a motorcycle: the impact of sample load on a chiral capillary electrophoresis separation using mixtures of neutral and sulfated cyclodextrins.

Chiral capillary electrophoresis (CE) separations are useful for monitoring the presence of a minor isomer at low levels (e.g., <0.5%) in the presence of the major form. In order to quantitate these low levels, it is necessary to inject large amounts of sample. Separations which appear to have more than enough resolution ("big enough to drive a truck through) for dilute, equal-concentration mixtures of isomers can become inadequately resolved when the necessary amount of sample is injected. This paper addresses some important considerations in maintaining adequate resolution at high sample loads for chiral separations involving a dual-cyclodextrin (CD) system. For hydrophobic compounds, the use of both a neutral and a sulfated CD can be helpful in achieving a chiral separation. In such a system, the migration time and resolution can be controlled by varying the ratio of neutral to charged CD concentrations. It is demonstrated here that not only the ratio, but also the total CD concentration can significantly affect the separation. In this paper, the impact of the total CD concentration in a dual-CD system (with the concentration ratio constant) is examined with respect to peak shape and resolution. The influences of temperature, capillary diameter, and current are also considered. The corresponding impact on the amount of sample which can be loaded and successfully separated determines the limit of quantitation of the minor isomer. Thus, this information is important in making such chiral separations applicable to determinations of low levels of minor isomer in the presence of large amounts of the major form.

Chiral separations in capillary electrophoresis.

The marked increase in the number of communications on the utilization of electrophoresis for practical chiral separations within the last three years is the most evident, and the most important fact. It reveals that the basic period of intensive research in the field is finished. The search for chiral selectors discriminating racemates in a reasonably analytical manner and the study of both the mechanism and physicochemical aspects of the chiral discrimination process were the main features of that period. Here, we review the state of the art in the field and state the references of the related literature up to the end of 1998.

Chiral separations in capillary electrophoresis

The marked increase in the number of communications on the utilization of electrophoresis for practical chiral separations within the last three years is the most evident, and the most important fact. It reveals that the basic period of intensive research in the field is finished. The search for chiral selectors discriminating racemates in a reasonably analytical mannerand the study of both the mechanism and physicochemical aspects of the chiral discrimination process were the main features of that period. Here, we review the state of the art in the field and state the references of the related literature up to the end of 1998.

Capillary Electrophoresis with Electrochemical Detection for Chiral Separation of Optical Isomers

The enantiomers of two amine derivatives were directly separated by capillary electrophoresis (CE), employing β-cyclodxtrin (β-CD) as a chiral additive in strongly alkaline solutions. The analytes were detected by electrochemistry, using a copper disk electrode at +675 mV vs Ag/AgCl reference electrode. Both the free enantiomers and the enantiomer-cyclodxtrin inclusion complexes could be detected using this approach, although the complexed forms gave lower oxidation currents than the free forms. Factors affecting the chiral CE separation of the analytes, such as working potential, concentration of running buffer and β-CD, and applied voltage, were extensively investigated. Under the optimum conditions, baseline separation of the enantiomers could be accomplished in less than 18 min. In addition, a successful application of the method to the enantiomeric purity determination confirmed its validity and practicability.

Nonaqueous capillary electrophoresis chiral separations with quaternary ammonium β-cyclodextrin

Chiral separation of nonsteroidal anti-inflammatory drugs (profens), 1,1′-binaphthyl-2,2′-diyl-hydrogen phosphate, N-[1-(1-naphthyl)ethyl]phthalamic acid, and derivatized amino acids by a cationic cyclodextrin, quaternary ammonium β-cyclodextrin (QA-β-CD), was investigated by capillary electrophoresis (CE) in both aqueous and nonaqueous media. Several profens and amino acids could only be separated by QA-β-CD in pure formamide system. No chiral separation of the profens was achieved in the following solvents: N-methylformamide, methanol, dimethyl sulfoxide, and water; however, chiral separations of most of the amino acids were obtained in all of these solvents. The effects of other experimental parameters such as the CD concentration and apparent pH (pH*) were also investigated. The first application of nonaqueous CE chiral separation of ketoprofen in a commercially available sample, Actron, was also examined. In addition, the reversal of electroosmotic flow by QA-β-CD was observed in water, formamide, N-methylformamide, methanol, and dimethyl sulfoxide media.

Use of chiral and achiral ion-pairing reagents in combination with cyclodextrins in capillary electrophoresis

The suitability of chiral and achiral acidic ion-pairing reagents to effect chiral separation in capillary electrophoresis in the presence of cyclodextrins (CDs) has been demonstrated previously. This method was applied to a great variety of analytes. Besides alkylsulfonates, alkanoic acids are able to improve chiral resolution in combination with CD. The dependence of the separation on alkyl chain length is described. The use of the cationic ion-pairing reagent quinine, is a powerful tool in influencing enantiomeric separation of acidic and basic analytes. Methods of direct and indirect UV detection are used in quinine containing running buffers. The influences of pH value, quinine and CD concentration on the separation factors are reported. l-Hyoscyamine showed the best capability to improve the enantioseparation of propranolol.

Training software for chiral separations in capillary electrophoresis

A previously published steady-state simulation program for CE was extended with a sub-menu for chiral interaction. The interaction was modelled with a hypothetical (neutral) selector with properties similar to cyclodextrins. A three-type chiral interaction model was implemented in such a way that it was valid for both anionic and cationic analytes. User-defined chiral variables in the model were: the concentration of this chiral selector, the complex formation constants (K) of both non-ionic and ionic analyte species, the relative K differences between the two optical isomers (also for both non-ionic and ionic species) and the mobility of the analyte–selector complex, relative to that of the fully dissociated free analyte. In addition the following non-chiral variables are added: the dependence of the bulk viscosity on the chiral selector concentration and the dependence of the mobilities on the viscosity. Chiral components in the data base are simulated as mixtures of optical isomers with variable enantiomeric concentration ratios. These chiral parameters, together with the original features of the program, provide a very flexible training tool for chiral separations in capillary electrophoresis. For example, determination of the optimum chiral selector concentration can be done in seconds, using the simulation software. Although the software is intended mainly for training purposes, in cases where sufficient details of existing chiral selector–analyte combinations are available, the program is also suitable for method development of chiral separations in CE.The performance of the program is illustrated with simulations, using chiral parameters obtained from literature (ibuprofen, fenoprofen, homatropine) and from our own experiments (m-fenfluramine). Simulation results were compared with experimental electropherograms.

Use of 1H-NMR spectroscopy to determine the enantioselective mechanism of neutral and anionic cyclodextrins in capillary electrophoresis

One-dimensional (1D) and two-dimensional (2D) 1H nuclear magnetic resonance (NMR) techniques have been used to investigate the chiral recognition process in capillary electrophoresis (CE) for seven different cyclodextrins (CDs) with the calcium channel blocker amlodipine as a model compound. These include five neutral CDs (α-CD, β-CD, γ-CD, hydroxypropyl-β-CD and hydroxyethyl-β-CD) and two anionic CDs (sulphobutyl-ether-β-CD and carboxymethyl-β-CD) where mixtures of amlodipine with each of the seven CDs were examined by 1D NMR in deuterated phosphate buffer at pD 3.4. The resonance shift of signals with added CD, relative to the CD-free position (shift displacement, Δδ) and shift non-equivalence (Δδ ∗) of enantiomeric signals shifted relative to each other after addition of CD were examined for non-overlapped protons of amlodipine. The possible correlations of NMR shift non-equivalence data with chiral separation in CE for amlodipine have been critically assessed. Qualitative differences in the 1D NMR shifts and enhanced enantioselectivity in CE were observed for amlodipine with sulphobutyl-ether-β-CD. Further experiments on the through-space interactions using 2D rotating frame nuclear Overhauser effect spectroscopy (ROESY) indicated that there was no association between internal glucopyranose hydrogen atoms and the aromatic hydrogens of amlodipine. This gives evidence for the aromatic ring not being included in this CD. Moreover, data from spin-lattice relaxation times ( T 1) measured for amlodipine in the free state and after addition of the anionic sulphobutyl-ether-β-CD indicate that the aromatic moiety of amlodipine is not included into the sulphobutyl-ether-β-CD cavity. There is evidence that it interacts with the sulphobutyl side chains, and may adopt a preferred orientation outside the sulphobutyl-ether-β-CD toroid itself.

Chiral separations in capillary electrophoresis using proteins as stereoselective binding agents.

Proteins are receiving increased attention in capillary electrophoresis (CE) for use as ligands in chiral separations. This review examines the types of proteins that have been employed in CE for the analysis of chiral compounds and provides a summary of applications for such an approach. Several formats for this type of analysis are discussed, including methods that use immobilized proteins (i.e., affinity capillary electrochromatography and capillary gel affinity electrophoresis) or techniques that use proteins as running buffer additives (i.e., affinity capillary electrophoresis). The role of various experimental factors in these separations are considered, and a comparison is made between the general analytical properties of CE systems that use immobilized versus solution-phase proteins.