2880 Ann Van Eeckhaut Yvette Michotte

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Pharmaceutical Institute, Vrije Universiteit Brussel, Brussels, Belgium

Electrophoresis 2006, 27, 28802895

Review

Chiral separations by capillary electrophoresis: Recent developments and applications

This paper provides an overview of the different classes of chiral selectors that are used in CE. The main properties of every class are described, together with the mechanism of enantioseparation. Newly introduced selectors are also discussed. Pharmaceutical and biomedical applications published from January 2004 till March 2005 are summarized. Keywords: Biomedical applications / Capillary electrophoresis / Chiral selectors / Pharmaceutical applications DOI 10.1002/elps.200500375

Received January 27, 2005 Revised December 21, 2005 Accepted December 28, 2005

1 Introduction
Many pharmaceutical compounds possess one or more chiral centers responsible for the optical activity of the drug. At the molecular level, biological systems are homochiral. Therefore, each enantiomer of a chiral compound can interact differently with chiral targets as receptors, enzymes and ion channels. As a result, both enantiomers can differ in their pharmacological and toxicological properties. Enantioselective analysis methods are therefore required not only for pharmacodynamic and pharmacokinetic studies but also for toxicological studies in development, and during quality control of drug substances and drug products. At present, LC still dominates chromatographic enantiomeric analysis in industry [1]. However, over the last two decades CE has proven to be a powerful alternative to chromatography. CE offers a tremendous flexibility for enantiomeric separations because a wide variety of chiral additives are available.
Correspondence: Professor Dr. Yvette Michotte, Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium E-mail: ymichot@vub.ac.be Fax: 132-2-477-4113 Abbreviations: 2-AHP--CD, 2-O-acetonyl-2-O-hydroxypropyl-bCD; CM--CD, carboxymethyl-b-CD; DM--CD, heptakis(2,6-di-Omethyl)-b-CD; DS, degree of substitution; HDAS--CD, heptakis(2,3-di-O-acetyl-6-O-sulfo)-b-CD; HP--CD, hydroxypropyl-bCD; ODAS--CD, octakis(2,3-di-O-acetyl-6-O-sulfo)-g-CD; QACD, 2-hydroxypropyl-trimethylammonio-b-CD; S--CD, sulfated-b-CD; SBE--CD, sulfobutyl ether-b-CD; TM--CD, heptakis(2,3,6-tri-Omethyl)-b-CD

The advantages of CE, compared to LC, GC and TLC, are its simplicity and its applicability for the separation of a wide range of compounds using the same instrument and, in most cases, the same capillary while changing only the composition of the BGE. In addition, in spite of a few disadvantages, CE possesses high resolving power due to its plug flow and minimal diffusion [2]. Because of the low injection volume, the concentration sensitivity is low and stacking procedures are therefore sometimes needed. In addition, precision of injection is worse compared to LC and GC. In the latter a known amount of sample is injected on column, which is not the case for CE due to the different injection mechanism. However, instrument manufacturers and scientists have worked on improving system performance. In recent years, many fully validated CE methods have been described and CE is becoming a wellestablished technique not only in academia but also in industry [3], especially for chiral separations. This is also proven by the amount of papers published on the subject. In this review, an overview of the different classes of chiral selectors used in CE is given and new developments in this field are discussed. In addition, the latest applications in pharmaceutical and biomedical analysis are presented, showing the potential of CE for chiral analysis. This technique has proven to be suitable for enantiomeric purity testing, for enantioselective analysis of drug products and also for the analysis of chiral drugs and their metabolites in biological samples.

2 Fundamental aspects
Chiral separation with CE requires the formation of diastereoisomeric entities. These can be stable diastereoisomers or transient diastereoisomeric complexes,
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depending upon the separation principle used [4]. The separation of chiral molecules in CE can be accomplished via two different principles: indirect or direct [46].

2.1 Indirect separations

Indirect separations are based on the formation of stable diastereoisomers with a chiral derivatization agent. These diastereoisomers can then be separated, based upon their different physicochemical properties, using an achiral BGE. However, there are several drawbacks to this method. A suitable, nearly 100% pure enantiomeric derivatization reagent is needed [7], the derivatization process is time-consuming and reacting groups (amino, carboxyl) are requested [8]. Furthermore, racemization can take place during the derivatization process. If possible, direct separation is usually preferred. However, the indirect method is a valid solution in those cases where direct separation cannot be accomplished or when the detectability of analytes should be increased [7, 9] and/or to modify the chemical structure of analytes for advantageous interactions [8].

where mE is electrophoretic mobility of both enantiomers in free solution, K1 and K2 are the complexation equilibrium constants of both enantiomers with the chiral selector, mE1S and mE2S are the electrophoretic mobilities of both enantiomerchiral selector complexes and [S] is the chiral selector concentration. A prerequisite for good chiral separation of enantiomers is that the equilibrium is reversible and fast [7]. Enantioseparation is based on differential mobilities of both enantiomers. These are achieved by either differential affinity of the enantiomers to the chiral selector (K1 = K2) and/or differential mobilities of the analytechiral selector complexes [10, 13, 14]. Vigh and co-workers have demonstrated that besides the CD concentration also the pH of the BGE influences the chiral separation. Three different types of CE enantiomer separations are presented. In desionoselective separations only the noncharged forms of the analyte interact selectively with the chiral selector. When only the charged form of the analyte selectively interacts with the chiral resolving agent, this is called ionoselective separations. A third possibility is that both the charged and noncharged form of the analyte interacts selectively with the chiral selector. This is known as duoselective separations [15 18]. The pH of the BGE influences not only the charge of the analyte but also the degree of ionization of charged chiral selectors and the EOF [1922]. Following their previous model for neutral chiral selectors, Vigh and coworkers [23] developed the charged resolving agent migration model (CHARM model) for permanently charged CDs. Thus, the pH of the BGE must be carefully selected to achieve chiral separations.

2.2 Direct separations

Chiral separation can also be performed directly by adding a chiral selector to the BGE. Hereby reversible diastereoisomeric complexes are formed between the enantiomers and the chiral selector. In this case relatively weak bonds are involved in the diastereoisomer formation process [9]. The primary basis of chiral recognition by direct methods is the difference between the intermolecular interactions of the enantiomers E1 and E2 and the chiral selector S. Wren and Rowe [1012] have developed a theoretical model relating mobility to the concentration of a CD selector. The following complexation equilibria exist for a pair of enantiomers (E1E2) and a chiral selector (S), assuming a 1:1 interaction between the enantiomer and the chiral selector, which is mostly the case [4, 7, 10]: E1 S E2 S
K1

3 Chiral selectors
3.1 CDs
The most frequently used chiral selectors in CE are the CDs [5, 9, 24, 25]. The mechanism of chiral discrimination using CDs is the inclusion of the analytes into the cavity of the chiral selector. CDs are often used for chiral separations in CE because of their good solubility in aqueous solvents, their low toxicity and low UV absorbance. The shape of a CD is similar to a truncated cone with a relatively hydrophobic cavity, able to host the molecule completely, or at least its hydrophobic part. The cavities of the three native CDs possess the same depth but have different widths [2628]. The outside of the CD is relatively hydrophilic due to the presence of primary and secondary hydroxyl groups. The inclusion complexes are stabilized by secondary bonds between the rim of the CD and subwww.electrophoresis-journal.com

! E1 S ! E2 S

(1) (2)

K2

The equation that allows determination of the mobility difference between the two enantiomers can be written as follows: Dm mE mE1 S K 1 S mE mE2 S K 2 S 1 K 1 S 1 K 2 S (3)

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stituent groups of the analyte. The hydroxyl groups present on the rim of the CD can easily be modified by chemical reactions with various functional groups. A wide number of CD derivatives are now used in CE for chiral analysis. Four different groups of CD derivatives can be distinguished.

3.1.1 Neutral CDs

Relatively weak interactions, such as hydrogen-bonding, pp and dipoledipole interactions are involved in the chiral discrimination process of native CDs [7]. Therefore, the capability to separate enantiomers is not so high for these CDs. The chiral discrimination capability can be increased through chemical derivatization of the hydroxyl groups. In this way, additional interaction points are introduced into the molecule and both the depth of the cavity and the free cross-section of its smaller opening are modified [7, 29]. Replacing hydroxyl groups with alkyl groups increases the solubility and flexibility of the CDs. These two factors help to accommodate the guest and increase the stability of the resulting inclusion complex [30]. Various neutral CD derivatives have been synthesized and applied for a great variety of compounds. Examples of these neutral chiral selectors are heptakis(2,6-di-Omethyl)-b-CD (DM-b-CD), heptakis(2,3,6-tri-O-methyl)-b-

CD (TM-b-CD) and hydroxypropyl-b-CD (HP-b-CD). Recently, Lin et al. [31] have described the synthesis of a new, highly water-soluble CD derivative, 2-O-acetonyl-2O-hydroxypropyl-b-CD (2-AHP-b-CD) (Fig. 1a). The enantioresolution properties of this CD derivative for several basic and acidic drugs was investigated and compared with those of b-CD, DM-b-CD and HP-b-CD. Chiral resolution was in most cases largely enhanced with 2AHP-b-CD compared to the other tested CDs. The substitution with the 2-O-acetonyl-2-O-hydroxypropyl group provides new interaction possibilities between the CD and the analyte enantiomers.

3.1.2 Negatively charged CDs

Anionic CDs, like sulfated b-CD (S-b-CD) and sulfobutyl ether-b-CD (SBE-b-CD), are negatively charged over the whole pH range and are mainly used for the chiral separation of neutral and basic compounds. The improved selectivity compared to neutral CDs is mainly attributed to the countercurrent mobility [24]. The presence of charged groups also increases the solubility of the CD and introduces electrostatic interactions [7, 9]. Carboxylated CDs contain chargeable carboxylic groups, and thus their charge depends upon the pH of the running buffer. Examples are carboxymethyl-b-CD (CM-b-CD),

Figure 1. Structures of some newly developed CD derivatives. (a) 2-AHP-b-CD; (b) heptakis(2-O-methyl-3-O-acetyl-6O-sulfo)-b-CD; (c) 2-O-(2-aminoethyl-imino-propyl)-b-Ohydroxypropyl-b-CD.
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carboxyethyl-b-CD and succinyl-b-CD. At low pH (2.5) they are not charged and behave like neutral CDs. At pH above 5 they are completely ionized. These carboxylic functions located on the rim of the CD can enhance selectivity and chiral resolution, probably due to the hydrogenbonding capacity of these functions and the higher polarity of these carboxylic functions [20, 30, 32, 33]. Since most of the developed CD derivatives consist of mixtures of different products, having different substitution patterns, separations are often difficult to reproduce [29]. Therefore, single isomers are being developed in order to produce selectively substituted derivatives [24]. Vigh and co-workers developed a family of single-isomer a-, b- and g-CDs [3440]. This group produced CD derivatives that were completely sulphated on their primary outer rim (position 6). On their larger, secondary rim they were completely substituted either with hydrophilic (e.g. heptakis(2,3-di-O-hydroxy-6-O-sulfo)-b-CD), moderately hydrophobic (e.g. heptakis(2,3-di-O-acetyl-6-O-sulfo)-b-CD (HDAS-b-CD)) or hydrophobic groups (e.g. heptakis(2,3-diO-methyl-6-O-sulfo)-b-CD). They also synthesized a single-isomer sulfated CD with nonidentical substitutions in position 2 and 3, namely heptakis(2-O-methyl-3,6-di-Osulfo)-b-CD [41]. Recently, they have reported the synthesis of the first single-isomer sulfated CD that carries nonidentical groups at all three positions, heptakis(2-Omethyl-3-O-acetyl-6-O-sulfo)-b-CD (Fig. 1b) [42].

3.1.4 Amphoteric CDs

Depending on the pH of the BGE these CD derivatives can be anionic, neutral or cationic [47]. Only two different zwitterionic CDs are described so far for CE enantioseparations [20], namely a CD containing 2-hydroxypropyltrimethylammonium chloride (degree of substitution, DS = 2.0) as well as sodium acetate (DS = 2.0) as substituents [48] and glutamylamino-b-CD [49].

3.1.5 Polymerized CDs

Apart from the monomeric CD derivatives, also polymerized CDs are available [13, 29]. They are mostly prepared by cross-linking the CDs with bifunctional reagents, like diepoxides and diisocyanates [50]. The high molecular mass of the polymer causes a reduced effective mobility of the enantiomers due to the lower mobility of the chiral selector, leading to a better enantioseparation. In addition, their structure is more rigid and has a different conformation compared to native CDs which can influence the enantiorecognition [51, 52]. Most importantly, polymerized CDs exhibit higher solubility than the parent CD [13, 50]. This is in particular the case for b-CD polymer (4050% w/w) in comparison to native b-CD (2% w/w) [51, 52]. Polymerized CDs can however be adsorbed to the capillary wall and change the magnitude and/or direction of the EOF.

3.1.3 Positively charged CDs

6A-Methylamino-b-CD, mono-(6-b-aminoethylamino-6deoxy)-b-CD and 2-hydroxypropyl-trimethylammonio-bCD (QACD) are examples of cationic CD derivatives. They are applied for the chiral separation of acidic and neutral compounds [20, 43]. The most common application is the separation of carboxylic acids and amino acids [20]. Lin et al. [44] developed a new charged highly water-soluble CD, 2-O-(2-aminoethyl-imino-propyl)-b-O-hydroxypropylb-CD (Fig. 1c) and successfully applied this cationic CD derivative for the chiral separation of some acidic compounds. CDs containing quaternary ammonium groups, like QACD, are charged over the whole pH range. Because of their strong ionic interactions, only very low chiral selector concentrations are necessary to enantioseparate acidic compounds [24, 30]. Besides single-isomer anionic CD derivatives, also heptasubstituted cationic CD derivatives were developed. Warner and coworkers [45, 46] have described the application of these polycationic b-CD derivatives for the chiral separation of nonsteroidal anti-inflammatory drugs.
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3.2 Chiral crown ethers

Crown ethers are cyclic polyethers that form hostguest complexes with several inorganic and organic cations like alkali- and earth alkali metal ions, as well as organic compounds with an amino group [5, 9, 24, 29, 53]. Due to this restriction, they are much less used than the CDs and therefore only described briefly in this review. The most used crown ether in chiral CE is (1)-(18-crown6)-2,3,11,12-tetra-carboxylic acid (18C6H4) [13, 30, 54]. Wang et al. [55] synthesized a new chiral crown ether, (S,S)-1,7-bis(4-benzyl-5-hydroxy-2-oxo-3-azapentyl)1,7-diaza-12-crown-4, and demonstrated its applicability for chiral separations with CE. Chiral crown ethers can stereoselectively include compounds containing primary amino groups. The complexation is different compared to CD complexation. Here, the analyte fits with its hydrophilic part (the protonated amino group) in the cavity on forming ion-dipole bonds with the oxygen atoms of the chiral selector. Secondary bonds between the substituent groups of the analyte and the carboxyl groups of the chiral
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selector stabilize the complex [9, 27, 30]. The BGE may not contain cations such as potassium or ammonium ions because they compete with the enantiomers for the crown ethers cavity [53].

network (pore size) will affect enantiorecognition. In ionic polysaccharides, electrostatic interactions also play an important role [57, 58]. Two different groups of carbohydrates can be distinguished: neutral and charged oligo- and polysaccharides. Examples of neutral carbohydrates are dextrins [56, 57, 59, 60] and dextrans [57, 60]. Negatively charged polysaccharides like heparin, dextran sulphate, chondroitin sulphate C and A have been shown to be suitable chiral selectors for basic drugs [24, 30, 58, 60]. Nishi et al. [61] introduced several positively charged polysaccharides like the aminoglycoside antibiotics that were applied for the resolution of some acidic analytes. Du et al. [62] introduced colominic acid as chiral selector. Colominic acid consists of a mixture of 2 ? 8 linked Nacetylneuraminic acid polymers (Fig. 2a). This polymer was able to separate the enantiomers of some basic drugs containing an amino/imino group adjacent to the chiral carbon atom. Recently, N-(3-sulfo,3-carboxy)-propionylchitosan (Fig. 2b) was investigated as new chiral selector by Budanova et al. [63]. The mechanism of enantiorecognition of this chiral selector appeared to be somewhat different as compared to other charged polysaccharides, due to the presence of an additional chiral centre in the monosaccharide residue.

3.3 Linear oligo- and polysaccharides

Besides CDs, which are cyclic oligosaccharides, also linear oligo- and polysaccharides can be used as chiral selectors in CE. The low UV absorption of these sugar derivatives and the often high efficiency of the achieved separations makes these resolving agents attractive [54]. They consist of D-glucose units linked by different bonds, i.e. a-(14), a-(16), b-(13) and b-(14). The conformational change from a flexible coil to a helix for polysaccharides having a-(14) linkages, in the presence of analyte and buffer salts, is believed to be of importance for enantioselectivity. The helical structure forms a hydrophobic cavity, more flexible than CDs, in which the analyte can be included [13, 5658]. The complexation between the analyte and the oligosaccharide is weaker than that with CDs, possibly due to the weaker hydrophobic interactions [13, 58]. In other polysaccharides that are linked by a-(16) bonds, the helical structure cannot be formed. Here, the polymer

Figure 2. Structure of colominic acid (a) and N-(3-sulfo,3-carboxy)-propionchitosan (b).

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3.4 Proteins
The strong and highly selective interactions of proteins with other compounds are called affinity interactions. The complexed compounds are usually bonded at particular selector binding sites with high selectivity. High selectivity and low separation efficiency are typical of separations based on affinity chiral selectors in free-solution electrophoresis [7, 64]. Several proteins have successfully been employed as chiral selectors in CE, e.g. bovine and HSA, a1-acid glycoprotein, ovomucoid, avidin, transferrin and pepsin for a broad spectrum of compounds [13, 24, 65]. There are two problems associated with the use of proteins as chiral selectors in CE. Firstly, the adsorption of the chiral selector to the capillary wall and secondly, they interfere with the UV detection of the analytes due to their high UV absorption [8, 13, 64, 65]. To eliminate interactions between the chiral selector and the capillary surface, the capillary wall must be modified. This can be achieved by dynamic modification, adsorption of polymers to the capillary wall or covalent derivatization of the silanol groups [13, 66]. To overcome the problem of UV interference, the partial-filling technique has been used. In cases where the partial-filling technique is applied, the EOF should be suppressed or largely reduced. The use of coated capillaries has been reported to prevent the EOF from forcing the chiral selector towards the detector [64, 67, 68]. Another possibility is the use of indirect UV detection [69].

stituted phenols with various sugars or saccharide moieties bound to them [5, 7, 29, 70]. Although glycopeptide antibiotics are amphoteric, they are highly effective in the chiral discrimination of neutral and negatively charged enantiomers [8, 29, 30]. Recently, erythromycin, balhimycin and derivatives were evaluated as chiral selectors in CE. Hou et al. [71] successfully applied erythromycin for the chiral separation of four antihepatitis drugs. On the contrary, Ha et al. [72] tested erythromycin and several derivatives for their performance as chiral selectors but did not observe any chiral separation. Balhimycin and bromobalhimycin were shown to be good chiral selectors for acidic analytes, with higher enantiorecognition capabilities for most tested compounds as compared to vancomycin [73]. Adsorption to the capillary wall and UV interference, problems observed for proteins, are also associated with the use of macrocyclic antibiotics as chiral selectors [30, 69, 70].

3.6 Chiral calixarenes

Calixarenes are macrocyclic compounds consisting of benzene rings linked by methylene groups forming a hydrophobic cavity that is able to form hostguest complexes [24, 54]. In principle, chiral calixarenes, in contrast to crown ethers, may be used not only for enantioseparation of chiral primary amines but for any kind of compound. However, a more universal chiral recognition ability than now available will be necessary. In addition, their strong UV absorbance may limit their use as chiral selector [74]. An example of this type of resolving agent is N-p-tertbutylcalyx[4]arene tetracarboxyloyl-L-alanine [54].

3.5 Macrocyclic antibiotics

These chiral selectors were first introduced by Armstrong et al. [69]. The macrocyclic antibiotics have several asymmetric centers and many functional groups, allowing multiple interactions between both enantiomers of an analyte and the chiral selector. They possess hydrophobic pockets and pendant polar arms. Therefore, the hydrophobic moieties of the analyte can be included into these pockets and hydrogen bonds can be formed with the polar arms [5]. Two different classes of macrocyclic antibiotics are available for chiral separations: ansamycins (e.g. rifamycin) and glycopeptides (e.g. vancomycin). Ansamycins possess an aromatic chromophore system spanned by an aliphatic bridge that gives a particular shape to the resulting molecule. Rifamycin B is negatively charged and is used to enantioseparate cationic compounds, especially hydrophilic amine-containing compounds. Neutral rifamycin SV resolves anionic racemates [5, 29, 30, 69, 70]. Glycopeptides consist of up to four fused macrocyclic rings, formed by linked amino acids and sub 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.7 Ligand-exchange CE
This mechanism can be used for compounds containing aminocarboxyl or hydroxycarboxyl groups, for example amino acids and hydroxyl acids [30, 75, 76]. The mechanism of separation is based upon the formation of complexes with a central ion (e.g. Cu21, Ni21) and at least two chiral bifunctional ligands. A chelate complex (hemicomplex) for enantioseparation is formed by mixing an amino acid (e.g. histidine) with a salt of heavy metal. Enantioseparation is based on the different stability of the complex formed between both enantiomers and the hemicomplex [13, 30, 75, 77]. The limited stability of the ligand-exchange selectors, the detection difficulties resulting from their UV absorption and the tendency of some analytes to be poorly resolved because of their rather slow ligand-exchange kinetics are the main practical disadvantages of these selectors [7, 30].
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The first application of the principle of ligand-exchange in CE was by Zare and co-workers [75, 76]. They applied histidine- or aspartame-copper(II) complexes for the enantiomeric resolution of dansylated amino acids. A comprehensive overview of the selectands involved and the selectors used is given by Schmid et al. [77] and Davankov [78]. Recently, Kodama et al. [79] have described chiral ligand exchange CE using borate anion as a central ion.

having a detergent nature like bile salts may form micelles. The chiral separation of analytes is based on their partition coefficients between the chiral micellar phase and the electrolyte bulk phase. Another possibility is the use of charged achiral detergents in combination with a chiral selector. An example is the combination of SDS with CDs. Separation is based on the distribution of the analyte between the micellar pseudostationary phase, chiral selector and aqueous phase [13, 92]. This principle was introduced by Terabe and co-workers [93] and is called CD-MEKC. Different classes of surfactants are applied as chiral selectors. Natural chiral surfactants are bile salts, saponines and digitonins. The synthetic chiral surfactants can be classified into the following families: N-alkanoyl-Lamino acids, N-dodecoxycarbonyl amino acids, alkylglucoside chiral surfactants, tartaric acid-based surfactants and steroidal glucoside surfactants [24, 30, 54]. An interesting approach is the use of polymerized micelles, like undecyl-L-valine. The elimination of the dynamic equilibrium between monomer and micelle may enhance chiral recognition. The polymer can be used over a larger concentration range because it has no CMC and organic modifiers can be added without disrupting micelle formation [54, 9498]. In addition, also the efficiency can be improved. The polymerized micelle is believed to have a more compact structure and the penetration of the solute is therefore less deep and faster, although the selectivity of the separation remains good [54, 99]. One of the disadvantages of MEKC using micelle-forming surfactants is the low migration range, which affects the separation. Using vesicle-forming surfactants as chiral selectors the migration range can be increased [100]. Recently, Mohanty and Dey [101] demonstrated the use of vesicle-forming anionic and cationic surfactants [100] as chiral selectors in MEKC. Microemulsion EKC (MEEKC) is often compared with MEKC due to their similar utilization of surfactant aggregates. However, there are some distinct differences between micelles and microemulsions. Microemulsions, mostly oil-in-water, are spherical aggregates of nanometre size that consists of a surfactant, an oil and a cosurfactant in a ratio that provides a single, optically transparent and thermodynamically stable phase. Surfactant molecules, like SDS, used in a concentration above their CMC lower the surface tension and thereby facilitate droplet formation. A small alcohol cosurfactant is usually added to bridge the oilwater surface for further stabilization of the microemulsion system [102105]. The presence of the oil core gives the microemulsions their
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3.8 Chiral ion-pairing reagents

Ion-pairing reagents have been applied successfully in NACE, but not in aqueous media. Apparently, water interferes with the intramolecular interactions responsible for chiral recognition and largely suppresses the formation of intermolecular hydrogen bonds [24]. Exceptions are the use of chiral and achiral counterions as supporting agents for separations with CDs in dual systems (see also Section 3.10) [24, 80]. One paper by Kodama et al. [81] describes the chiral resolution of tartaric acid using an aqueous BGE with (1R,2R)-(2)-1,2-diaminocyclohexane as chiral counterion. However, 65% v/v ethanol was present in the BGE. The first ion-pairing reagent reported for CE separations was (1)-(S)-camphor-10-sulfonic acid [82]. Quinine [83], tert-butyl carbamoylated quinine and other cinchona alkaloids and derivatives were also investigated for the chiral separation of N-protected amino acids [8487]. Higher enantioselectivity was observed using the cinchona alkaloid derivatives, especially those with a carbamate function [85]. Moreover, the presence of a bulky substituent on the carbamoylated derivatives or the use of dimeric forms of carbamoylated quinine and quinidine derivatives had a favorable effect on the enantioselectivity [86, 87]. In addition, N-protected amino acids, like (R)and (S)-3,5-dinitrobenzoyl-leucine, can also be used as chiral ion-pairing reagent for the enantioseparation of cinchona alkaloids and structurally related compounds [88]. Carlsson et al. [89] introduced (2)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid ((2)-DIKGA) as chiral ion-pairing reagent for the enantioseparation of pharmacologically active amines. Recently, also N-benzoxycarbonylglycyl-L-proline has been introduced for the chiral separation of amines in NACE [90].

3.9 Chiral surfactants

The principle of MEKC was introduced by Terabe et al. in 1984 [91]. It can be used for charged and uncharged analytes and for a wide range of substances with hydrophilic or hydrophobic characteristics. Chiral selectors
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unique properties when compared to micelles [102]. The separation principle is a combination of electrophoretic separation and chromatographic partitioning with the microemulsion droplets. Only a few applications to chiral separations have been described. For this purpose, a chiral oil [106] or surfactant [102, 104, 107, 108] can be applied or chiral selectors, like CDs, can be added to the microemulsion system [103].

3.10 Dual-selector systems

In some cases enantioseparation in CE cannot be achieved using one single chiral selector. However, the simultaneous addition of two chiral selectors can often lead to a successful enantioseparation. So far, several approaches have been studied [109]. The most frequently used dual selectors are combinations of different CDs [109, 110]. The use of CDs with chiral surfactants such as bile salts or polymeric surfactants was shown to improve resolution [6, 97, 111, 112]. Mertzman and Foley [103] have combined for the first time CDs with MEEKC for the chiral separation of some basic drugs. Resolution was found to be highly dependent on buffer identity and concentration. The combination of CDs with chiral or achiral crown ethers also proved to be synergistic [5, 6, 53, 113, 114]. Horimai et al. [115] investigated the chiral resolution of some quinolone drugs using ligand-exchange and hostguest interactions. No optical resolution was observed when one of the running solution components was omitted. The use of chiral and achiral ion-pairing reagents in combination with CDs has also been investigated in aqueous [80, 116, 117] and nonaqueous BGEs [118, 119].

dansylated amino acids and quinolone antibacterial agents was performed with amphiphilic aminosaccharides. They possess a glucosamine chiral head group carrying three carboxylic functions as the hydrophilic region and three long hydrocarbon chains as the hydrophobic part [126]. Thormann et al. [127] used a Pirkle-type chiral selector, R-(2)-N-(3,5-dinitobenzoyl)-a-phenylglycine, for the electrokinetic separation of the enantiomers of albendazole sulphoxide. Chiari and co-workers [128130] evaluated a series of cyclopeptides synthesized by combinatorial chemistry as chiral selectors. Ephedrine enantiomers were separated and analyzed using the amino acid L-leucine as chiral discriminator [131]. Dowling et al. [132] applied a guanosine gel as a chiral selector in CE for the enantioseparation of propranolol. Guanosine gels are reversible organized media that are formed through the self-association of guanosine compounds. An overview of the new chiral selectors used in CE is also given in the recent review by Gbitz and Schmid [133].

4 Applications
In this section papers from 2004 till the beginning of 2005, dealing with applications in pharmaceutical and biomedical analysis, are presented.

4.1 Pharmaceutical applications

In most pharmaceutical applications covered by this review neutral and/or charged CDs are used as chiral selector. In two cases HSA is used as chiral discriminator. Sometimes CDs are combined with achiral or chiral surfactants. An overview of the different chiral separations is given in Tables 1, 2. The pharmaceutical applications can be divided into two parts: content assays to determine the active single enantiomer in pharmaceutical preparations and chiral impurity determination.

3.11 Other chiral selectors

Recently, cyclosphoraoses were introduced as chiral selectors by Lee and Jung [120]. Cyclosphoraoses are a family of unbranched cyclic b-(1 ? 2)-D-glucans. The exact molecular mechanism of chiral recognition is not yet elucidated. Hemispherodextrins are capped CDs and have been tested as chiral selectors for the enantioseparation of phenoxyacids [121] and profens [122]. In contrast to other capped CDs, the cap of these hemispherodextrins is saccharidic in nature, which makes it similar to the CD cavity [122]. Enantioseparations of 18 different compounds was achieved by Nair et al. [123] using (1)-tubocurarine chloride as chiral selector. Ingelse et al. [124] used 1-allylterguride, a positively charged ergot alkaloid, for the enantioseparation of organic acids. Different terguride-based chiral selectors were investigated by Honzatko et al. [125] using dansyl amino acids as model compounds. The optical resolution of some
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4.1.1 Content assays

Studies that have been published from January 2004 till March 2005 are summarized in Table 1. Unfortunately, most of the assays developed for label claim control describe the determination of the enantiomers in formulations containing the racemic drug. More appropriate would be to use these methods for the analysis of singleenantiomer formulations. Wang et al. [134] investigated different CDs for the chiral separation of cefoperazone, an antibiotic drug. Baseline separation was obtained using b-CD in a phosphate
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Table 1. Pharmaceutical applications content assays Analyte Cefoperazone Etodolac Omeprazole Balaglitazone Pioglitazone Rosiglitazone Ragaglitazar Sertraline Bupivacaine Propranolol Buffer Chiral selector pH (concentration) 4.0 7.0 4.0 8.0 b-CD (0.04 mM) HP-b-CD (20 mM) S-b-CD (3%) SBE-b-CD (2%) 1 DM-b-CD (0.7%) SBE-b-CD (2%) 1 DM-b-CD (0.7%) HP-b-CD (20 mM) 1Na-deoxycholate (30 mM) HSA (partial-filling technique) (160 mM) HSA (complete-filling technique) (100 mM) Application Content determination in injection vials containing racemic cefoperazone Content determination in tablets containing racemic etodolac Content determination in tablets containing racemic omeprazole Determination of a stable 50:50 equilibrium of R- and S-balaglitazone in active pharmaceutical ingredient and in tablets Content assay of ragaglitazar and arginine (counterion) Determination of sertraline enantiomers in bulk drug, tablets and capsules containing racemic sertraline Content determination of both bupivacaine enantiomers in injectable solutions containing racemic bupivacaine Determination of the content of propranolol enantiomers in tablets and an injectable solution Reference [134] [135] [136] [137]

8.0 11.5 8.0 7.4

[138] [139] [140] [141]

Table 2. Pharmaceutical applications enantiomeric purity testing Analyte Calcium levofolinate SLV307 Lisuride Nucleosides Baclofen Substituted imidazole p38 MAP kinase inhibitor Organic disulfates R209130 Methotrexate Buffer pH 9.9 2.5 2.5 2.5 2.5 2.2 2.4 3.0 9.3 Chiral selector (concentration) DM-b-CD (20 mg/mL) HP-a-CD (50 mM) g-CD (20 mM) Highly S-a-CD (3%) Highly S-b-CD (3%) Highly S-b-CD (3%) Randomly S-b-CD (2%) QACD (5 mM) S-b-CD (2%) 1 a-CD (5 mM) b-CD (45 mM) 1 SDS (100 mM) LOQ 0.1% 0.25% 0.05% 1.112 mg/L depending on the compound 0.5% 0.3% 0.5% 0.1% 0.1% Reference [148] [149] [150] [151] [152] [153] [154] [156] [157]

buffer pH 4.0. The method was applied to measure cefoperazone enantiomers in pharmaceutical doses. The separation of the nonsteroidal anti-inflammatory drug etodolac was achieved using HP-b-CD as chiral selector [135]. The effect on the enantioresolution of the buffer concentration, the DS and concentration of the CD was studied, as well as the capillary temperature and the applied voltage. The optimized method was validated and applied for the quantification of both enantiomers in tablets. Bonato and Paias [136] have described the enantioselective analysis of omeprazole in pharmaceutical formulations using S-b-CD as chiral selector. They also developed a chiral LC method for the same purpose and compared both techniques. They concluded that chiral CE was more versatile and less expensive than LC. However, the LC method was more sensitive and resulted in a better separation of omeprazole.
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SBE-b-CD in combination with DM-b-CD was used for the chiral separation of three glitazone compounds, namely balaglitazone, pioglitazone and rosiglitazone [137]. The validated method was applied for routine tests during the drug and formulation development work of balaglitazone. The same separation buffer was used when this group developed an enantioselective method to assess the chiral purity of ragaglitazar in drug substance and in tablets [138]. The paper of Chen et al. [139] deals with the separation of the cis-trans isomers and the enantiomers of sertraline. The combination of a chiral surfactant, DOC, with neutral HP-b-CD was used to determine sertraline enantiomers in drug substance, tablets and capsules.
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Bupivacaine enantiomers were separated using HSA as chiral selector. The partial-filling technique was applied to avoid the high background UV absorption of the selector. The cationic surfactant CTAB was used as a dynamic capillary coating in order to reduce the EOF, in that way achieving the detection of both bupivacaine enantiomers out of the selector plug. The method was validated and applied for the analysis of both enantiomers in an injectable solution [140]. The same group investigated the chiral separation of propranolol, using the complete-filling technique. In this case the capillary was completely filled with HSA and the propranolol enantiomers were detected in the selector plug. The method was applied to determine the content of both propranolol enantiomers in tablets and in an injectable solution [141].

related substances of calcium levofolinate, including the diastereomeric impurity (6R,2S)-folinic acid. Separation of all related compounds was obtained with DM-b-CD in a borate buffer pH 9.9. This assays allowed the detection and determination of the related compounds at the 0.1% level. A CE method for the determination of the enantiomeric purity of SLV307, a potential antipsychotic drug, in capsules was developed and validated. HP-a-CD was used as chiral selector and tris(hydroxymethyl)aminoethane was added as coion to reduce electrodispersion [149]. Lisuride is a chiral compound derived from natural ergot alkaloids. Kvasnicka et al. [150] were able to detect 0.02% of the distomer L-lisuride using an acidic BGE containing g-CD. The method was employed for batch analysis of the drug substance. Baseline separation of five new acyclic nucleosides was achieved using highly sulphated CDs and a capillary dynamically coated with polyethylene oxide [151]. The method was validated in terms of repeatability, LOD and LOQ. This assay could be used for enantiomeric purity testing after full validation of the method. The same group investigated the chiral resolution of baclofen, using approximately the same methodology. Good enantiomeric separation was obtained using highly S-b-CD and a capillary dynamically coated with polyethylene oxide [152]. Zhou et al. [153] investigated the chiral separation of a substituted imidazole p38 MAP kinase inhibitor and two intermediates with various sulfated-CDs. Baseline separation could only be achieved using randomly substituted S-b-CD. The validated method allowed the detection of 0.1% and quantification of 0.3% of chiral impurity. The enantiomers of the drug candidate, 3-(p-isothiocyanatophenoxy)-3-(p-isothiocyanatophenyl)propane-1,2-disulfate, were separated with positively charged QACD [154]. The nature of the buffer was quite important for the separation. Using a glycine buffer complete separation was obtained; however, no separation was observed using a phosphate buffer. Both enantiomers were obtained by enantioselective and regioselective syntheses. Enantiomeric purity of both enantiomers could be determined. Jimidar et al. [155] have described a screening strategy that utilizes design of experiments methodology for the enantiomeric separation of basic, neutral and acidic compounds. This screening approach was used for the chiral separation of a basic drug with three chiral centres [156]. The eight stereoisomeric compounds could be separated using a combination of neutral a-CD with charged S-b-CD. The method was used for the determination of the chiral purity of the compound.
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4.1.2 Enantiomeric purity testing

When a new drug substance consists predominantly of one enantiomer, then the other enantiomer will be considered as an impurity. However, this minor enantiomer is excluded from the ICH guidelines on impurities in new drug substances [142] because of practical difficulties that can occur when quantifying at low levels. Anyhow, as stated in the ICH guideline Q6A [143], the impurity in the chiral new drug substance should otherwise be treated according to the principles established in the guideline on impurities in new drug substances. Limits of 0.1% enantiomeric impurity are widely accepted as threshold in the testing of single enantiomer drug substances [22, 144, 145]. Thus, the analysis of the enantiomeric purity of a single enantiomer drug is a great challenge for analytical techniques. Reversal of the enantiomer migration order is an important topic to be considered during method development for enantiomeric purity testing. Peak dispersion as a result of sample overload can result in both peak tailing as well as peak fronting. Therefore, depending on the peak symmetry characteristics, the minor peak should elute in front or after the major peak [146, 147]. In most cases, the minor peak preferably migrates in front of the major one, especially in cases where the resolution between both peaks is not so high [22, 144146]. Most common chiral selectors, like CDs, are natural compounds, available in only one enantiomeric form. Reversal of the migration order can therefore not be accomplished by a change in stereochemical configuration [146, 147]. Several methods to reverse the enantiomer migration order are described in an excellent review by Chankvetadze [146]. The enantiomeric purity assays described in recent literature are summarized in Table 2. Sss et al. [148] have described a validated method for the determination of
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A CD-MEKC method was developed for the determination of the most important potential impurities of methotrexate, including the distomer D-methotrexate. The addition of b-CD to the MEKC system based on SDS was essential for the enantioresolution of methotrexate and for the separation of achiral impurities. The application of this method for the analysis of tablets and injections showed that the major impurity was D-methotrexate [157].

4.2 Biomedical applications

CDs are the most widely used chiral selectors in biomedical applications, as was also observed for pharmaceutical applications. An overview of the methods described in recent literature is given in Table 3. Glowka and Karazniewicz [158] developed a stereospecific method for the determination of ibuprofen, flurbiprofen and ketoprofen in human serum. The profen enantiomers were extracted from acidified serum samples with methylene chloride. Separation of a mixture of the three racemic compounds, including the internal standard Snaproxen, was obtained using TM-b-CD as chiral selector. The validated method was used for pharmacokinetic and bioavailability study of ketoprofen enantiomers in humans after administration of standard and sustained release tablets containing racemic ketoprofen. In a second study, the chiral separation of indobufen was investigated [159]. Complete enantioseparation was achieved using 25 mM TM-b-CD in a BGE pH 5.0. The validated method was used in a pharmacokinetic study. The enantiomeric separation of carvediol in human plasma samples was achieved using HP-b-CD as chiral discriminator [160]. The method was validated for linearity, precision and accuracy. Pharmacokinetic parameters for both enantiomers were calculated after oral administration of racemic carvediol. Ha et al. [161] developed a method for the enantioseparation of apomorphine. HP-b-CD was chosen as chiral selector instead of SBE-b-CD, because with the latter four peaks were observed due to different complexes that were formed. The method was validated and the transport of apomorphine enantiomers was studied using an in vitro cell culture system of the intestinal mucosa. No special sample preparation was necessary, despite the high-ionic strength of the sample. Chiral separation of lactic acid was obtained in a BGE containing HP-b-CD as chiral selector and tetradecyltrimethylammonium bromide to reverse the EOF by dynamically coating the capillary [162]. The method has been applied for the determination of L- and D-lactic acid in plasma from humans and rats.
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Enantiomeric separation of dopamine-derived neurotoxins was performed with b-CD as chiral selector [163]. This group showed that the addition of a surfactant improved the recovery rates of these compounds from the deproteinization procedure. The developed method in combination with electrospray ionization MS/MS (direct infusion into the MS) was used to investigate the in vitro formation of tetrahydroisoquinoline derivatives from catecholamines and aldehydes. However, the method was not validated. The same group developed an enantioselective analysis method for the determination of D/L-serine, fluorescently labelled with naphthalene-2,3-dicarboxyaldehyde. Chiral separation was achieved by employing a dual-selector system consisting of HP-g-CD and D-(1)glucose [164]. The addition of a saccharide enhanced the enantioresolution and was necessary to obtain baseline separation. Coupled with LIF detection, this method could be used for sensitive determination of D-serine in neural samples. The method was only validated in terms of linearity and LOD. Esteban et al. [165] have developed a CE method for the stereoselective quantification of methadone and its major metabolite in human serum samples. Highly S-b-CD was used to resolve both racemic compounds. The validated method was applied to ascertain whether antiretroviral therapy altered the plasma levels of the enantiomers of methadone. Mandrioli et al. [166] aimed to enantioseparate mirtazapine and its active metabolite in human plasma. For this purpose, CM-b-CD was selected as chiral discriminator in a BGE of pH 2.5 and short-end injection was applied. The validated method was applied to plasma samples from patients treated with mirtazapine tablets. A chiral CE method for the simultaneous analysis of the four stereoisomers of anisodamine in plasma samples was developed and validated [167]. Complete separation of the four peaks was obtained using a BGE pH 2.5 containing CM-g-CD as chiral selector. The method was applied to study the stereoselective pharmacokinetics of anisodamine in rabbits after oral and intraveneous administration of racemic anisodamine. Servais et al. [168] described a validated NACE method for the determination of salbutamol enantiomers in urine samples. The single-isomer, negatively charged HDAS-bCD was used as chiral selector and SPE was used for sample cleanup prior to the CE separation. Simultaneous chiral analysis of methamphetamine and five related compounds in urine samples was achieved using HDAS-b-CD as chiral selector [169]. The BGE consisted of 1 M formic acid (pH 1.7). Detection was perwww.electrophoresis-journal.com

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Table 3. Biomedical applications Analyte Buffer Chiral selector pH (concentration) 5.0 5.0 4.0 3.0 7.0 3.0 TM-b-CD (50 mM) TM-b-CD (25 mM) HP-b-CD (10 mM) HP-b-CD (14 mM) HP-b-CD (220 mM) b-CD (12 mM) Biological fluid Serum Serum Plasma Caco-2 cells Plasma Fetal bovine serum Neural samples LLE SPE LLE SPE LLE LLE Sample prep LLE LLE LLE PP PP LOQ Application Reference [158] [159] [160]

Ketoprofen Indobufen Carvediol Apomorphine Lactic acid Tetrahydroisoquinoline derivatives Serine

0.5 mg/mL 0.2 mg/mL 7.5 ng/mL 0.5 mM 20 mM Not validated

Pharmacokinetic-bioavailability study in human volunteers Pharmacokinetic-bioavailability study in human volunteers Pharmacokinetic study in human volunteers

Transport study of apomorphine enantiomers [161] in an in vitro cell culture Determination of D-lactic acid in patients with [162] short bowel syndrome

In vitro study of the formation of neurotoxines [163]

10.0

HP-g-CD (20 mM) 1 D-(1)-glucose (15%) CM-b-CD (0.24%) CM-g-CD (25 mM)

0.3 mM

Determination of D-serine in neural samples

[164]

Methadone Mirtazapine Anisodamine Salbutamol Methamphetamine

5.0 2.5 2.5

Highly S-b-CD (0.2%) Serum Plasma Plasma

Not specified 5 ng/mL 0.040.06 mg/mL 375 ng/mL 5 ng/mL 1.25 ng/mL

Quantification of drugdrug interactions Determination of mirtazapine and its active metabolite in plasma

[165] [166] [167]

In vivo pharmacokinetics in rabbits

NACE HDAS-b-CD (15 mM) Urine 1.7 HDAS-b-CD (0.85 mM) SBE-b-CD (0.8 mM) Urine Plasma Urine Plasma Urine

Determination of both enantiomers in urine of [168] human volunteers Analysis of urine samples of drug addicts Pharmacokinetic study in human volunteers [169] [170]

trans-tramadol 2.5 trans-O-desmethyltramadol Labetalol 2.5

Deprenyl metabolites 2.7

ODAS-g-CD (10 mM) DM-b-CD (4 mM) 1 CM-b-CD (2 mM) HP-b-CD (10 mM)

SPE SPE

Not validated 0.10.5 mM 2.5 mM

Spiked plasma samples

[171] [172] [173]

In vivo metabolism studies in rats In vitro metabolism studies (humans)

N-Oxygenated 3.6 metabolites of: Deprenyl Amphetamine Methamphetamine

Primaquine car- 3.0 boxyprimaquine

In vitro SPE incubations

Maltodextrin (10%)

Homogenate

LLE

100 ng/mL 40 ng/mL

In vitro metabolism studies (rats)

[174]

LLE: liquidliquid extraction; PP: protein precipitation; SPE: solid-phase extraction.

formed using ESI-MS/MS, conducted in the positive-ion mode. The method was applied to the analysis of urine samples of methamphetamine addicts. The pharmacokinetics of the enantiomers of trans-tramadol and its active metabolite, trans-O-desmethyltramadol, were studied in healthy volunteers [170]. Plasma and urine samples were analyzed using CE and
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SBE-b-CD as chiral selector. The pharmacokinetics of both compounds were stereoselective in men and women. Gender difference were observed for trans-tramadol, but not for its active metabolite. Goel et al. [171] compared the use of HDAS-b-CD and octakis(2,3-di-O-acetyl-6-O-sulfo)-g-CD (ODAS-g-CD) for the separation of all four stereoisomers of labetolol.
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The effect of the ODAS-g-CD concentration and the effective capillary length were examined. The method was applied to plasma samples spiked with labetalol. Szko et al. [172, 173] have studied the N-oxide metabolites of deprenyl in rat urine and in in vitro studies using recombinant human flavin-containing monooxygenase enzymes and human liver microsomal preparations. In a first paper [172], they validated a chiral CE method for the quantitative analysis of R-(2)-deprenyl, also called selegiline, and seven of its metabolites in rat urine. A dualselector system, consisting of DM-b-CD and CM-b-CD, was applied for this purpose. The validated method was applied for in vivo metabolism studies in rats treated with either single or repeated doses of selegiline or its N-oxide. Stereoselective N-oxidation of selegiline and rapid urinary excretion of selegiline-N-oxides was observed. In a second study, HP-b-CD in a BGE pH 3.6 enabled the quantification of deprenyl-N-oxide, methamphetamine-hydroxylamine and amphetamine-hydroxylamine enantiomers [173]. This method was used to compare the in vitro metabolism of the enantiomers of deprenyl, methamphetamine and amphetamine by two recombinant human flavin-containing monooxygenase enzyme isoforms. All previous described chiral separations were acquired using CDs as chiral selectors. Bortocan and Bonato [174] have used maltodextrin as chiral discriminator for the enantioselective analysis of primaquine and its metabolite carboxyprimaquine. The maltodextrin concentration, the buffer pH, the applied voltage and the temperature were optimized. The validated method was applied to study the in vitro metabolism of primaquinine in rat liver mitochondria.

be a suitable technique for enatiomeric purity testing, where the minor enantiomer could be determined at the 0.11% level in the presence of a large excess of the eutomer. CE has also shown to be a good alternative to the widely used and well-established LC methods for the enantioselective analysis of chiral drugs and their metabolites in biological samples. The major concern in CE when applied to enantioselective bioanalysis is the lack of sensitivity. Several approaches have been developed and studied to overcome these problems. These include the use of more sensitive detection systems and sample stacking techniques. Further investigation of chiral CE-MS seems promising for the stereoselective analysis of drugs in biological fluids. Miniaturization of analysis systems is an emerging new technology. Microchip electrophoresis (MCE) can be regarded as a further miniaturized version of CE. Recently, chiral separations on microchip devices have been reported. As in classical CE the most commonly used chiral selector are the CD derivatives [175177]. However, in the last year also chiral separations with ligand-exchange [178] and crown ether [179] have been described. MCE has great potential for chiral highthroughput screening and also for real-time process control [180]. The major drawback is the lack of detection sensitivity as in classical CE. Therefore, sample stacking applied to microchip devices and the development of sensitive detection methods applicable to a wide variety of analytes will be challenging research topics in the near future.

6 References 5 Concluding remarks and future trends

In recent years, CE has proven to be a well-established analytical technique, especially in the field of chiral separations. It has several advantages compared to LC, including simplicity, short analysis times, high efficiencies, different separation mechanisms, small volumes and low-running costs. In addition, CE offers a tremendous flexibility for enantiomeric separations, because a wide variety of chiral additives are available. Nevertheless, CDs are by far the most widely used chiral selectors in CE. Even so several new chiral selectors have been developed in recent years. As shown in previous sections of this review, CE can be easily applied to drug analysis. However, in many cases the enantioselective analysis methods were applied to drug products containing the racemate instead of a single enantiomer. Electromigration techniques have proven to
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