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Anal. Chem.

2004, 76, 4635-4644

Chiral Separations
Timothy J. Ward* and Daisy-Malloy Hamburg

Department of Chemistry, Millsaps College, 1701 North State Street, Box 150306, Jackson, Mississippi 39210

Review Contents metabolites in biological fluids by CE-mediated microanalysis were


Reviews 4635 summarized by Bonato (2), who observed an increase in the use
Capillary Electrophoresis (CE) 4635 of negatively charged β-CD derivatives as chiral selectors. The
High-Performance Liquid Chromatography 4635 use of combinations of different chiral selectors was also noted.
(HPLC) The direct chiral separation of short-chain organic acids by CE
Gas Chromatography (GC) 4636 was reviewed by Barbas and Saavedra (3), with existing methods
Thin-Layer Chromatography (TLC) 4636 using various techniques discussed. These techniques included
Capillary Electrochromatography (CEC) 4636
CE utilizing macrocyclic antibiotics, CDs, ion-pair method, transi-
Multiple Technique Reviews 4636
tion metal complexes, and exchange CE. Strategies to advance
Capillary Electrophoresis 4636
Cyclodextrins 4636 the chiral separations in these areas were also described. Altria
Antibiotics 4637 and Elder summarized the status and applications of CE to the
Micelles 4637 analysis of small molecules, with an application area devoted to
Microchip CE 4638 chiral separations (4).
Miscellaneous 4638 CE moved into microscale with microchip electrophoresis
Thin-Layer Chromatography 4638 (MCE) showing great promise for enantiomeric separations.
Supercritical Fluid Chromatography and Related 4638 Chiral MCE was reviewed by Belder and Ludwig (5) with an
Techniques
introduction given into the principles of chiral separation with
Gas Chromatography 4639
MCE regarding instrumentation and methodology. Emphasis in
Liquid Chromatography 4639
Cyclodextrin CSPs and Mobile-Phase Additives 4639 this review was on approaches to improve detection and resolution
Macrocyclic Antibiotic CSPs 4639 in chiral MCE.
Polysaccharide CSPs 4640 Several approaches used to couple CE and electrospray
Protein-Based CSPs 4640 ionization-mass spectrometry (ESI-MS) for the analysis of chiral
Micellaneous CSPs 4640 compounds were summarized by Shamsi (6). A short compilation
Capillary Electrochromatography 4641 of commercially available CE-MS instruments and interface
Miscellaneous Techniques 4642 design was given, followed by an extensive discussion on the
Literature Cited 4642 various modes of chiral CE-MS. These modes included chiral
capillary zone electrophoresis-mass spectrometry (CZE-MS)
This fundamental review article covers developments and
with neutral derivatized cyclodextrins, chiral electrokinetic chro-
applications in chiral separations from January 2002 to January
matography-mass spectrometry (EKC-MS) with a charged chiral
2004 and is restricted to the English language literature. With
selector, micellar electrokinetic chromatography-mass spectrom-
the tremendous number of publications in this field, a compre-
etry (MEKC-MS), and capillary electrochromatography-mass
hensive review of all published papers is not feasible; rather this
spectrometry (CEC-MS).
fundamental review focuses on major developments in the field
CE-MS was again discussed in regard to affinity CE and CD-
of chiral separations as well as representative applications. Several
EKC, with particular attention paid to method development in a
excellent general reviews covering chiral separations are pre-
review by Tanaka (7).
sented first, followed by a examination of the methods and
High-Performance Liquid Chromatography (HPLC). The
techniques utilized in each area.
HPLC methods for the enantiomeric separation of amphetamine
and related compounds were reviewed by Herraiz-Hernandez et
REVIEWS
Capillary Electrophoresis (CE). Capillary electrophoresis al. (8), with discussions of the direct enantioseparation of non-
remains a popular technique for the enantioseparation of biologi- derivatized amphetamines using β-cyclodextrin as the chiral
cally active compounds. A review discussing the determination selector, both immobilized on the stationary phase and added to
of the enantiomeric excess in enantiomerically pure drugs by the mobile phase. Other chiral stationary phases such as Pirkle
cyclodextrin (CD)-modified CE, compiled by Schmitt et al. (1), type, cellulose based, or protein based were also discussed. The
reviews strategies of method development, sensitivity of detection, use of immobilized proteins as chiral stationary phases (CSPs) in
and optimization of separation parameters. The recent develop- HPLC and in CE for the enantioseparation of drugs was sum-
ments in the determination of enantiomeric drugs and their marized by Millot (9), including the main procedures for protein
immobilization onto matrixes and factors affecting enantiosepa-
* Corresponding author: (phone) (601) 974-1405; (fax) (601) 974-1401; ration. Clarke and Hage reviewed the clinical application of affinity
(e-mail) wardtj@millsaps.edu. chromatography including chiral separations (10, 11).
10.1021/ac040093t CCC: $27.50 © 2004 American Chemical Society Analytical Chemistry, Vol. 76, No. 16, August 15, 2004 4635
Published on Web 06/19/2004
Gas Chromatography (GC). The separation of enantiomers were reported. Egger et al. demonstrated that sulfated β-CDs used
by GC on CSPs was extensively reviewed by Schurig (12), with at low pH in the reversed polarity mode were found to give the
discussion of the method development, applications, and ancillary best separation of chiral dihydrofurocoumarin compounds in a
techniques of chiral separations using GC. CSPs with amino acid study comparing four different chiral methods using CE and
derivatives, terpene-derived metal coordination compounds, and micellar CE (22). Sulfated β-CD was used as the chiral selector
modified cyclodextrins were included. Schurig also comprehen- in the simultaneous enantiomeric CE separation of citalopram and
sively reviewed the practice and theory of enantioselective its metabolite desmethylcitalopram (23).
complexation GC on optically active metal(II) bis[3-perfluoroacyl)- A CE method for the chiral separation of racemic methotrexate
(1R)-camphorate] selectors (13). Applications extend to chiral using hydroxypropyl-β-CD was developed and validated by Kuo
analysis in asymmetric synthesis, enzymic reactions, pheromone, et al. (24). Various commercially available CDs were used in a
and flavor chemistry. The elucidation of thermodynamic param- study concerning the CE separation of aminophosphonic acid
eters of enantioselectivity and the investigation of the enantiomer- enantiomers, with variation of pH of background electrolyte,
ization of configurationally labile enantiomers was also discussed. concentration of CD, and type of CD used as the chiral selector
Thin-Layer Chromatography (TLC). The development of (25). Neutral CDs were used in the CE enantioseparation of
stationary phases for TLC in the last 10 years was compiled by propoxyphene, in which baseline resolution was achieved in ∼6
Gocan (14), and included chiral separation and recent advances min (26). A dual CD system of polyanionic heptakis-6-sulfato-β-
in chiral stationary phases. The CSPs discussed included nonpolar CD with neutral heptakis(2,3,6-tri-O-methyl)-β-CD was utilized in
bonded stationary phases impregnated with transitional metal ions, the CE enantioseparation of nonsteroidal antiinflammatory drugs
cellulose, modified cellulose, chitin, chitosan, and their derivatives. such as fenoprofen, flurbiprofen, ibuprofen, and ketoprofen (27).
Cyclodextrin and macrocyclic antibiotics were reported to have Selectivity for the enantioseparation of these drugs was predicted
very good results for enantioseparation by TLC, with molecular using mathematical models. Carboxymethylated β- or γ-CDs were
imprinting polymers also finding use as CSPs in TLC. used in a CE method to achieve the rapid separation of a set of
Capillary Electrochromatography. The applications of CEC 12 basic amino-containing drugs (28), but neither of the two CDs
were reviewed by Remcho et al. (15). Chiral separations with used was able to separate the entire set of drugs. A new CE
cyclodextrins and their derivatives, biomolecules, molecularly method was reported for the detection of enantiomeric purity of
imprinted polymers, “brush”-type phases, ion exchangers, anti- imidazo(2,1-i)purin-5-ones and related tricyclic water-soluble purine
biotics, polysaccharide derivatives, and other CSPs were included derivatives using native and modified β-CDs as chiral selectors
in this review. A summary of recent progress in open-tubular CEC (29).
for chiral and achiral separations included stationary-phase The distribution of enantiomers of methamphetamine, meth-
preparation (16), with the major developments, potential applica- cathinone, ephedrine, and pseudoephedrine in clandestine tablets
tions, technical difficulties, and advantages of each wall coating and urine samples was analyzed by a β-CD-modified CE method
discussed. and described by Liau et al. (30). The chiral separation method
Multiple Technique Reviews. The use of cyclodextrins in of 3,4-methylenedioxymethamphetamine and related compounds
chiral chromatography was compiled by Juvancz and Szejtli (17). by CE with β-CD and fluorescence spectroscopy was developed
The role of cyclodextrins in methods using capillary columns such and used to determine the distribution of isomers in clandestine
as GC, supercritical fluid chromatography (SFC), and CE was tablets and urine samples (31). Highly sulfated γ-CD was used in
detailed, as well as their use in other forms of chromatography the simultaneous chiral separation of nine amphetamine-type
such as HPLC and TLC. The mechanism of chiral recognition stimulants by CE (32), and highly sulfated β-CD was the chiral
using cyclodextrins was also discussed. Williams and Wainer (18) selector in the enantioseparation of warfarin (33).
reviewed the role of chiral chromatography in therapeutic drug A chiral CE method using heptakis(2,6-di-O-methyl)-β-CD was
monitoring and in clinical and forensic toxicology. Enantioselective reported for the resolution of the six enantiomers associated with
GC and HPLC were used as tools to unravel complex phenomena the metabolism of methadone (34). Native CDs and neutral and
associated with drug transport and metabolism. charged CD derivatives were evaluated in the development of the
chiral separation of diastereomeric flavanone-7-O-glycosides in
CAPILLARY ELECTROPHORESIS citrus by CE (35). The chiral separation of deprenyl N-oxide
Cyclodextrins. Cyclodextrins and their derivatives remain one isomer using CE in the presence of various CD derivatives was
of the most widely used chiral selectors for CE chiral separations. reported by Tabi et al. (36). Anionic and neutral compounds were
Sulfated β-CDs and carboxymethyl-β-CD were used as chiral enantioseparated using a heptasubstituted cationic β-CD as chiral
selectors in the separation of 41 chiral sulfoxides and sulfinate selector in CE (37). Chiral separation was found to be highly
esters (19). The sulfated β-CDs were reported to separate a dependent on pH, with the resolution enhanced at higher pH. A
greater number of compounds and had better separating capabili- β-CD substituted by an imidazole-bound histamine was used to
ties than carboxymethyl-β-CD. Hydroxypropylated, dimethylated, separate underivatized tryptophan in CE in the presence of copper-
and sulfated CDs were used as chiral additives in CE to evaluate (II) ion in what is reported to be the first ligand-exchange chiral
their effectiveness as chiral selectors (20). Eleven different CDs separation by a CD derivative added to the background electrolyte
including neutral and charged derivatives were used as chiral (38).
selectors in the chiral CE separation of fluoxetine and four A chiral CE method using highly sulfated CDs in a low-pH
analogues (21). Optimized separation conditions including type phosphate buffer and the short-end injection technique to enan-
of CDs, CD concentration, and pH of the background electrolyte tioseparate a basic, neutral, and acidic compound was evaluated
4636 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
for robustness, and the statistical interpretation was presented by EKC with carboxymethylated γ-CD and β-CD was used to
Perrin et al. (39). The chiral separation of enantiomers of a plant monitor the stereoselectivity of biodegradation of chiral polychlo-
growth regulator, abscisic acid, by CE with CD additives was rinated biphenyls (56). A comparison of charged CD derivatives
reported (40), with dimethyl-β-CD, hydroxypropyl-β-CD, and γ-CD for the enantioseparation of atropisomeric polychlorinated biphe-
giving satisfactory enantioselectivity. A novel assay method for nyls by CZE was reported by Garcia-Ruis (57), and the results
enantiomeric separation was developed using a combination of were compared with those obtained by the dual CD system
CE and electrospray tandem MS connected with a homemade reported above. Cyclodextrin-based EKC was used with quenched
interface (41). Nonaqueous CE using an ion-pairing reagent in phosphorescence detection to monitor the stereoselective bio-
combination with an anionic CD derivative enantioresolved basic degradation of camphorquinone by yeast (58). CZE employing
pharmaceuticals, with the enantiomeric resolution lost or strongly vancomycin as a chiral selector was used in the chiral separations
reduced in the absence of the ion-pairing reagent (42). of a nonsteroidal antiinflammatory drug candidate (59) and
CE is often compared with or used in conjunction with HPLC racemic N-acetyl derivatized amino acids (60).
methods. A comparative study on the enantiomeric separation of Antibiotics. The macrocyclic antibiotics were also used as
1,1′-binaphthyl-2,2′-diyl hydrogen phosphate and 1,1′-binaphthol chiral selectors for CE enantioseparations during this review
by HPLC and CE, using CDs and bile salts as single and dual period. A CE study including a theoretical approach was used to
selectors, was reported by Bielejewska et al. (43). The chiral determine the binding constant of acid herbicide enantiomers-
separation of an M3 antagonist was investigated using CE with vancomycin complexes, and the dependence on salt concentration
various sulfated CDs and by reversed-phase HPLC with derivatized was discussed (61). Several strategies for using vancomycin as a
cellulose, derivatized amylose, and two protein stationary phases chiral selector in CE were explored, including the dynamic coating
(44). Based on the comparison of techniques, a practical CE technique, the coelectroosmotic flow technique, and the partial
method using sulfated γ-CD was selected and validated. The chiral filling technique (62). The vancomycin analogue A82846B pro-
vided excellent selectivity for some acidic test solutes, with its
separation of bioactive cyclic Mannich ketones was performed
dimerization in solution proposed as the reason it showed
using HPLC with cellulose derivative CSPs or CD-CSPs, and in
enhanced enantioseparations compared with vancomycin (63).
CE, different CDs such as β-CD, γ-CD, carboxymethyl-β-CD, and
Micelles. Micellar methods continue to be used for chiral
succinyl-β-CD were added to the background electrolyte as chiral
separations in CE. A MEKC method was developed for the
selectors (45). Two independent methods using HPLC on polysac-
detection of chiral amino acids in orange juice (64). This method
charide-type CSP and CE with native and derivatized CDs were
utilized β-CDs as chiral selectors and laser-induced fluorescence
developed for the chiral resolution of the vasodilator drug
for detection. A system using 1-S-octyl β-D-thioglucopyranoside,
isoxsuprine (46). The methods were compared for efficiency,
SDS, and CD was used in the chiral separation of amino acids by
sensitivity, analysis time, and costs. The enantioseparation of
Tran and Kang (65), with the three-component system showing
tetramisole by CE and HPLC was reported, and these techniques
better separations than 1-S-octyl β-D-thioglucopyranoside alone as
were applied to the determination of the enantiomeric purity of a
chiral selector. Another three-component system utilized 3-((3-
veterinary drug formulation of L-levamisole (47). The CE methods
cholamidopropyl)dimethylammonio)-1-propanesulfonate with SDS
evaluated various CDs as chiral selectors, while the HPLC method
and CD for the separation of dansyl amino acids (66). Organo-
used a polysaccharide-type CSP. CE was determined to be the
phosphorus pesticides were enantioseparated in a capillary mixed-
preferred method.
mode EKC method using surfactants and neutral and charged
CZE was used with sulfated β-CD for the simultaneous CDs (67). A neutral CD derivative, hydroxypropyl-γ-CD, and an
determination of DOPA and carbidopa enantiomers (48). The ionic derivative, heptakis-6-sulfato-β-CD, were used as an additive
separation and migration behavior of 13 structurally related in CD-modified MEKC for the enantioseparation of triadimenol,
phenothiazines in CD-modified CZE was reported by Chen et al. a component of systemic agricultural fungicide (68). Pemoline
(49), with β-CD and hydroxypropyl-β-CD evaluated. A method for enantiomers were separated with CD-modified MEKC, with the
the enantiseparation of synthetic tetrahydronaphthalenic deriva- chiral separation dependent on the type of CDs used as chiral
tives using anionic CDs as chiral selectors and CZE was developed selectors (69).
(50). A CZE method for the detection of 0.1% of (R)-levochlor- Two amino acid-based alkenoxy micelle polymers were syn-
pheniramine maleate in samples of (S)-dexchlorpheniramine thesized and used as a pseudostationary phase in MEKC for the
maleate was reported (51), using carboxymethyl-β-CD in an acidic simultaneous separation of eight chiral β-blockers (70), with the
background electrolyte. The complete chiral separation of meth- polysodium N-undecenoxycarbonyl-L-leucinate polymer giving
oxamine and lobeline was achieved by CZE on a ethylbenzene- overall better chiral resolution and a wider chiral window. A
deactivated fused-silica capillary column, with dimethyl-β-CD and synthetic chiral polymer was utilized as a pseudo stationary phase
hydroxypropyl-β-CD giving the best results (52). Carboxyethyl- in MEKC in aqueous buffers in methods showing high efficiencies
β-CD dissolved in the operating buffer in CZE was used for the with the potential for rapid separations (71).
enantioseparation of dioxopromethazine in eye drops (53). Xu et The separation of benzoporphyrin derivative mono- and diacid
al. reported the chiral separation of 2,3-allenoic acid by CZE using enantiomers was achieved using 25 mM sodium cholate as the
CD derivatives of hydroxypropyl-β-CD and hydroxypropyl-γ-CD chiral selector in a method using laser-induced fluorescence-
(54). Abscisic acid enantiomers were enantioseparated by CZE capillary electrophoresis (72). The chiral separation of 20 pairs
using CDs and their derivatives (55), with γ-CD, hydroxypropyl- of amino acids derivatized with fluorescein-5-isothiocyanate was
β-CD, and dimethyl-β-CD forming strong complexes. studied with a mixture of β-CD and sodium taurocholate as chiral
Analytical Chemistry, Vol. 76, No. 16, August 15, 2004 4637
selectors (73), using CE and laser-induced fluorescence detection. SUPERCRITICAL FLUID CHROMATOGRAPHY AND
Resolution with the dual selectors was reported to be considerably RELATED TECHNIQUES
A variety of CSPs have been used for chiral separations in SFC
superior to that achieved with either selector alone.
during this review period. Phinney and Sander (89) studied the
Microchip CE. Microchip electrophoresis coupled with chemi-
polar additive concentration effects on CSPs containing either a
luminescence detection was used with hydroxypropyl-β-CD as a
macrocyclic glycopeptide or a derivatized polysaccharide. The
chiral selector for the enantioseparation of chiral dansyl amino
polar additives were found to significantly decrease retention, and
acids (74) and dansylphenylalanine (75). Sulfated CDs were used many analytes failed to elute in the absence of the polar additives.
in fast chiral separations of a variety of basic and acidic compounds The commercially available column Chiralpak was used in the
on a microchip electrophoresis instrument with linear imaging enantioseparation of triadimenol and triadimefon in SFC (90), with
UV detection (76). It was reported that a mixture of three chiral the separation occurring in 15 min. A Whelk-O1 column was used
drugs could be separated in less than 11 s. Chiral crown ether in a SCF method to determine the enantiomerization energy
was used as chiral selector in a CE and microchip CE method for barrier for some 3-hydroxy-1,4-benzodiazepine drugs (91).
the enantioseparation of gemifloxacin in a sodium-containing The enantioseparation of albendazole sulfoxide was achieved
media (77). on two columns, Chiralpak AD and Chiralcel OD, and the effects
Miscellaneous. Enantioseparations have also been carried out of different modifiers were examined (92). This enantioseparation
using a number of miscellaneous chiral selectors, with a few was also achieved at the semipreparative scale (93), using an
selected examples reported here. Nonaqueous ion-pair CE using adapted SFC chromatograph and a Chiralpak AD (250 mm × 10
quinine and tert-butylcarbamoylquinine as chiral selectors was mm) column. The effect of different injection volumes on purity
used in the enantioseparation of N-protected amino acids (78, 79) and throughput of the individual enantiomers was studied.
and peptides (80). Chiral separations in CE using cinchona Three macrocyclic glycopeptide chiral selectors, teicoplanin
(Chirobiotic T), teicoplanin aglycon (Chirobiotic TAG), and
alkaloid derivatives as chiral selectors were reviewed by Lam-
ristocetin (Chirobiotic R), were exhaustively evaluated for enan-
merhofer and Lindner (81). A new chiral receptor containing 1,7-
tioseparations with supercritical and subcritical fluid chromatog-
diaza-12-crown-4 was synthesized, and its application in chiral
raphy by Armstrong et al. (94). A set of 111 chiral compounds,
separations by CE was described by Wang et al. (82). A novel
including hetercycles, nonsteroidal antiinflammatory compounds,
chiral selector, N-benzoxycarbonylglycyl-L-proline, was introduced
β-blockers, sulfoxides, N-protected amino acids, and native amino
by Hedeland et al. (83) for enantioseparation of pharmacologically acids were separated on the three CSPs. Chirobiotic TAG and
active amines in nonaqueous CE. The enantioseparation of Chirobiotic T were found to fully or partially resolve 92% of the
mepivacaine was achieved in 72 s using short-end injection with enantiomers in the compound set, while Chirobiotic R separated
an effective capillary length of 8.5 cm. Underivatized amino acids only 60%. All separations were performed in less than 15 min, with
were enantioseparated by ligand-exchange CE using L-lysine as the majority of compounds separating in less than 4 min.
the ligand and copper(II) as the central ion (84). SDS was Twenty-four chiral dihydrofurocoumarin derivatives and struc-
necessary for simultaneous resolution of the amino acids but turally related compounds were enantioseparated on three mac-
caused precipitation at less than 32 mM at room temperature. It rocyclic glycopeptide CSPs using supercritical and subcritical fluid
was postulated that the precipitation might be caused by the chromatography (95), with the Chirobiotic T CSP reported to
formation of a neutral substance from the SDS monomer and the demonstrate the best enantioselectivity for 21 of the compounds.
copper(II)-lysine complex. Thermally unstable furan derivatives were enantioseparated by
SFC and HPLC with a derivatized cellulose or amylose CSP (96).
THIN-LAYER CHROMATOGRAPHY This report also included the separation of volatile furan ethers
Thin-layer chromatography remains a reliable technique for using GC/MS.
rapid screening of chiral separations. Some 2-arylpropionic acids Packed-column subcritical fluid chromatography was used in
were enantioresolved using silica TLC plates impregnated with the enantioseparation of a thiazolbenzenesulfonamide compound,
optically pure L-(-)-serine, with the method applied to commercial using a Chiralpak AD CSP (97). The effects of temperature and
ampules of ketoprofen dosage formulation (85). Silica TLC plates alcohol modifier were also reported. A ristocetin CSP was
evaluated for chiral separations in subcritical fluid chromatogra-
impregnated with L-(-)-serine and L-(-)-threonine and a mixture
phy, with solutes of differing structures and pKa values tested (98).
of these chiral selectors were prepared for the enantioseparation
The effects of modifiers, additives, temperature, and flow rate on
of more 2-arylpropionic drugs (86). Detection limits were reported
the enantioseparations were presented.
to range between 0.25 and 0.5 µg/mL, and the effect of temper-
SFC-MS was utilized in various applications during this review
ature, pH, and chiral selector concentration was studied.
period. A wide variety of pharmaceutical racemates were separated
Three commonly used β-blockers, atenolol, metoprolol, and and characterized using SFC coupled to a hybrid MS (Q-TOF)
propranolol, were enantioseparated using normal-phase TLC on equipped with an electrospray ion source (99). Three different
silica gel plates impregnated with L-aspartic acid with iodine CSPs and different pressure/temperature working conditions were
detection (87). Molecularly imprinted polymers of S-timolol were used. A technique for rapid method development for chiral
prepared and used in the direct chiral separation of some separations in drug discovery using sample pooling and SCF-MS
cardiovascular drugs, including propranolol, atenolol, timolol, was reported by Zhao et al. (100). Four CSPs and eight different
nadolol, nifedipine, and verapamil (88). modifier concentrations were used in a fully automated process
4638 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
lasting 15 h to attain optimal chiral separations for multiple on silica packed capillary columns using β-CD and 2-hydroxypro-
compounds. pyl-β-CD as mobile-phase modifiers (112). Separations were
accomplished in less than 2 min using this method.
GAS CHROMATOGRAPHY Macrocyclic Antibiotic CSPs. A set of 42 chiral sulfoxides
Cyclodextrin-based GC columns were found to have wide- were enantioseparated by HPLC using 5 different macrocyclic
spread use for the direct resolution of enantiomers during this glycopeptide CSPs, ristocetin A, teicoplanin, teicoplanin aglycon,
review period. Four derivatized CD-based CSPs, Chiraldex-G-TA, vancomycin, and vancomycin aglycon, and 7 eluents, three normal
G-PN, G-BP, and B-DM, were used in GC to enantioseparate 17 phase, two reversed phase, and two polar organic (113). The
chiral sulfoxides and 8 chiral sulfinate esters (101), with the G-TA teicoplanin and teicoplanin aglycon CSPs were reported to be the
and B-DM CSPs generally giving opposite elution orders for most most effective, with 35 and 33 of the 42 compounds studied
of the compounds. Alkylated β-CD and γ-CD CSPs were used in resolved, respectively. The enantiomers of 28 substituted dihy-
the GC separation of enantiomers of seven N-TFA-O-alkylamino drofurocoumarins were separated by HPLC using CSPs containing
acid derivatives (102). An evaluation of the nonpolar interactions ristocetin A, teicoplanin, and teicoplanin aglycon (114), with the
of the separations was also given. teicoplanin CSP exhibiting the broadest enantioselectivity with 24
The 2-O-methyl-3-O-acetyl and 2-O-acetyl-3-O-methyl derivatives compounds baseline resolved.
of 6-O-tert-hexyldimethylsilyl-γ-CD were synthesized as a GC CSP A study of D,L-tryptophan enantiomer retention on a teicoplanin
and tested for chiral separation (103). Enantioseparations were CSP was reported, using the perturbation technique to determine
compared with those of 2,3-di-O-methyl- and 2,3-di-O-acetyl-6-tert- the solute distribution isotherms while varying the mobile-phase
hexyldimethylsilyl-γ-CD CSPs, with the 2-O-methyl-3-O-acetyl-6- sodium perchlorate concentration (115). Phenoxypropionic acid
O-tert-hexyldimethylsilyl-γ-CD exhibiting the highest enantio- herbicides were enantioseparated by teicoplanin CSP, and the
selectivity. The chiral GC separation of 2-alkyl-2-keto-γ-butyrolac- perturbation method was used to calculate the solute distribution
tone derivatives and their alcohol analogues using a 2,3-di-O- isotherms (116). The effects of both temperature and methanol
methyl-6-O-tert-butyldimethylsilyl-β-CD CSP was reported by Ra- were described by a bi-Langmuir approach. A HPLC-MS assay
mos et al. (104). using a teicoplanin CSP for the determination of albuterol
A chiral dual-column GC system was used in the chiral enantiomers in human plasma was described (117). This method
separation of N-TFA-O-Me esters of six amino acids in a study to allows adequate sensitivity and reproducibility for the application
vary the selectivity by adjusting the individual carrier gas flow of studies of inhaled albuterol.
rates (105). Two columns, Chirasil-L-Val and Chirasil-D-Val, which The enantioseparation of secondary amino acids by HPLC was
demonstrate opposite enantioselectivities, were coupled in series studied using teicoplanin and ristocetin A as CSPs (118). An online
for this study. coupled HPLC method for the determination of diperodon enan-
A method suitable for the enantioseparation of D-amino acids tiomers in blood serum used a teicoplanin CSP for enantiosepa-
in a wine sample was reported using a GC capillary column coated ration after the reversed-phase separation of diperodon from the
with immobilized poly(dimethylsiloxane) anchored to (S)-(-)-tert- matrix (119). Teicoplanin and teicoplanin aglycon CSPs were used
Leu-(S)-(-)-1-R-naphthyl)ethylamide (106). The amino acids were in a study of the influence of carbohydrate moieties of teicoplanin
converted into N-pivaloyl methyl esters in two reaction steps. on the separation of some phenylcarbamic acid derivatives, with
the teicoplanin aglycon CSP achieving better separations (120).
LIQUID CHROMATOGRAPHY An enantioselective HPLC method for the determination of
Direct chiral separations using macrocyclic antiobiotics CSPs arotinolol in human plasma using teicoplanin CSP was validated
in HPLC continue to be very common, with cyclodextrin- and (121), and a method for the determination of baclofen in human
polysaccharide-based columns also finding much use. plasma using teicoplanin CSP was developed (122).
Cyclodextrin CSPs and Mobile-Phase Additives. The A semipreparative HPLC method for the enantioseparation of
HPLC chiral analysis of alkoxy-substituted esters of phenylcar- two 2-arylpropionic acids on novel CSPs containing teicoplanin
bamic acid was performed on β- and γ-CD CSPs (107). The A2-2 and A-40,926 was described by Alcaro et al. (123).
enantioselective separation of 28 racemic dihydrofurocoumarins A vancomycin CSP used with a polar organic solvent in HPLC
was studied using three native and six derivatized CD CSPs in was used in an investigation of the enantiorecognition process of
reversed-phase mode, polar organic mode, and normal-phase derivatives of substituted phenylcarbamic acid, with a discussion
mode (108), with the hydroxypropyl-β-CD reported as the most of the interaction mechanism of the separation included (124). A
effective CSP in the reversed-phase mode. Native and derivatized displacement study on a vancomycin CSP using N-acetyl-D-alanine
CD CSPs were evaluated for the enantioseparation of aromatic as a competing agent for the aglycon pocket was reported,
and aliphatic sulfoxides (109), and many sulfoxide enantiomers showing that dansyl amino acids bind to the active aglycon pocket
were baseline resolved using the derivatized CD CSP in the of the selector, with additional enantioselective sites at the
reversed-phase mode. vancomycin surface also involved in chiral discrimination (125).
The enantioseparation of 42 derivatized amino acids and A vancomycin CSP was used in a method for the enantioseparation
biogenic amines was accomplished with an amino-β-CD CSP of promethazine by HPLC (126), with vancomycin showing the
(110). A novel urea-covalent-bonded methylated β-CD CSP was best resolution over teicoplanin and ristocetin A CSPs. The effects
used in the chiral resolution of flavor and fragrance compounds of temperature and solute molecular size effects on the retention
(111). Ultrahigh-pressure liquid chromatography was demon- and enantioselectivity of D,L-dansyl amino acids were studied using
strated for fast and efficient chiral separations of pharmaceuticals a vancomycin CSP (127). Enantioseparation of fluoxetine (Prozac)
Analytical Chemistry, Vol. 76, No. 16, August 15, 2004 4639
in human plasma on a vancomycin CSP by HPLC-MS was dose pharmacokinetic studies (142). The enantiomers of 14
reported by Shen et al. (128). Vancomycin CSP was used in the organophosphonate derivatives were directly separated on Chiral-
HPLC method for the enantiomeric separation of 1,4-dihydro- pak AD, with all of the selected compounds being baseline
pyridines (129), with the method suitable for semipreparative separated (143).
separation. A comparison of the enantioseparation of methylphenidate
A ristocetin A CSP was used in a HPLC study of the effects of (Ritalin) was achieved in normal-phase mode on three different
temperature on retention of tryptophan, 1,2,3,4-tetrahydroiso- CSPs, Chiralpak AD, Chiralcel OD, and Chiralcel OB, and the
quinoline, and γ-butyrolactone analogues (130). Enantioselective role of benzoic acid and phenol as mobile-phase additives was
ion-exclusion chromatography on teicoplanin aglycon and (+)- also discussed (144).Two chiral methods for the separation of the
(18-crown-6)-2,3,11,12-tetracarboxylic acid CSPs was reported by enantiomers of 25 racemic 4-aryl-7,7-dimethyl- and 1,7,7-trimethyl-
Steffeck and Zelechonok (131). 1,2,3,4,5,6,7,8-octahydroquinazoline-2,5-diones were developed us-
Polysaccharide CSPs. An amylose tris(3,5-dimethylphenyl)- ing a Chiralpak AD and a Chiralcel OD CSP (145), with the
carbamate CSP was used in a study of the mechanistic aspects of resolution obtained on the two columns found to be complemen-
chiral discrimination (132). The direct chiral determination of tary. The enantiomeric resolution of o,p-DDT and o,p-DDD was
metyrapone and metabolites in plasma was accomplished using achieved on Chiralpak AD-R, Chiralcel OD-R, and Chiralcel OJ-R
a multidimensional achiral-chiral LC method (133). The CSP (146). A comparison of the enantioseparation of several tetralone
used was amylose tris(3,5-dimethoxyphenylcarbamate). The un- derivatives was accomplished using Chiralcel OB, Chiralcel OD,
usual effect of column temperature on the enantioseparation of Chiralpak AD, Chiralpak AS, and Chiralcel OF, with most of the
dihydropyrimidinone acid and its methyl ester on Chiralpak AD compounds being completely separated (147).
was summarized, with data suggesting that the CSP was under- Protein-Based CSPs. An enantioselective HPLC-MS method
going a thermally induced irreversible conformational change that was developed and validated for the simultaneous detection of
altered the separation mechanism between the heating and cooling saliva concentrations of the enantiomers of methadone and its
cycles of the method used (134). This change was dependent on metabolite EDDP using a CSP based on immobilized R 1-acid
the type of polar mobile-phase additive used. These thermal glycoprotein (AGP). The method was deemed accurate and
changes were not observed when the cellulose-based CSP Chiral- precise and was used to successfully analyze saliva obtained from
cel OD was used. patients enrolled in a methadone maintenance program. A Chiral-
The memory effect of mobile-phase additives in chiral separa- AGP CSP was used in the investigation of the heterogeneous
tions on Chiralpak AD was studied, and a procedure to remove adsorption behavior of selected enantiomers by Goetmar et al.
bound additives was developed (135). The unusual equilibrium (148). Reboxetine and O-desethyl reboxetine enantiomers were
behavior of the Troger’s base enantiomers on Chiralpak AD was simultaneously resolved by three stereoselective chromatographic
reported (136), with the finding that the adsorption of the more- methods, using Chiral-AGP, ChiraGrom 2, and Chiral-CBH,
retained (-)-enantiomer was not competitive and the amount respectively, in the reversed-phase mode (149). The chiral
adsorbed onto the CSP was independent of the concentration of resolution of mosapride enantiomers was achieved on Chiral AGP,
the (+)-enantiomer. However, the adsorption of the less retained and temperature studies were performed to investigate the
(+)-enantiomer was cooperative, with the amount adsorbed thermodynamics of the reversal in retention order (150).
increasing with increasing concentrations of the (-)-enantiomer. Amlodipine in human plasma was enantioseparated by a Chiral
The chiral HPLC separation of nucleoside analogues of d4T AGP CSP in an HPLC-tandem MS method (151). The method
and acyclovir was accomplished on the silica-based amylose CSPs was validated, and the limit of quantitation was 0.1 ng/mL for
Chiralpak AD and Chiralpak AS, with the effect of structural each amlodipine enantiomer. The enantiomers of ketamine and
features studied in relation to retention, selectivity, resolution, and norketamine in human plasma were determined using LC-MS
elution order (137). The HPLC enantioseparation of hydroxy- with a Chiral AGP CSP. The method was validated and applied to
mebendazole in human plasma was achieved on a Chiralpak AD, samples from a clinical study of ketamine in pain management
and a concurrent CE method using sulfated β-CD as chiral selector (152).
was also developed (138). Both methods were validated. A CSPs obtained by immobilization of human serum albumin on
validated chiral HPLC normal-phase method for the enantiosepa- various polymer-coated silicas were investigated for the enantio-
ration of linezolid on Chiralpak AD was reported (139). The separation of racemic mixtures of tryptophan, oxazepam, warfarin,
resolution of the enantiomers of dialkylaminoalkylnaphthalenes and NBP (153). An immobilized human serum albumin CSP was
was achieved by a preparative HPLC method using Chiralpak AD used in the study of the stereoselective binding of 2-(4-biphenylyl)-
CSP (140). For the chiral resolution of flurbiprofen and its major 3-substituted 3-hydroxypropionic acids (154). CSPs based on
metabolites, four methods were developed using Chiral-AGP and human serum albumin and bovine serum albumin were utilized
Chiralpak AD CSPs, one was developed using a reversed-phase to determine the species dependency in chiral drug recognition
HPLC method using hydroxypropyl-β-CD as a chiral mobile-phase (155).
additive, and another utilized a precolumn derivitization method Micellaneous CSPs. Novel CSPs based on cinchona alkaloids
(141). Only the method utilizing the Chiralpak AD CSP showed were used in the chiral HPLC separation of 3,5-dinitrobenzoyl
the separation and enantioresolution of all three analytes within amino acids (156), all-R- and all-S-enantiomers of oligoalanines
45 min. The enantioseparation of ibuprofen in human plasma was with N-terminal protection groups (157), N-acylated amino acids
performed using Chiralpak AD-RH in reversed-phase mode by (158), and N-protected peptides (159). Enantiomeric discrimina-
HPLC-MS, with the method reported to be suitable for single- tion for these CSPs was exceptionally high. A quinine-based chiral
4640 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
anion-exchange CSP achieved the enantioseparation of 23 N- using a high-sensitivity UV detection cell (172). Basic compounds
acylated amino acids, and the effect of temperature on the were enantioseparated using CEC with packed silica capillaries
performance of the separation was investigated (160). This CSP derivatized with vancomycin with polar organic solvents as mobile
was also used in a micro-HPLC method for the separation of phases (173). It was reported that the enantiomeric resolution,
peptide enantiomers (up to 6 amino acid residues) (161). Resolu- electroosmotic flow, and number of theoretical plates were
tion of the enantiomers of diphosphines and of the corresponding strongly influenced by the type and concentration of the organic
phosphine oxides was accomplished using four different CSPs, solvent.
Supelcosil LC-(S)-naphthylurea and LC-(R)-phenylurea, Chiral- Strong cation-exchange-type CSPs based on sulfodipeptide
pak AD, and Whelk-O1 (162). After optimization of the mobile chiral selectors were reported by Lindner et al. (174), and the
phases, resolution factors ranged from 0.35 to 3.61 for the synthesized CSPs were used to separate chiral bases by nonaque-
Supelcosil CSPs and from 0.37 to 6.57 for the Chiralpak AD and ous CEC. New CSPs derived from enantiomerically pure deriva-
Whelk-O1 columns. The Whelk-O1 CSP was utilized in the HPLC tives of cysteine carrying sulfonic acid groups were evaluated for
resolution of enantiomers of 2-methoxy-1-((4-methylpiperazino)- the nonaqueous CEC enantioseparation of chiral bases including
methyl)ethyl esters of N-(2-,3-, and 4-alkoxyphenyl)carbamic acid β-blockers, β-sympathomimetics, and other basic drugs (175). A
in the polar organic and reversed modes (163) and in a study of CEC method for the determination of the enantiomeric excess of
the mechanism of enantioseparation. A method using HPLC-MS D-ephedrine was developed using a novel low-molecular-weight
with a Crownpak CR+ CSP was used for high-throughput strong chiral cation exchanger based on penicillamine sulfonic
screening of enzymatic racemase activity (164). Antibody-based acid, which was immobilized on thiol-modified silica particles
CSPs were shown to be useful for routine enantiomeric separations (176). The high loadability of the CSP and good peak sensitivity
in HPLC in an immunoaffinity chromatography method (165). allowed the determination of less than 0.1% enantiomeric impurity
with good accuracy. Cation-exchange-type CSPs based on 3,5-
CAPILLARY ELECTROCHROMATOGRAPHY dichlorobenzoylamino acid and aminophosphonic acid derivatives
The popularity of capillary electrochromatography remained as chiral selectors and silica as the chromatographic support were
high in this review period for the separation of chiral racemates, used in the enantioseparation of chiral bases by nonaqueous CEC
with various separations performed using macrocyclic antibiotics (177). A wide variety of chiral bases were reported resolved,
and other CSPs. The enantioseparations of 12 glycyl dipeptides including β-blockers and other amino alcohols, local anesthetics
by CEC on a teicoplanin aglycon-packed capillary were compared such as etidocaine, antimalarial agents such as mefloquine,
to results obtained using micro-HPLC with the same stationary Troger’s base phenothiazines such as promethazine, and antihis-
phase (166). Efficiency and resolution were generally found to taminics. A new chiral monomer derived from cinchona alkaloid
be higher in CEC than with micro-HPLC. Two different types of was used in an in situ preparation of an enantioselective monolithic
fused-silica capillaries were evaluated for the enantioseparation capillary column that was comparatively evaluated with previously
of several basic compounds by CEC (167). The first capillary was described analogues used as CSPs in CEC (178). The new CSP
a CSP containing teicoplanin mixed with silica microparticles, showed enhanced enantioselectivities and faster separations than
while the second capillary contained only the teicoplain. Several the analogues, with derivatized amino acids being enantiosepa-
β-blockers were fully enantioseparated with both capillary types rated with resolution values between 2 and 4.
in very short times. A capillary packed with teicoplanin aglycon Pressure-assisted CEC was used with a Pirkle-type CSP, the
was used as a CSP in CE for the enantioresolution of diastereo- Whelk-O1, and normal-phase solvents with small percentages of
meric di- and tripeptides (168). While all compounds studied could water to separate a number of chiral analytes (179). The effects
be baseline resolved under optimized conditions, it was not of the different contributions of pressure and voltage were
possible to find a uniform mobile phase showing optimal results reported. Another pressure-assisted CEC application was reported
for all peptides. by Honzatko et al. (180) in the enantioseparation of neutral amino
A new CSP was prepared by Fanali et al. (169)by reacting MDL acids and amino alcohol derivatives. Two Pirkle-type columns were
63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the used.
teicoplanin family, with silica particles. These particles were used Chiral CEC-MS was used in the enantioseparation and
to pack a capillary column for only 6.6 cm. This short-end injection determination of warfarin enantiomers in human plasma (181),
capillary achieved separations in 1-3 min in CEC, but relatively using a Whelk-O1 CSP and electrospray ionization MS. A CSP of
long retention times were observed for the same CSP capillary avidin was prepared by physical adsorption to a monolithic silica
using capillary liquid chromatography. Fanali et al. (170) also column and used for chiral separations in CEC and capillary HPLC
reported the separation of hydroxy acid enantiomers using CEC (182) for 12 chiral test compounds. An avidin CSP was used in
using the CSP reported above. Separation parameters such as an evaluation of extended light path capillary and etched capillary
organic solvent type and concentration, buffer pH, and tempera- for use in open tubular CEC (183). A novel method to synthesize
ture on the enantioresolution factor, retention time, and retention a human serum albumin CSP was reported, and its use in CEC
factor were studied. The silica-based packed capillary teicoplanin and capillary HPLC was detailed (184). Affinity CEC with zonal
derivative (Hepta-Tyr) CSP and a vancomycin CSP were employed elution method was used to investigate the competitive binding
to enantioseparate some selected acidic nonsteroidal antiinflam- of enantiomers to a bovine serum albumin column (185).
matory drugs by CEC (171). A packed capillary with vancomycin A polyelectrolyte multilayer (PEM) coating was used to modify
CSP was used to achieve the enantioseparation of the antidepres- fused-silica capillaries for open tubular CEC (186), with the PEM
sant drug fluoxetine and its main metabolite norfluoxetine by CEC coating constructed in situ with alternating rinses of positively
Analytical Chemistry, Vol. 76, No. 16, August 15, 2004 4641
and negatively charged polymers. Optimal conditions for the Daisy-Malloy Hamburg, currently a graduate student at the Uni-
versity of Cincinnati, received her B.S. degree from Millsaps College
coating procedure were reported, and the PEM-coated capillary (Jackson, MS).
was used for the enantioseparation of 1,1′-binaphthyl-2,2′dihydro-
gen phosphate, 1,1′-bi-2-naphthol, secobarbital, pentobarbital, and
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