114 2010 114-130 chiral separation of pharmaceuticals by ......for separation of new...

114 Current Pharmaceutical Analysis, 2010, 6, 114-130

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Chiral Separation of Pharmaceuticals by High Performance Liquid

Chromatography

Yiwen Zhang, Shun Yao, Hao Zeng and Hang Song*

Department of Pharmaceutical and Biological Engineering, Sichuan University, Chengdu 610065, P. R. China

Abstract: The increasing demand for enantiopure drugs has led to the development of a variety of stereoselective separa-

tion technologies. Among them, high performance liquid chromatography (HPLC) is well recognized as a powerful, fast

and efficient technique, which has been successfully employed for analysis and preparation of enantiomers of drugs.

Nowadays, liquid chromatographic techniques are the focus of intensive research, which lead to the rational design and

production of highly selective and efficient chromatographic materials. This review focuses on various HPLC methods

and related hyphenated techniques (including LC-MS, LC-OR detector, LC-CD detector) for chiral separation of pharma-

ceuticals, many new developments and applications are introduced in chiral HPLC separations in recent years. In this pa-

per, the usage of new materials as chiral stationary phases (CSPs) in liquid chromatography for enantiomeric discrimina-

tion is investigated in detail. Moreover, hyphenated techniques are very useful for chiral separation of HPLC, especially

for separation of new pharmaceuticals.

Keywords: High performance liquid chromatography, Chiral separation, Enantiomeric purity, Hyphenated techniques.

1. INTRODUCTION

Molecular chirality is a fundamental consideration in drug discovery, it is very necessary to understand and de-scribe biological targets as well as to design effective phar-maceutical agents (2005) [1]. A visual context was ever pro-vided to position chiral technology in the highly complex and evolving drug discovery environment with its numerous challenges, hurdles, and tools (see Fig. (1)) (2007) [2].

Fig. (1). Positioning Chiral Technology in a highly complex and

evolving drug discovery environment [2].

*Address correspondence to this author at the School of Chemical Engi-neering, Sichuan University, Chengdu 610065, P. R. China; Tel: +86-28-8540-5221; Fax: +86-28-8540-5221; E-mail: [email protected]

Nowadays, chiral separation covers an important area of analytical chemistry of relevance to a wide variety of scien-tific professionals. Many enantiomeric forms of drugs have been recognized to have different physiological and thera-peutic effects. Very often, only one form is pharmacologi-cally active in an enantiomeric pair. It is, thus, hardly sur-prising that the pharmaceutical industry needs effective methods to determine enantiomeric purity and obtain effec-tive and safe drugs with single steroconfiguration (2006) [3]. Ward et al. (2006, 2008) [4, 5] ever reviewed various devel-opments and applications in chiral separations. A large number of approaches have been used to isolate enantiomers (such as gas chromatography, thin-layer liquid chromatog-raphy, capillary electrophoresis, liquid chromatography, su-percritical fluid chromatography, electrokinetic chromatog-raphy, etc.), among the approaches enantioselective liquid chromatography has become the most popular technique for quickly obtaining limited quantities (from mg to multi-grams) of pure single enantiomers, especially in drug discovery [1]. Moreover, HPLC is well recognized as a powerful, fast, highly efficient and selective technique, suc-cessfully employed for preparation and analysis of enanti-omers of drugs (2007) [6]. Okamoto et al. (2008) [7] re-ported about chiral HPLC for efficient resolution of enanti-omers and mentioned that more than half of the determina-tions have been carried out by chiral HPLC in all the relative reports. In recent years, some new HPLC methods have been developed for the analytical and semipreparative resolution of new drug enantiomers (2005) [8]. Although HPLC may be considered as a mature technology, many advances have been made to improve the separation capability and the effi-ciency of method development and sample analysis con-tinuously. Ever increasing demands for chiral analysis and for higher laboratory efficiency have driven the development

Chiral Separation of Pharmaceuticals Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 115

of various options for high-speed analysis with the desire to improve chiral resolution (2006) [9].

HPLC can be used either indirectly with chiral derivati-zation reagents (CDR) or directly with chiral stationary phases (CSPs) or chiral mobile phase additives (CMPA) to separate chiral compounds. The characteristics of these three chromatographic methods are summarized in Table 1. At present, large kinds of new materials have been developed as chiral stationary phases (CSPs) in liquid chromatography. Also, some new chiral derivatization reagents and new chiral mobile phase additives have been used for enantiomeric separation successfully. Moreover, large amounts of applica-tions of several hyphenated techniques (LC-MS, LC-CD and LC-OR, etc.) were reported succesively in chiral analysis (2000-2007) [10-14]. For example, when an enantiomer ex-presses exciton coupling that is observed in the CD spec-trum, the absolute stereoconfiguration can be assessed in combination with molecular modeling studies of the lowest energy conformers (2005-2008) [15-22].

This review focuses on the current developments in col-umn technology and method development approaches, then a brief discussion of advances is given in hyphenated tech-niques. Related methods of rapid analysis include mi-cro-columns, small particle columns and multidimensional chiral high performance liquid chromatography.

2. NOVEL CHIRAL STATIONARY PHASES (CSPS)

As is well known, chiral separation by HPLC with CSPs has become a powerful tool in the development of chiral drugs (2009) [23]. The HPLC-CSPs demonstrated great suc-cess for performing chiral separation in pharmaceutical in-dustry, because the HPLC can be used both for analytical or preparative purpose in a very efficient and flexible way (2005, 2005) [8, 24]. So far, there are numerous commer-

cially available chiral selectors or CSPs for the resolution of racemic drugs. Several new selectors for HPLC applications have been developed in recent years such as cyclodextrin, polysaccharide, protein, macrocyclic antibiotics, crown ethers, etc. Table 2 presents various typical kinds of chiral selectors, including their characterizations, structures and possible separation analytes.

2.1. Brush-type CSP

Brush-type CSPs, also called ‘Pirkle CSPs’, were in-vented by Pirkle’s group since the early 1980s (1980, 1991) [25, 26]. The chemical structural characteristics of this type of CSPs are based on the presence of -donor groups,

-acceptors group or the groups that could form multiple hydrogen bonds, and these groups can give a good fit to the “three point interaction principle” theory. This type of CSPs (such as DNB-Leu, DNB-PG, Whelk-O 1, Whelk-O 2, ULMO, -Burke, -Gem 1, all from Regis Technologies, Inc. USA) have been extensively used for chiral analysis (2003-2008) [27-34]. Some structural improvement of exist-ing brush-type CSP have been carried out for improving lim-ited separating capability of the CSPs. These novel CSPs show strong separating capability for many specific chiral compounds. Landek et al. (2006) [35] adopted brush type chiral stationary phases derived from L-leucine for enanti-omeric separation of 1,1’-binaphthyl-2,2’-diol enantiomers. A novel chiral packing material for HPLC was prepared by connecting (R)-1-phenyl-2-(4-methylphenyl) ethylamine (PTE) amide derivative of (S)-isoleucine to aminopropyl silica gel through 2-amino-3, 5-dinitro-1-carboxamido-ben-zene unit (2007) [36]. A Pirkle-type chiral stationary phase for column chromatography based on N-p-Amino ben-zoyl-L-glutamic acid has been synthesized for resolution of

-methylphenylethylamine (2010) [37].

Table 1. The Characterization of Three Chromatographic Methods

Method Advantages Disadvantages

Chiral

deriva-

tization

reagents

(CDR)

(1) Less expensive, i.e., convention nal chromatographic

columns can be used;

(2) Flexible, because various achiral columns and mobile

phase conditions, as in HPLC, can be used;

(3) Numerous types of derivatization chemistry are

available and the cost of each reagent may be less expen-

sive than for a chiral column;

(4) Different selectivities can be achieved.

(1) Long analysis time that include sample preparation and verification of

the derivatization chemistry;

(2) Inconvenience, specifically in preparative chromatography, when re-

versal of derivatization is needed to recover the pure enantiomers;

(3) Yhe need to synthesize noncommercially available pure derivatizing

reagent;

(4) Biased results for enantiomeric composition due to partial racemization

of derivatizing agent or unequal reaction rates.

Chiral

stationary

phases

(CSPs)

(1) A large number of commercially available chiral

columns can be selected;

(2) Convenience, specifically in preparative chromatog-

raphy;

(3) The mode of operation is easy;

(1) Many commercially available chiral columns are expensive;

(2) One specific chiral column sometimes, can only separate very few

kinds of enantiomers;

(3) It is difficult to select CSPs for efficient resolution of specific enanti-

omers

Chiral

mobile

phase

additives

(CMPA)

(1) Less expensive conventional LC columns can be used;

(2) A wide variety of possible additives are available;

(3) Different selectivities from the chiral phases can be

obtained.

(1) Many chiral additives are costly, and sometimes, have to be synthe-

sized;

(2) The mode of operation is complex,

(3) Incovenient for preparative applications because the chiral additive

must be removed from the enantiomeric solutes.

116 Current Pharmaceutical Analysis, 2010, Vol. 6, No. 2 Zhang et al.

Table 2. Most Popular Commercially Available LC CSPs

CSP’s

Type Characteristics

Symbolic

Column Structures of the CSP

Separation

Analytes

Brush-

type

Based on “three point

interaction principle” for

chiral discrimination

Whelk-O 1

NH

O

O2N

NO2

Si

CH3

CH3

O

Acids, amines,

alcohols,

amides, esters,

sulfoxides, thiols,

amino alcohols,

alkaloids, cyclic

ketones,

alkaloids,

Cyclo-

dextrin

Cyclodextrin cavity con-

tains hydroxyls for

H-bonding with polar

groups of analyte; hydro-

phobic portion of analyte

fits into non-polar cavity

(inclusion complexes)

CYCLOBOND

2000AC O

OHRO

OH

O

O

OH

RO

ORO

OOH

OH

OH

O

O

OH

OR

OH

OO

OR

OH

HO

O

OOH

OH

RO

O

O

OH

HO

HO

O

R=CH3CO-

Immobilised on silica gel

Amino acids,

amines, alcohols,

amides, crown

ether, metallo-

cenes, polycyclic

aromatic hydro-

carbons, lactones,

Poly-

saccharide

Enantiomers form

H-bonds with carbamate

links between side chains

and polysaccharide back-

bone

CHIRALPAK IC

O

O

n

O

HN

OCl

O

HN

O

Cl

Cl

O

NH

Cl

Cl

Cl

O


Acids, amino

acids, amines,

alcohols, amides,

esters, sulfoxides,

lactams, lactones,

cyclic ketones,

NSAIDS,

Protein-

based

Natural proteins bonded

to a silica matrix; proteins

contain large numbers of

chiral centers and interact

strongly with small chiral

analytes through hydro-

phobic and electrostatic

interactions, H-bonding

CHIRAL-AGP 1-acid glycoprotein


Acids, amines,

alcohols, amides,

sulfoxides, cyclic

drugs, aromatic

drugs,NSAIDS,

peptides


Table 2. contd….

CSP’s

Type Characteristics

Symbolic

Column Structures of the CSP

Separation

Analytes

Macro-

cyclic antibi-

otics

Contain a large number of

chiral centers together

with cavities for analytes

to enter and interact

CHIROBIOTIC R

OH

O

NH HN

HO

OH

O

HO

CH2OHOHHO

HN

HN

CH3

OHH2N

O

O

NH

HN

O

O

O

COOCH3

OO

O

O

O

CH3

NH2

OH

OO

O OCH2OH

OHOH

O

O OH

OH

OH

OO

HO

HO

H3C

HO

HO

HO


Acids, amino

acids, amines,

amides, esters,

sulfoxides, amino

alcohols,

Crown ethers Crown ethers are macro-

cyclic polyethers that can

form host-guest com-

plexes

CROWNPAK CR

O

O

O

OO

O

CR+=(S)-18-crown-6-ether


Amino acids,

amines, alcohols,

crown ether,

metallocenes,

polycyclic aro-

matic hydrocar-

bons,

2.2. Cyclodextrin CSP

The first CSP containing cyclodextrin (CD) bonded to silica gel was developed by Armstrong and DeMond in 1984 (1984) [38]. Recently, both the native CD CSPs and the modified CD CSPs with improved enantioseparation per-formances have been used in many cases (2008) [39]. Berkecz et al. (2007) [40] employed a kind of -CD based chiral stationary phase for enantioseparation of 1-( -amino-benzyl)-2-naphthol and 2-( -aminobenzyl)-1-naphthol analogs. Despite the numerous CD derivatives were developed as chiral selectors in the past decades for the purpose of chiral separation, a great need still exists for further effort to synthesize novel CD-CSPs with improved chiral recognition ability towards a large pool of racemic analytes with shorter time and greater accuracy. Tang et al. (2008) [41] provided a meaningful overview of recent work carried out in their laboratory on the synthesis of four series of mono-substituted positively charged CDs which were applied as chiral selectors in the separation of derivatized amino acids, anionic pharmaceutics and neutral analytes by

and neutral analytes by chromatographic techniques. These novel cyclodextrin CSPs showed good chiral recognition ability in actual separation.

2.3. Polysaccharide CSP

Polysaccharide type CSPs have proven to be one of the most useful CSPs because of their versatility, durability, and loadability (2001) [42]. In recent years, a broad range of commercially available polysaccharide phases have been produced by Daicel group (Osaka, Japan), based on cellu-lose, amylose esters or carbamates. The cellulose-based columns (e.g. Chiralcel OD) and the amylose-based columns (e.g. Chiralcel AD) are frequently used for the enantiosepa-ration (2001-2009) [42-46]. Immobilised polysaccha-ride-based CSPs are a new generation of chiral chroma-tographic materials combining the remarkable enantioselec-tive performance of the polysaccharide derivatives with sol-vent versatility in enantiomeric resolution. Since 2004 three immobilised polysaccharide-derived CSPs have become commercially available: Chiralpak IA, Chiralpak IB and


Chiralpak IC by Daicel Chemical Industries, Ltd. (Tokyo, Japan) (2008) [47]. Zhang et al. (2008) [48] employed Chiralpak IC for separation of 70 chiral molecules. The re-sults indicated that Chiralpak IC exhibited high enantioselec-tivity for a large number of enantiomers. In addition, the versatility of mobile phase is an important factor for chiral separation on Chiralpak column. The chromatographic per-formances of Chiralpak IA under various mobile phases were investigated (2005) [49]. It was found that four solvents (methyl tert-butyl ether, dichlormethane, tetrahydrofuran and ethyl acetate) were particularly versatile in mobile phase. So it was highly recommended to screen these solvents first for enantioselectivity.

Jin et al. compared several polysaccharide-derived chiral stationary phases for the enantiomeric separation of N-fluorenylmethoxycarbonyl -amino acids (2007) [50] and N-protected -amino acids (2009) [51]. The result indicated that all the CSPs resulted in the greatest separation factor. However, Chiralpak AD and Chiralcel OD of the typical coated-type CSPs are not compatible with all solvents of the mobile phase (normal phase mode is mainly used), because the chiral selectors of polysaccharide derivatives for these CSPs are coated on a silica matrix. Moreover, owing to the compatibility with a wide range of solvents of the covalently

bonded CSPs, it is expected some new application of Chiralpak IA and IB in enantiomer separation, especially for preparative purpose. Some literatures reported the technique for immobilizing the polysaccharide derivatives onto the silica gel formerly (2008, 2007) [52, 53].

2.4. Protein-based CSP

The advantages of protein-based CSP include enantiose-lectivity to a wide range of compounds because all proteins have certain ability to discriminate chiral molecules. CSPs based on bovine serum albumin (BSA), human serum albu-min (HAS), 1-acid glycoprotein (AGP), ovomucoid from chicken egg whites (OMCHI), avidin (AVI), cellobiohydro-lase I (CBH I) and pepsin are now commercially available (2001) [54]. So far, many protein-based CSPs have been developed, and some studies (2000-2003) [55–57] were pub-lished on the mechanisms and applications of enantiosepara-tion of chiral drugs.

In recent years, serum albumin of other species, penicillin G-acylase, antibodies, fatty acid binding protein and strep-tavidin were newly introduced as the chiral protein-based selectors in CSPs (2008, 2008) [58, 59]. Fig. (2) shows good enantioseparation of tryptophan, warfarin and ibuprofen on

Fig. (2). Enantioseparations of (a) D/L-tryptophan, (b) R/S-warfarin and (c) R/S-ibuprofen on HSA columns prepared by the SMCC and SIA methods. The HPLC conditions were as follows: sample concentration, 20μM tryptophan, ibuprofen or warfarin; sample volume, 20μL; mo-bile phase for tryptophan, pH 7.4, 0.067M potassium phosphate buffer; mobile phase for ibuprofen, pH 7.0, 0.067M potassium phosphate buffer containing 8% 2-propanol and 1mM octanoic acid; mobile phase forwarfarin, pH 7.0, 0.067M potassium phosphate buffer containing 5% 2-propanol and 1mM octanoic acid; flow rate for the SMCC HSA column, 1.5 mL/min for tryptophan, 1.0 mL/min for ibuprofen and warfarin; flow rate for the SIA HSA column, 0.3 mL/min for tryptophan and warfarin, 0.5 mL/min for ibuprofen; column size, 5 cm 4.6mm i.d.; temperature, 25 [58].


HSA columns prepared by N-(4-carboxycyclohexylmethyl) maleimide (SMCC) and succinimidyl iodoacetate (SIA) methods [58]. The chiral recognition mechanisms of these proteins were persumed by molecular modeling, ligand docking, and X-ray crystallography, which can effectively promote the rapid development in designing new pro-tein-based CSPs with specific group proteins used for chiral selectors.

2.5. Macrocyclic Antibiotics CSP

Macrocyclic antibiotics used as chiral selectors were first

introduced by Armstrong’s group (1994) [60] and represent a relatively new class of chiral selectors in separation science (2001, 2006) [61, 62]. The selectors have a large number of stereogenic centers and functional groups, which could real-

ize multiple interactions with chiral molecules. So far, sev-eral macrocyclic antibiotics (including rifamycins B and SV, avoparcin, teicoplanin, ristocetin A and vancomycin) have been used as chiral selectors widely (2009) [63]. A vanco-

mycin degradation product-based CSP has been adopted for chiral separation of racemic compounds (2007, 2008) [64, 65]. Honetschlagerova-Vadinska et al. (2009) [66] made satisfying evaluation of the enantioselective performance of

two teicoplanin-based CSPs Chirobiotic T and Chirobiotic T2, and then the authors discussed the possible interaction mechanisms on the basis of the separation results and some theories. Pataj et al. (2009) [67] compared three teico-

planin-derived CSPs (Chirobiotic T, T2 and TAG) for the enantioseparation of

2-homoamino acids. The result indi-

cated that Chirobiotic TAG, whose structure contain no sugar units, may promote the interactions of the enantiomers

of 2-homoamino acids with branched alkyl or aryl

side-chains, whereas 2-homoamino acids with alkyl

side-chains Chirobiotic T and T2 seem to be more favorable. Vancomycin-CSP was used for determinating thalidomide

enantiomers in biological samples (2006) [68].

2.6. Other CSPs

Chiral synthetic polymers, crown ethers, chiral imprinted polymers, ionic compounds, chiral ionic liquid, and some natural products have also been used as CSPs for the enanti-oseparation of pharmaceuticals and their unique structure always result in uncontemplated separation ability for chiral drugs.

A comprehensive review on the synthesis and application of chiral synthetic polymers was reported by Nakano (2001) [69]. Two new synthetic polymeric CSPs based on trans-(1S, 2S)-cyclohexanedicarboxylic acid bis-4-vinylphenylamide and trans-N,N-(1R,2R)-cyclohexanediyl-bis-4-ethenylben-zamide monomers were prepared and evaluated by HPLC in normal phase system (2008) [70]. Further, a new synthetic polymeric CSP for liquid chromatography was prepared via freeradical-initiated polymerization of trans-9,10-dihydro-9, 10-ethano-anthracene- (11S,12S)-11,12-dicarboxylic acid bis-4-vinylphenylamide. This new polymeric CSP showed special enantioselectivity for many chiral compounds in multiple mobile phases (2007) [71].

Crown ethers are macrocyclic polyethers that can form host-guest complexes. The CSPs of crown ethers have an

excellent chiral recognition ability and are easily bound co-valently to the support. Berkecz et al. (2008) [72] employed a chiral stationary phase containing (+)-(18-crown-6)-2,3,11, 12-tetracarboxylic acid for chiral separation of several

-amino acids. A novel CSP prepared by immobilizing a chiral pseudo-18-crown-6-type host onto 3-aminopropyl silica gel was used for the enantioseparation of 18 common natural amino acids efficiently (2005) [73]. In addition, a CSP based on (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was evaluated for the direct resolution of the enanti-omers of dipeptides and tripeptides (2005) [74].

Molecularly imprinted polymers (MIP) have been ex-ploited as CSPs since the pioneering work of Wulff in the 1970s (2005) [75]. Several excellent reviews were given in past years on the use of molecularly imprinted stationary phases in HPLC (1999-2001) [76-78]. Molecularly imprinted polymers CSPs can be extremely successful in most chiral for many certain compounds.

CSPs with ionizable groups have been developed in past few years. Hoffmann et al. (2008) [79] investigated the syn-ergistic effects on enantioselectivity of zwitterionic CSPs for the separation of chiral acids, bases, and amino acids. For example, a novel strong cation-exchange-type of chiral sta-tionary phase has been used for separation of cinchona alka-loids (2009) [80].

Ionic liquids (ILs) are a group of organic salts that can keep liquid-state at room temperature. They have unique chemical and physical properties, including air- and mois-ture-stable, a high dissolving capacity and virtually no vapor pressure. Because of these properties, they can serve as “green” recyclable alternatives to the volatile organic com-pounds which are traditionally used as industrial solvents (1999-2003) [81-84]. Advances in ILs have made the syn-thesis of chiral ILs become a subject of intense study in re-cent years (1999-2006) [85-96]. The popularity stems from the fact that it is possible to use chiral ILs as a chiral station-ary phase in chromatography for the determination of phar-maceutical compounds (2005) [97]. Tran et al. (2006) [3] successfully synthesized both enantiomers of a novel chiral ionic liquid, (R)- or (S)-[(3-chloro-2-hydroxypropyl) trimethylammonium] [bis((trifluoromethyl)-sulfonyl) amide] ((R)- and (S)-[CHTA]

+[Tf2N]

-) in optically pure form which

are commercially available by simple ion exchange reaction from corresponding chloride salts, and they successfully developed a novel method based on the near-infrared tech-nique with this chiral IL serving both as solvent and as a chiral selector for the determination of enantiomeric purity. Yu et al. (2008) [98] also synthesized a series of structurally novel chiral ionic liquids with an either chiral cation or chiral anion, or both. Structurally novel and strong intra- and in-termolecular chiral recognition ability of these chiral ILs showed that they could be used in a variety of applications including as chiral solvents for asymmetric synthesis and as CSPs for chromatographic separation.

In addition, a large number of natural products have been

used as novel chiral stationary phases. A novel chiral sta-

tionary phase based on the L-RNA aptamer was a kind of

highly biostable stationary phase due to the intrinsic insensi-

tivity of L-RNA to the RNase degradation. Such an approach


allowed one to reverse the elution order of enantiomer rela-

tive to that obtained with the corresponding D-RNA CSP

(2005) [99]. Wang et al. (2008) [100] adopted glucose, cel-

lobiose, lactose and raffinose as chiral stationary phases in

HPLC, and the novel chiral stationary phases also possess

high enantioseparation selectivity, which may be used in

normal-phase and reverse-phase mode, and there is a great

chiral discriminating complementary to present study.

3. CHIRAL DERIVATIZATION REAGENTS AND CHIRAL MOBILE PHASE ADDITIVES

3.1. Chiral Derivatization Reagents (CDRs)

The indirect method involving the derivatization process with some suitable chiral tagging reagent is an efficient way for the separation of many enantiomers, especially when suitable CSPs were not available. The mechanism of indirect separation is based on the use of chiral derivatization rea-gents to form a complex of diastereomers, the diastereomers differing in their physicochemical properties can hence gen-erally be separated by conventional chromatographic col-umns. So far, enormous chiral derivatization agents are available. A review described the application of chiral de-rivatization agents in the HPLC separation of amino acid enantiomers (2008) [101]. A new chiral derivatization agent, 2,3,4,6-tetra-O-acetyl- -D-galacto-pyranosyl isothiocyanate (GATC) was adopted for chiral separation of several

-blockers (2006) [102]. Ten novel CDRs have now been used for HPLC enantioresolution of some -amino alcohols. The method was also found successful for the separation of 20 diastereomers from a mixture (2009) [103]. Kurosu et al. (2009) [104] reported unprecedented chiral derivatizing agents that allowed for the determination of the absolute configurations of a wide range of amino acids by only ana-lyzing the carbamate nitrogen protons in 1H NMR spectra. Bhushan et al. (2008) [105] synthesized chiral hydrazine reagents for enantioseparation of carbonyl compounds. An-other new chiral derivatizing reagent synthesized from s-triazine chloride was employed for enantioseparation of

-amino acids by reversed-phase liquid chromatography (2008) [106]. With more and more new chiral derivatizing agents appear for selection, the methodology of chiral de-rivatizing agents will play a more important role in chiral separation.

3.2. Chrial Mobile Phase Additives (CMPAs)

It is possible to effect an enantiomeric separation using

conventional HPLC stationary phases by adding a chiral se-

lector in the mobile phase. In this method, enantiomeric

separation is accomplished by the formation of a pair of

transient diastereomeric complexes between racemic analyte

and the chiral mobile phase additive. The advantages of this

technique are so many, the most obvious of them are sim-

plicity and flexibility; moreover there is no need for chiral

derivatization. The frequently used CMPAs are ion-pairing

reagents, ligand-exchanging reagents, protein-affinity rea-

gents and cyclodextrin inclusion reagents. Among them,

cyclodextrin was used for chrial mobile phase additives gen-

erally (1997-2001) [107-111]. Ma et al. (2009) [112] em-

ployed sulfated -cyclodextrin (S- -CD) as CMPA for enan-

tiomeric separation of chiral amine and explored the chiral

recognition. The results showed that S- -CD could undergo

a conformational change with increasing temperature. Par-

ticularly, the aromatic part of the molecule was included

inside the cyclodextrin through hydrophobic interactions

with the interior of the macrocycle, which could reflect the

interactions between the CMPA and analytes. Moreover,

norvancomycin and vancomycin were adopted as CMPAs in

chiral separation and gained expected affect for several cer-tain analytes (2006, 2000) [113, 114].

4. HYPHENATED TECHNIQUES

4.1. LC-MS

During the last decade, separation techniques coupled with mass spectrometry became an essential and powerful tool in molecular biochemistry, especially in the analysis of chiral compounds of pharmaceutical, clinical, environmental and/or agro-chemical interests. In order to evaluate the pharmacokinetics of a single enantiomeror of enantiomer mixture, manufacturers should develop quantitative assay for the individual enantiomers in vivo samples early in the drug development process. Currently, there is a trend toward con-verting chiral HPLC methodologies into more sensitive mass spectrometry-based approaches without losing the enanti-oselectivity of the assay (2004) [115]. The methodology of chiral LC-MS is becoming increasingly important for the routine analysis of chiral drug and their metabolites in bio-logical matrices (2005-2009) [116-121], also for toxicology research (2005) [122].

The recent developments in chiral liquid chromatography

coupled with atmospheric pressure ionization tandem mass

spectrometry (LC-API-MS/MS) for the analysis of pharma-

ceuticals were reviewed by Chen et al. (2005) [123]. Various

new ion sources including electrospray ionization (ESI), at-

mospheric pressure chemical ionization (APCI) and atmos-

pheric photoionization (APPI) have been employed for chiral

separation of pharmaceuticals. Among these soft ionization

techniques, only APPI source is compatible with both re-

versed phase and normal phase conditions without the safety

concerns of a potential explosion. The schematic of APPI

with a chiral column for the pharmaceuticals is shown in Fig.

(3). D’Orazio et al. (2008) [124] employed a novel chiral

nano-LC-MS for analysis amino acids in orange juice profil-

ing. The complete set-up used for nano-LC-MS analysis of

amino acid enantiomers is shown in Fig. (4). Luo et al.

(2010) [125] adopted chiral liquid chromatography and tan-

dem mass spectrometry for simultaneous analysis of bam-

buterol and its active metabolite terbutaline enantiomers in rat plasma, the typical chromatograms are shown in Fig. (5).

4.2. LC-CD

It is always required to determine the enantiomeric purity

in the quality control of chiral drugs production. At present,

the importance of the circular dichroism (CD) spectroscopy

is increasing importance in pharmaceutical analysis because

of the continuous appearance of commercially available CD

detector in liquid chromatography (2007) [126]. Fig. (6)

shows the basic components of the CD detector (2007) [127].


HPLC with CD detector analysis (LC-CD) can establish a correlation between the absolute configuration of separated enantiomers and their characteristic CD transitions. So, LC-CD hyphenated technique is playing a gradual key role for determination of absolute configuration of chiral drug,

especially in drug discovery. There are lasting LC-CD ap-plications reported about determination of absolute configu-ration for enantiomers (2001-2009) [127-134]. Iwasa et al. (2009) [130] employed on-line HPLC-CD in combination with HPLC-MS for analysis of benzylisoquinoline alkaloids,

Fig. (3). Schematic diagram of an atmospheric pressure photoionization (APPI) source [123].

Fig. (4). Schematic representation of the column-switching system coupled with MS [124].

Fig. (5). Typical MRM chromatograms of R-bambuterol, S-bambuterol, R-terbutaline, S-terbutaline, and S-propranolol (I.S.) in Wistar rat plasma. The retention times were approximately 19.5, 21.6, 15.1, 16.9, and 20.1 min, respectively. (A) Blank plasma sample; (B) plasma spiked with 1 ng/mL of bambuterol and terbutaline enantiomers and 1.5μg/mL S-propranolol; (C) plasma sample 4 h after intravenous infu-sion of 5mg/kg rac-bambuterol. Panels marked are (1) for bambuterol (m/z 368 294); (2) for terbutaline (226 152); and (3) for I.S. (260183) [125].


the chromatogram is presented in Fig. (7). A novel chiral detector, a circular-dichroism thermal lens microscope (CD-TLM), was developed to realize sensitive and selective detection of small volume chiral samples on a microchip (2006) [135]. Thermal lens spectrometry (TLS) is a kind of photothermal spectrometry which holds promise for over-coming the low sensitivity of absorption-based detection methods, and the principle of CD-TLM is illustrated in Fig. (8). This novel chiral detector is more than 250 times higher sensitivity versus that in a CD spectrophotometer for pure enantiomer detection.

Ranjbar et al. (2009) [136] introduced circular dichroism techniques for biomolecular and nanostructural analyses. The result indicated circular dichroism is a powerful technique for evaluating the conformation of polypeptides and proteins. In a word, the development tendency of CD detector is aim-

ing to improve its sensitivity to detect smaller amounts of enantiomers.

4.3. LC-OR

Some applications for OR detectors have received the most attention to date. The most fundamental is to verify the optical activity of the compounds of interest and to assign either a positive or negative rotation of the structure. LC-OR can also be adopted for the identification and characteriza-tion of impurities. However, this application has received little attention to date, primarily due to the relatively low sensitivity of most OR detectors and the need for optically pure standards to facilitate identification (2000) [137].

Kott et al. (2007) [127] made a meaningful evaluation of four commercial HPLC chiral detectors (three polarimeters and a circular dichroism detector). Moreover, LC-OR hy-

Fig. (6). Optical components of a CD detector [127].

Fig. (7). LC-CD data and stop-flow LC-CD of (+)-reticuline and (±)-reticuline. CD detector: Jasco CD-2095Plus; pump: Jasco PU-2SOOPlus; column: chiralcel OJ-H (4.6 250 mm); fluent: N-hexane/ethanol/diethylamine: 50/50/0.1; flow rate: 0.5 mL/min. LC/CD detection: 236 nm; sample injection: 5 μg [130].


phenated technique has been developed for determining the change of enantiomeric excess in the chiral synthesis in order to confirm the reaction termination (2006, 2008) [138,139].

5. SOME NEW DEVELOPMENTS AND APPLICA-TIONS IN HPLC CHIRAL SEPARATIONS

Today, rapid analysis for chiral pharmaceuticals has be-come an inevitable trend. Fmoc- and Z-derivatives of natural and unnatural sulfur containing amino acids were separated by micro-HPLC with very short time (2004) [140]. The separation was carried out in microbore columns packed with a new material based on Ristocetin A bonded to 3.5 μm silica gel. The major advantage of micro-HPLC is much less consumption of expensive packing material, solvent and sample. Furthermore, the small particle size leads to higher efficiency and ideal resolution with shorter retention times.

Currently, particle size can be decreased even more, and the use of columns packed with sub-2 μm particles working under ultra-high pressure have been developed over the last 5 year (2005, 2006) [141, 142]. So, the use of columns packed with sub-2 μm particles in liquid chromatography with very high pressure conditions (known as Ultra High Pressure Liq-uid Chromatography, UHPLC) was investigated for the fast enantioseparation of drugs. Guillarme et al. (2010) [143] employed two alternative approaches for evaluation for rapid chiral analysis by UHPLC using amphetamine derivatives and -blockers as model compounds. Using this strategy, it was possible to achieve the robust separation presented in Fig. (9). As a result 10 enantiomers from 5 different

-blockers were well separated in less than 3 min, which demonstrated that the developed method could be applied for the establishment of optical purity of pure compounds at low levels in very short analysis times.

Fig. (8). Principle of CD-TLM [126].

Fig. (9). Enantioseparation of a mixture containing 5 rac- -blockers after derivatization with AITC reagent. Column: Acquity CB18B BEH (50 3 2.1 mm I.D., 1.7 μm); Mobile phase: acetate buffer 20 mM pH 5-MeCN, gradient from 23.5 to 56.5% MeCN in 2.34 min. (slope of 14.1%/min); F=1000 μl/min; Injection: 2 μl; Temperature: 30 . UV detection: 254 nm. Each rac- -blocker was between 100 and 500 μg/ml before derivatization [143].


CSPs associated with simulated moving bed (SMB)

technology have also shown excellent performance in the

separation of racemates. This technology has been mainly

used in the area of chiral purification (2007, 1997)

[144,145], where the resolution of enantiomers is usually a

binary separation task with low selectivity and high opera-

tional cost (2007) [144]. Therefore, SMB is not only the

method to prepare few grams of the chiral drug required by

preliminary clinical tests but even more so most suitable way

for the large-scale production. A laboratory-scale SMB unit

made in home was adopted in an eight-column configuration

(two column per section), as shown in Fig. (10) (2008) [146].

Rajendran et al. (2009) [147] reviewed simulated moving

bed chromatography using for the separation of enantiomers.

After realizing its potential for enantiomer separations, now

the SMB technology is an important component of the sepa-

ration toolbox in the pharmaceutical industry, particularly to manufacture single pure enantiomers of racemic drugs.

Nowadays, multidimensional high performance liquid chromatography technique is a powerful tool for analysis of complicated samples especially biological fluids. It is a powerful tool for chiral analysis too, which can be used to determine not only drugs’ metabolites in plasma but also value of enantiomeric excess (e.e.%). Mullett et al. (2007) [148] provided an overview of the existing literatures with emphasis on advances in automated sample preparation methods for liquid chromatography. The column configura-tions including multi-dimensional-column mode can provide additional dimensions of selectivity. Albendazole metabo-lites were analyzed by direct injection of bovine plasma on multidimensional high performance liquid chromatography

(2008) [149], and the column-switching system employed is illustrated in Fig. (11).

Shi et al. (2009) [150] reviewed a recently developed technique that couples HPLC with on-line, post-column (bio)chemical assays and parallel chemical analysis to screen and identify bioactive compounds from complex mixtures without the need for cumbersome purification and subse-quent screening. Those strategies have proved to be very useful for rapid profiling and identification of individual active components in mixtures, hence to provide a powerful method for natural product-based drug discovery. This sys-tem coupled with chiral columns can be employed for analy-sis of complicated samples with the high selectivity and sen-sitivity of detection. The on-line assays can always be adopted in a qualitative way and known active compounds can be screened and identified rapidly, while novel active compounds could be further investigated after target purifi-cation.

6. MECHANISM OF CHIRAL RECOGNITION

Although there are so many commercially available chiral columns used for chiral analysis, it is difficult to select suitable CSPs for efficient resolution of specific enantiomers rapidly. Therefore, it is very important for us to understand the mechanism of enantioseparation. Today, the intermo-lecular interactions between chiral selector and enantiomers can be calculated with quantum mechanics, molecular me-chanics and molecular dynamics. The various molecular modelings have been established by X-ray crystallography, NMR and computer simulation in order to study chiral dis-crimination mechanism (2004) [151]. Among them, molecu-

Fig. (10). Simulated moving bed chromatographic unit placed on the UNICAMP Bioseparation Laboratory. (A) Detectors controller module, (B) raffinate pump, (C) feed pump, (D) UV detector, (E) extract pump, (F) inlet desorbent pump, (G) polarimeter, (H) valves system, (I) columns, (J) valves controller system, (K) sampling valve [146].


lar modeling is considered as a practical tool for evaluating the complex interactions taking place as analytes migrate through a chromatographic column, which could provide detailed information at the atomic level about how chroma-tographic process undergoes. Several molecular simulation theories have been employed to simulate the liquid chroma-tography process in enantioseparation. Chemometric analysis of enantiospecific retention data was first described by Francotte and co-workers (1988, 1991) [152, 153]. These authors applied multiple regression analysis to relate enanti-oselective separation on cellulose triacetate and cellulose tribenzoate columns to structural descriptors of analytes (2007) [154]. In order to study the retention and chiral rec-ognition mechanism, the method of quantitative struc-ture-enantioselectivity retention relationships (QSERR) was investigated from the quantitative equations established be-tween the chromatographic retention of enantiomers and their molecular descriptors of physico chemical properties. QSERR was first introduced as a special term in a report on studies of HPLC data determined on human serum albumin (HSA) column (1992) [155]. Then, QSERR has been exten-sively studied since decades (1996-2001) [156-159]. Heber-get (2007) [160] reviewed Quantitative structure (chroma-tographic) retention relationships (including QSERR) from 1996 to 2006. QSERR models can be used for successful classification of chiral drugs of various compound classes and/or chromatographic columns (systems), which is very useful for HPLC practitioners. Consequently, in QSERR studies one can address either variation in the non-enantiospecific strength of solute-CSP interactions due to differences in the chemical structure and properties (such as polarizability, number of hydrogen atoms, etc.) which increases the overall retention of both isomers or the forces which lead to chiral discrimination (2009) [161]. Moreover, Lipkowitz (2001) [162] introduced the atomistic modeling of enantioselection in chromatography, and the mechanism of chiral recognition for five types of CSPs has been studied and direct interactions are calculated between CSPs and analytes. The chromatographic process can be well analyzed using molecular dynamic model theories. The determination of the adsorption sojourn times and the number of adsorp-tion–desorption or mass-transfer events can give a practical view of the molecular process of separation (2008) [163]. The studies of various kinds of molecular modeling for chiral

separation by LC have been investigated extensively (2004-2009) [164-172]. With researching the chiral recogni-tion mechanisms at the molecular level, we can not only ex-plain the experiment phenomena but also predict the results. It can guide us to adjust chromatographic conditions in order to improve the resolution of enantioseparation. What is more important, the new types of CSPs can be designed to realize the expected separation.

7. CONCLUSION

A major trend in LC for the separation of drug enanti-omers is clearly heading toward faster and more efficient separation with comparable or improved separation capabil-ity. The development of liquid chromatographic procedures focuses on usage of efficient novel materials in column chromatography and hyphenated techniques with highly se-lectivity.

After the rapid development in many years, enantioselec-tive liquid chromatography, particularly HPLC on CSPs, has become an indispensable part of drug discovery not only in daily chiral analysis but also in the fast preparation of drug molecules. As more chiral selectors are being developed and commercialized as CSPs, a systematic and deeply under-standing of the chiral recognition mechanisms at the mo-lecular level is essential for the future, so that researchers can rapidly select or even create suitable CSPs for efficient reso-lution of specific enantiomers in one day. Moreover, by us-ing hyphenated techniques (LC-MS, LC-CD, LC-OR), we can know more information about molecule structure, phar-macokinetics, spatial configuration, eluting order for drug enantiomers. With the advances and promotion of related techniques, recent developments in HPLC enantiomeric separation, including micro-columns, small particle columns, simulated bed moving chromatography and multidimen-sional chiral high performance liquid chromatography, have been reviewed in this paper. Collectively, these advances will lead to persistent improvement of chiral separation ca-pability.

ABBREVIATIONS

API = Atmospheric pressure ionization

APPI = Aatmospheric pressure photoionization

Fig. (11). Schematic diagram of the column-switching system [149].


AVI = Avidin

AGP = 1-acid glycoprotein

BSA = Bovine serum albumin

CBH I = Cellobiohydrolase I

CD = Circular dichroism

CD = Cyclodextrin

CDR = Chiral derivatization reagents

CD-TLM = Circular-dichroism thermal lens micro-scope

CMPA = Chiral mobile phase additives

CSPs = Chiral stationary phases

HAS = Human serum albumin

HPLC = High performance liquid chromatogra-phy

ILs = Ionic liquids

MIP = Molecularly imprinted polymers

MS = Mass spectra

OMCHI = Ovomucoid from chicken egg whites

OR = Optical rotation

SMB = Simulated moving bed

TLS = Thermal lens spectrometry

UHPLC = Ultra high performance liquid chroma-tography

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Received: 29 November, 2009 Revised: 29 January, 2010 Accepted: 03 March, 2010

114 2010 114-130 chiral separation of pharmaceuticals by ......for separation of new...

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