Chiral discrimination and enantioselective ... - Wiley Online Library

REVIEWS Chiral Discrimination and Enantioselective Analysis of Drugs: An Overview EMAD L. IZAKE Forensic Chemistry Section, Pathology and Scientific Services, Queensland Health, Queensland Government, Australia

Received 14 September 2006; revised 2 October 2006; accepted 3 October 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20820

ABSTRACT: Molecular recognition of different enantiomers of a drug has become of increasing importance in the last decade due to the racemic switch strategy adapted by the pharmaceutical industry. Different analytical techniques to carry out enantioselective analysis of chiral compounds have been suggested in the literature. In the following, a brief overview of different techniques used for enantioselective analysis is given. Challenging aspects of these techniques, such as the quality of analytical information received from each technique, advantages, and disadvantages are discussed. Alternatives (enantioselective membranes, amperometric biosensors, molecularly imprinted polymers (MIPs)), capable of meeting the requirements of industrial processes, in terms of productivity, cost-effectiveness, and environmental issues are critically reviewed. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:1659–1676, 2007

Keywords: analytical chemistry; polymers; cyclodextrins; chromatography; chirality; sensors and biosensors; enantioselective analysis; molecularly imprinted polymers

INTRODUCTION Chiral discrimination between enantiomers is becoming one of the most important fields in analytical chemistry especially for pharmaceutical industry, clinical analysis, food analysis, and forensic analysis. Many compounds are known to be a racemic mixture of enantiomers. One enantiomer may influence desirable physiological, pharmacological, pharmacodynamics, and pharmacokinetic properties while other enantiomers often exhibit a very different physiological role, different interactions with living

Correspondence to: Emad L. Izake (Telephone: þ6132749031; Fax: þ6132749007; E-mail: [email protected]; [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 1659–1676 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association

organisms, and different activity in chemical and biotechnological processes. Furthermore, the debate ‘‘racemate-versus-enantiomer’’ has opened a new market strategy, the so called racemic switch. A racemic switch stands for the development in single-enantiomer form of a drug that was first approved as a racemate. This means that a company can get in this way a patent on an individual enantiomer. A number of examples of new stereo-chemically pure drugs patented in the last years are shown in Figure 1. For these reasons, the enantiomeric purity of various compounds is important in stereo-specific synthesis, production of pharmaceuticals, pesticides, and some food additives, where only one enantiomer may interact satisfactorily. Molecular recognition of the enantiomers at an asymmetric center is described through a minimum of three-point interaction which may

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are molecularly imprinted polymers (MIPs). Molecular imprinting allows for the creation of specific, tailor-made recognition sites for a target molecule in synthetic polymers, that is, applying the lock and key principal. The elution order of this material is predefined; the enantiomer originally used as template is always more strongly retained; no sophisticated design and laborious synthetic protocols have to be elaborated, and the required components are readily available and inexpensive bulk chemicals.

Figure 1. Chemical structures of some stereochemically pure drugs patented as single enantiomers.

CHIRAL SELECTORS IN MOLECULAR RECOGNITION OF ENANTIOMERS

be attractive or repulsive. Numerous types of chiral selectors such as crown ethers, cellulose and amylose, polysaccharides, cyclodextrins (CDs), maltodextrins, and macrocyclic antibiotic classes have been described for quantitative enantiopurity tests of chiral drugs. Separation and determination of enantiomers is currently performed most commonly, by high-performance chromatographic and electro-migrating techniques. The sampling process in enantioselective analysis can introduce a lot of uncertainties, especially when a separation method using inadequate chiral selectors, is proposed.1 The introduction of different types of enantioselective sensors and biosensors increased the reliability of the assay as the enantiomer can be determined without prior separation, directly from the matrix, with only dissolution and dilution steps being involved1–6 and can be used as on-line detectors in flow injection (FIA) and sequential injection analysis systems (SIA). Carbon paste electrodes have the potential to be used as detectors in on-chip microfluidic plate forms used for a wide variety of applications. Enantioselective immunosensors are the only type that may be considered to be enantiospecific, because the antibody is not reacting with the other enantiomer (the key for the lock theory), which makes it the highest between the enantioselective sensors. Artificial receptors have become of increasing importance as a possible alternative to natural systems. The most promising materials in the field of artificial molecular recognition systems

One of the most critical tasks in molecular recognition of enantiometric pairs is the selection of the chiral selector. There are numerous compounds widely used as chiral selectors in chromatographic, electro-migrating, and electrochemical techniques. They have been reviewed in a recently published handbook by Gu¨bitz et al.7 Table 1 shows the main groups of chiral selectors used in enantioselective analysis.8 The first class of compounds used as chiral selectors is the crown ethers, macrocyclic polyethers with relatively polar cavity, which can form host–guest inclusion complexes with metal ions. They have been used as chiral selectors for primary amines, amino acids, and peptides.9,10 This class of organic selectors did not resolve the enantiometric pairs adequately and the accuracy of the separation was poor. Natural polysaccharides (e.g., cellulose, amylose, chitosan, chitin, amylopectin, and dextrans) and their derivatives have been widely used as stationary phases in HPLC and TLC11 and as additives to background electrolytes in capillary electrophoresis (CE).12 For this class, selectivity results from interaction with analytes including hydrogen bonding, p p interactions, and dipoleinduced interactions. Polyether antibiotics have been examined as chiral selectors in membranes for detection of chiral ammonium salts.13 The values of chiral selectivity coefficients were found to depend on the structure of ionophore used. Macro cyclic antibiotics, such as ansamycins and glycopeptides have been successful as chiral selectors in all techniques of liquid chromatography, CE, and capillary electro chromatography (CEC).14 They have multiple stereogenic groups,


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AN OVERVIEW OF DRUGS

Table 1. Main Groups of Chiral Selectors Arranged to Their Origin and Applicable Separation Techniques Source

Type

Chiral Selector

Techniques

Scale

Natural

Proteins

Oligosaccharides

Polysaccharides

Antibiotics

Low M molecules

Serum albumin Orosomucoid (a1-acid glycoprotein) Ovomucoid Cellobiohydrolase I Avidin Chymotrypsin Ovotransferrin a-, b-, and g-cyclodextrins Disaccharides Maltodextrins Cellulose Amylose Starch Dextran Heparin Pectins Vancomycin Teicoplanin Ristocetin Avoparcin Amino acids Cholic acids/bile salts Alkaloids Tartaric acids

LC, CE, CEC Membranes extraction

LC, CE, CEC, GC, TLC, PEME CE CE, PEME CE

CE CE LC, CE, GC SFC

A A A A A A A A A/P A A A A A A A A A/P

LC, CE, SFC Crystallization

Semisynthetic Modified oligosaccharides Modified polysaccharides

Derivatized cyclodextrins

LC, CE, CEC, GC, TLC

A/P

Cyclodextrin polymers Polysaccharide carbamates

CE LC, CE, SFC, TLC, membranes, extraction LC, CE, SFC, TLC, membranes, extraction CE

A A/P

Polysaccharide esters Polysaccharide sulfates

Modified low M molecules

Dextran sulfate l-Carrageenan Chondroitin derivatives Ion-exchange selectors

CE CE LC, CE, CEC, SFC, extraction

Pirkle type selectors

A/P

A/P

Synthetic Synthetic low M molecules

LEC selectors

Crown ethers Proline derivatives

Helical synthetic polymers

Polyacrylamides

LC, CE, GC, TLC, SFC, membranes LC, CE, extraction, crystallization LC, CE, extraction, LC, CE, TLC, extraction, membranes LC, CE, SFC,

Polyacrylates Crosslinked tartaramides MIPs

LC LC, CE, SFC, membranes

Receptor molecules

A/P A A/P A/P A/P A/P A/P A

A, analytical scale; P, preparative scale. DOI 10.1002/jps


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including aromatic rings, hydrophobic cavities, groups forming hydrogen bonds, and also ionizable groups influencing chiral recognition and electrophoretic behavior. The two most successful macrocylic antibiotics are vancomycin and teicoplanin.15,16 Teicoplanin, as chiral selector, assures the integrity of the chiral stationary phase (CSP). Teicoplanin has excellent enantioselectivity for native amino acids, peptides, a-hydroxycarboxylic acids, and a variety of neutral analytes including cyclic amides and amines.17 Maltodextrins with different dextrose equivalent (DE) per molecule have been proposed as novel chiral selectors for the construction of potentiometric enantioselective membrane electrodes (PEME).18,19 The higher the DE number, the higher is the extent of starch hydrolysis and consequently, the shorter are the oligomeric chains present in a mixture.18 DE does not affect the microstructure of the maltodextrins, but it affects the macrostructure.20 In CE, maltodextrins enabled highly efficient chiral separations of a broad range of acidic and basic compounds.21–23 The chiral separation was found to increase substantially with increasing maltodextrin concentration and decreasing DE value with Low-DE maltodextrins being the most efficient at separating enantiomers.21,23 Different interactions of chiral solutes with the helical structure of the maltodextrin were proposed to be the cause of enantioselectivity. This notion is supported by 1 H-NMR and 13C-NMR experiments18,20 which revealed that the helical structure of the maltodextrins mimics the cavity responsible for chiral recognition by CDs. The most widely used chiral selectors are the lipophilic CDs, naturally occurring cyclic oligosaccharides, often additionally derivatized to form neutral or charged species.24 They have been used in PEME, as ionophores incorporated in a plasticized PVC or graphite paste. In chromatographic techniques, CDs and modified CDs can be used as either the CSPs, as chiral mobile phase additives, or as chiral counter ions. CDs stationary phases have been termed a multimodal CSP as they have been used in three different chromatographic modes (normal, reversed, and polar organic).25 1 H-NMR and 13C-NMR have been used to explore the mechanisms of enantioselectivity by CDs.26–28 Studies showed that the superior performance of CDs as chiral selectors is because the enantioselectivity of the molecular interaction is based on:

Some efforts to create chromogenic chiral selectors have been made. Chiral azophenolic acerands were employed as hosts to judge the absolute configuration of chiral amines (Fig. 3a).29 These chiral hosts were shown to be capable of discriminating between the enantiomers of chiral amines by shift of bands in the visible spectra. In a more recent contribution, Kubo and coworkers described an enantioselective chromogenic sensor based on a calix[4]arene scaffold (Fig. 3b).30 In this molecular sensor, two indophenol units attached to the upper rim of the calixarene served as chromophores, while the chiral information was introduced by a crown ether attachment integrating a binaphthol unit at the lower rim. The use of different spacer length for the crown ether allowed positioning of the chiral binaphthyl unit closer to one of the indophenols than to the other, rendering the chromophores distinct. On binding of a chiral substrate, the chromophores are affected to different extents to create a strong chromogenic response. Low concentration of (R)-phenylglycinol has been reliably detected even in presence of 500 equivalents of (S)-enantiomer using this type of selector. James et al. designed a solution based enantioselective fluorescence selector for monosaccharides (Fig. 3c).31 The selector consisted of a fluorescent binaphthol unit, modified with side chains containing tertiary amino groups and boronic acid functions in close proximity. The molecular recognition is based on the interaction of the boronic acid with the amine. The latter is acting as an intra-molecular quencher of the binaphthol fluorescence. The binding of monosaccharides to the sensor in aqueous buffer gave rise to a significant increase of the fluorescence intensity, allowing chiral discrimination between the enantiomers of fructose, mannose, glucose, and galactose. The change in fluorescence intensity was explained as a consequence of two interrelated mechanisms: (1) formation of the 1:1


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1. Inclusion of chiral analyte into hydrophobic open cavity (internal enantioselectivity) and 2. Secondary interactions based on formation of hydrogen bonds or dipole–dipole interactions with the hydroxyl groups on the CD rim (external enantioselectivity). The external enantioselectivity is affected by the arrangement, size, and type of the radicals or ions bound to the external chain of the chiral selector (Fig. 2a–c).


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ing efficiency, which is induced due to enantioselective steric interaction enforcing a twist around the binaphthyl bond on binding of the D- or L-monosaccharides.

CHROMATOGRAPHIC AND ELECTRO-MIGRATING TECHNIQUES IN CHIRAL ANALYSIS

complex with the monosaccharide, which leads to a stronger nitrogen–boron interaction and, thus fixes the nitrogen in a certain orientation relative to the aromatic system, determining its quenching efficiency; (2) additional modulation of the quench-

In chromatographic methods used for chiral separation, the flow rate controls the time of contact between the enantiomers flowing through the column and the chiral selector bound on the column. Therefore, the separation factor depends on the flow rate, that is, the separation of enantiomers using chromatographic techniques is based mainly on the kinetics of the reaction between the chiral selector and enantiomer, rather than on the thermodynamics of the same reaction. The chromatographic separation can be performed directly or after derivatization using gas chromatography (GC),32,33 thin-layer chromatography (TLC),34 high-performance liquid chromatography (HPLC),35 supercritical fluid chromatography (SFC), CE,36–39 micellular electrokinetic chromatography (MEKC), and capillary electro-chromatography (CEC),40,41 where the chiral selector can be deployed in the shape of a stationary phase, chiral mobile additives, or chiral counter ion. Chromatographic methods have been utilized in enantioselective analysis for forensic purposes. Sadeghipour and Veuthey42 applied Native and derivatized b-CDs as CSP for the forensic investigation of enantioselective metabolism of the illicit drug 3,4-methylenedioxymethamphetamine in postmortem blood samples. These chiral selectors were used for the simultaneous enantiomeric separation of four methylenedioxylated amphetamines (MDA, MDMA, MDEA, and MBDB) by liquid chromatography with fluorimetric detection. This method was applied to the stereoselective analysis of illicit tablets (23 samples) and of human whole blood samples (spiked samples and 2 postmortem cases). In a more recent study, Chilmonczyk et al.43 used cellulose tris(4-methylbenzoate) CSP for the enantioselective chromatographic separation of 19 chiral 1,4-disubstituted piperazine derivatives. The chiral resolution has been obtained for 14 derivatives. Theoretical calculations were carried out for the CSP model, which stressed the

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Figure 2. a: Proposed interaction between the (þ)ephedrinium ion and poly-O-octyl-a-cyclodextrin (view down the CN bond). b: Model of the unfavorable interaction in complexation of the ()-(1R,2S)-ephedrinium ion with poly-O-octyl-a-cyclodextrin. c: Model of the favorable binding of the (þ)-(1S,2R)-ephedrinuim ion with poly-O-octyl-a-cyclodextrin.

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Figure 3. Chemical structures of chiral molecular sensors: (a) azophenolic acerands showing chromogenic enantioselective response for the chiral amines; (b) chromogenic sensor discriminating the enantiomers of phenylglycinol; and (c) fluorescent sensor showing enantioselectivity for monosaccharides.

between enantiomers and chiral selector, the lower sensitivity of detection systems (e.g., CE and MEKC need amplification system for diode array detector), and the sampling process can introduce a lot of uncertainties. For example, sample preparation in CE can be laborious and the separation step may have lower accuracy than electrochemical sensors.

importance of a solute and the stationary phase aromatic ring interactions. In MEKC, the quality of the analytical information obtained in enantiometric analysis of drugs depended on the chiral selector concentration in the background electrolyte rather than the concentration of the analyte.44 CE has proven to be the most successful chromatographic technique for enantiometric separation,45 especially when sulfobutyl etherb-CD is chosen as a chiral selector since it has a large countercurrent mobility, making it inherently advantageous as selector when compared with neutral CDs.46 In a recent study by Yang et al.,47 the enantiomeric separation of 37 clinically used racemic basic drugs among 50 drugs was achieved using sulfated b-CD (S-b-CD) as chiral selector at pH (2.5) and in the reversed polarity mode. The chiral discrimination depended on the appropriate interaction between the analyte and the sulfated b-CD. Sulfated b-CD increased the mobility of the inclusion complex which allows a potentially better resolution (Fig. 4). Although chromatographic techniques are the most used, yet they are not always the best choice for quantitative purity tests of chiral compounds, as they cannot always ensure the best precision and the working conditions must be improved for every enantiomeric pair. Chromatographic techniques have some other critical disadvantages. These are: necessity of derivatization processes (especially in HPLC and GC), low differences of stabilities of complexes obtained

Figure 4. The interaction between cicletanine and sulfated b-CD reported by Yang et al. Asterisk* denotes the position of the chiral carbon.


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PREDICTION OF ENANTIO-SEPARATION Most investigations on enantio-separation mechanisms have been performed on low molecular mass selectors, including p-donor/acceptors, crown ethers, and CDs. The most popular strategy to establish chiral recognition models for a given selector involves the collection of a representative body of chromatographic enantio-separation data with a series of analytes displaying incremental structural modifications. A systematic interpretation of these data may provide some mechanistic information on the contributions of the individual structure elements to enantioselective selector–analyte binding. Another strategy is to develop chemometrically driven predictions of retention and enantioselectivity by constructing quantitative structure– enantioselective retention relationships (QSERRs) through combining quantitatively comparable retention data for a set of solutes and of molecular descriptors reflecting the structural features of these solutes and establishing statistically significant equations. Several investigations have been performed in this direction.48–50 Studies on chiral recognition showed that the resolution of chromatographic methods is given by the apparent binding constants of enantiomers and by the chemical shift differences at saturation using 1H-NMR and 13C-NMR spectroscopies.51 Based on these studies, Bodenho¨fer set up a model for the chiral discrimination process by superimposing preferential and nonpreferential sorption mechanisms. He suggested that the ‘‘molecular recognition’’ effect can be observed by simply dissolving the supramolecular unit in an isotropic polymer.52 The model is applicable to any matrix containing preferential sorption sites. A more sophisticated approach is to model and optimize chiral separations based on the threepoint contact model where the chiral recognition at asymmetric center is described through a minimum of three points of interaction such as multiple electrostatic, hydrogen bonding, p p, dipole– dipole, attractive/repulsive van der waals and ionic interactions (the same interactions might be used in design of chemical or biochemical enantioselective sensors).53 Predictive models of chiral chromatographic separation have been generated by applying multivariate regression analysis combined with multilayer feed forward neural networks trained with error back-propagation.50 Combinations of charge transfer, electrostatic, lipophilic, and dipole interactions, DOI 10.1002/jps

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identified by multivariate regression, were found to describe retention, and enantioselectivity with highly predictive models. High molecular mass selector type molecules, including aggregates may generate different binding sites with different affinities to the analytes. Proteins express their chiral recognition properties preferably in aqueous medium in which ligand binding is governed by a highly delicate balance of electrostatic and hydrophobic interactions. In many cases, the contributions of hydrophobic forces to molecular recognition processes are considered to be important.54

ENANTIOSELECTIVE ELECTROCHEMICAL SENSORS AND BIOSENSORS By assuring a sensitive enantioselective analysis system, the separation step can be cancelled from the sampling process. One of the most reliable alternatives of chromatographic methods is the utilization of electrochemical sensors. The main advantages of these systems are: no separation steps required, assay of enantiomers is carried out directly from the matrix with only dissolution and dilution steps needed, high precision of the assay of the target enantiomer,1–6 can be used as on line detectors in, the nonequilibrium, FIA, and SIA designed for enantioanalysis,55–58 where the buffered sample is flowing through the electrochemical cell that contains the chiral selector, the enzyme or the antibody at an optimum flow rate. The measurement is done in a very short time (less than 2 min for a single enantiomer). Many systems have been reported including systems for molecular recognition of only one enantiomer by FIA1,52 and SIA57 as well as for the simultaneous molecular recognition of both enantiomers using SIA coupled with two amperometric biosensors59 or with a potentiometric, enantioselective membrane electrode for the S-enantiomer assay, and with an amperometric biosensor for the R-enantiomer assay.58

CLASSIFICATION OF ELECTROCHEMICAL SENSORS AND BIOSENSORS The main principle behind using these types of sensors is to find the key for the lock, where the lock is the target enantiomer and the key can be a substance with special architecture, in its skeleton and/or functional groups arrangement that can bind enantioselectively with the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 7, JULY 2007

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enantiomer. A second possibility for the key is to be an enzyme that only catalyzes a certain reaction of the main enantiomer. Accordingly, the process leading to enantioselectivity is either enantioselective binding or enantioselective catalysis. Based on these two main processes, different types of sensors have been developed. Potentiometric Enantioselective Membrane Electrodes (PEME) Enantioselective binding is the principle behind developing PEME where a chiral selector, such as

CDs, maltodextrins, quinine, and quinidine derivatives, accommodates the enantiomer.1–6 Some examples of the currently available PEME electrodes are shown in Table 2. Two main types of matrices have been described for the design of these sensors: a PVC-based matrix60 and a carbon paste-based matrix.5 In PVC-based types, the electro-active membranes were prepared by incorporating 1.2% chiral selector, 65.6% o-nitrophenyl octyl ether (oNPOE), or dioctyl sebacate (DOS) (plasticizer), 0.4% tetrakis{[3,5-bis(trifluoromethyl)phenyl]borate} (TKB)

Table 2. Examples of Potentiometric Enantioselective Membrane Electrodes (PEME) Reported in the Literature

Analyte a-Phenylethyl-ammonium ion Leucine methyl ester Ephedrine (þ)-(S)-amphetamine HCl Ephedrine Propranolol Norephedrine L-Proline S-Cilazapril S-Enalapril S-Pentopril S-Perindopril S-Ramipril S-Ramipril S and R-captopril Nitrophenylethyl-amine Ephedrine Ephedrine Ephedrine Pipecolic acid L-Carnitine Propanolol S-Perindopril S(þ)-Ibuprofen

Chiral Selector

Enantioselectivity Coefficient Log Kpot

Reference

Macrocyclic crown ether

0.41

Monensin 3-Methyl-2,6-di-octyl-a-CD 2,6-Di-octyl-a-CD Poly-o-octyl-a cyclodextrin

0.88 0.47

Bussmann and Simon (1991)9 Maruyama et al. (1992) Bates et al. (1994)

3.6

Bates et al. (1994)

2,3,6-Tri-octyl-a-CD 2,3,6-Tri-octyl-b-CD 2,3,6-Tri-octyl-a-CD 2-Hydroxy-3-trimethylammniumpropyl-b-CD 2-Hydroxy-3trimethylammoniopropyl -b-cyclodextrin (chloride salt) 2-Hydroxy-3trimethylammoniopropyl b-cyclodextrin (chloride salt) 2-Hydroxy-3trimethylammoniopropyl -b-cyclodextrin (chloride salt) 2-Hydroxy-3trimethylammoniopropyl -b-cyclodextrin (chloride salt) 2-Hydroxy-3trimethylammoniopropyl -b-cyclodextrin (chloride salt) 2-Hydroxy-3trimethylammoniopropyl -b-cyclodextrin (chloride salt) Maltodextrine (DE ¼ 13–17) (MeO)-b-CD 2,6-Didodecyl-b-CD, Carboxymethyl-b-CD Per-octyl-a-CD Vancomycine Teicoplanine 2-Hydroxy-3-trimethylammoniopropyl -b-cyclodextrine a, b, g-Cyclodextrin Maltodextrine I, II, III


2.6 2.7 1.5 3.96 4 3.18

Kataky et al. (1995)60 Kataky et al. (1995)60 Kataky et al. (1995)60 Stefan and van Staden (1998) Stefan et al. (1999)4

3.16

Aboul-Enein et al. (1999)5 Stefan et al. (1999)4

4

Stefan et al. (1999)4

4


4


2.55, 3.85 0.53, 2.11 0.38 0.31 0.39 3.52 2.38

Stefan et al. (2000)58 Wcisło (2003) Wcisło (2003) Wcisło (2003) Wcisło (2003) Ratko et al. (2004) Ratko et al. (2004) Sun et al. (2004)

4.00, 3.47, 3.21

Ozemena et al. (2005) Van Staden and ash (2006)

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in a tetrahydrofuran solution containing the poly(vinyl chloride) (PVC) (32.4% of the membrane composition).60 An inner solution containing the enantiomer to be determined at a certain concentration (usually 103 mol/L) was used to establish electric contact. The main disadvantage of this design is the nonreproducibility of PVCbased matrices which is caused by the nonuniformity and nonreproducibility of the repartition of the electro-active material in the matrix. In carbon paste types, Paraffin oil and graphite powder were mixed in a ratio of 1:4 (w/w) followed by the addition of a solution of the chiral selector (103 mol/L) (100 mL chiral selector solution to 100 mg carbon paste). Blank graphite–paraffin oil paste was filled into the electrode body followed by carbon paste that contained the chiral selector on top. A silver wire was inserted in the carbon paste to maintain electric contact.5 Before each use, the surface of the electrode was wetted with deionized water and then polished with an alumina paper. When not in use, the electrode was immersed in a 103 mol/L S- or R-enantiomer solution. Carbon paste electrodes have the potential to be used as detectors in on-chip microfluidic plate forms used for a wide variety of applications (e.g., point of care decentralized clinical testing, security analysis in the field, and environmental analysis).61–63 In general, carbon-based electrodes has proved to be very suitable detector to be used in micro-total analysis systems (m-TASs) because of their minimal fouling, lower noise, and larger potential range for organic compounds. A variety of types of carbon electrodes were used, such as thick-film, screen-printed,64–68 micro-molded ink,69 carbon fiber,70 carbon-paste composite,71 or rigid graphite-epoxy composite.72 The composite electrodes act as arrays of microelectrodes, they have a response comparable to conventional electrodes but with a low noise level,72 and they allow the electrode material to be modified biochemically.71,73

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1. Extraction of the enantiomer from the solution into the membrane–solution interface. 2. Complexation of the enantiomer with the chiral selector. 3. De-complexation of the enantiomer from the complex occurs when the concentration of the enantiomer in the membrane–solution interface is becoming higher than in the solution. 4. Re-extraction of the enantiomer from the membrane–solution interface after its decomplexation. The principal step responsible for the potential development is the complexation one. If L is the chiral selector and S and R the enantiomers to be determined, the following complexes between chiral selector and enantiomers are formed: L þ S $ LS

ð1Þ

L þ R $ LR

ð2Þ

The stability constants of the formed complexes are given by the following equations: DGS

KS ¼ e RT

DGR

KR ¼ e RT

ð3Þ

where KS and KR are the stability constants of the complexes formed between chiral selector and the S- and R-enantiomer, respectively, DGS and DGR are the free energies of the reactions (1) and (2), respectively, R ¼ 8.31 J/K mol and T is the temperature measured in Kelvin. The enantioselectivity of the chiral selector is given by the difference between the free energies of reactions (1) and (2): DðDGÞ ¼ DGS ¼ DGR

ð4Þ

For PEME, the thermodynamics of the reaction between the enantiomer and chiral selector play the main role in the enantioselectivity. This interaction is responsible for the potential development. The main processes that are taking place at the membrane–solution interface can be summarized as follows:

The difference in the stability of the complexes formed between chiral selector and the S- and R-enantiomers will result in a difference in the free energies of the reactions. The stability of the complexes is directly correlated to the response (slope) of the PEME.2 Accordingly, a large difference between the free energies of reactions (1) and (2) will give a large difference in enantioselectivity expressed as a difference between the slopes when the S- and R-enantiomers are assayed. The slope is the criterion for molecular recognition of the enantiomer, when the electrode is enantioselective. The minimum value admissible for a 1:n stoichiometry between

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Enantioselectivity Mechanism in PEME

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the enantiomer and chiral selector is 50/n mV/ decade of concentration. Enantioselective Immunosensors

of anti -L-T3 on a carbon paste matrix for the assay of -L-T3 with high enantioselectivity versus -D-T3. Carbon-paste-based immunosensors using antibodies and without preliminary thermal treatment for the graphite powder were also reported.5

It has been long known that the antibody can recognize the chirality center of a given antigen. Immunosensors are based on this reaction between antigen and antibody. Each enantiomer (as antigen) will have its pair (as antibody). This type of reaction is the only one that may be considered to be enantiospecific, because the antibody is not reacting with the other enantiomer which makes it the highest between the enantioselective sensors. The immunosensor can be coupled with an amperometric transducer. The main advantages of using such a sensor in enantioselective screening analysis are the highest sensitivity, enantioselectivity, precision, and accuracy. If the complex formed between the enantiomer (antigen) and antibody is not electrochemically active, then the antibody must be coupled with an enzyme (enzyme-linked immunosorbent assay (ELISA) technique). Through the coupling reaction, the reactivity of each compound is modified, and therefore, it can be decreased for the given antibody, and as a result, the sensitivity of the amperometric immunosensor will decrease, and also the accuracy of the measurements, as the determination of the enantiomer will not be a direct one. The possibility of constructing electrochemical immunosensor was first indicated by Hofstetter et al.74 who constructed immunosensors having stereo-selective antibodies sensitive to the chiral center of a-amino acids. These antibodies have been used for the determination of trace amounts of enantiomeric impurities.75 They have been used as well for enantioselective sequentialinjection chemiluminescence immunoassay of triiodothyronine (T3) and thyroxine (T4)76, and in micro-fabricated cantilevers for enantioselective detection of amino acids.77 Most of the strategies applied to develop separation-free electrochemical immunosensors, are based on heterogeneous immunoassay procedures.78 Electrochemical immunosensors have been developed for various classes of chemical compounds, including for example, immunosensors for immunoglobulin G,79 aflatoxin B-1,80 progesterone,81 various pesticides,82–84 polychlorinated biphenyls,85 and food pathogens.86 Aboul-Enein et al. reported an immunosensor for triiodothyronine (T3),87 based on immobilization

These types of sensors are based on enantioselective catalysis. The biosensor is an analytical device in which biological or biologically derived material, providing suitable step of molecular recognition, is in close proximity to a physicochemical transducer to measure the concentration of the target analyte. Three kinds of transducers can be used for the construction of electrochemical biosensors: potentiometric, amperometric, and piezoelectric. Potentiometric biosensors are known to give lower sensitivity. A higher sensitivity is assured by piezoelectric biosensors, but at the same time, their selectivity decreases significantly. Amperometric biosensors are the most preferred because the amperometric transducers can maintain the equilibrium between high sensitivity and high selectivity at the same time. The selectivity response of some developed enantioselective amperometric biosensors is summarized in Table 3. Amperometric biosensors are more precise for the complex molecules than PEME sensors.4 The main disadvantage of amperometric biosensors is the short life time (some times can be as low as 1–2 days) compared to that of PEME which has more than 6 months average life time. The most used receptors in constructing amperometric biosensors are isolated natural enzymes.88 The enantioselectivity of the sensor is given by the type of enzyme (L or D) involved in the enzymatic reaction. The enzyme that is able to catalyze only one of the enantiomer reactions must be chosen in concordance with the chirality (R(D) or S(L)) of the analyte. The source of the enzyme is very important. The same enzyme isolated from different media can show different response characteristics such as different sensitivity and detection limits. Enantioselective biosensors are constructed in the form enabling its immersion into solution or gas phase or enabling the placement of small volume of liquid sample on its sensing surface. Another category of such devices is biochemical flow-through detectors, where detection process takes place in the course of the sample flow through the detector. The most often developed


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Chiral Amperometric Biosensors


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Table 3. Enantioselectivity of Amperometric Biosensors Reported in the Literature

Analyte Methionine, leucine D

or L-Lactic acids

S-Phenylalanine b-Hydroxy acid esters S-Captopril D and L-Methotrexate

Pipecoilic acid S-Cilazapril S-Enalapril S-Pentopril S, R-Perindopril S-Ramipril S-Tandolapril L-Ascorbate D-Fructose L-Glutamate L-Lactate

Enzyme(s) D-Amino

acid oxidase, horseradish peroxidase D-Lactate dehydrogenase or L-lactate dehydrogenase Methyl ester esterase, a-chymotrypsin Esterase, lipase L-Amino acid oxidase L-Amino acid oxidase, glucose oxidase, horseradish peroxidase L-Amino acid oxidase, horseradish peroxidase L-Amino acid oxidase L-Amino acid oxidase L-Amino acid oxidase L-Amino acid oxidase L-Amino acid oxidase L-Amino acid oxidase Poly-L-histidine–copper complex D-Fructose dehydrogenase L-Glutamate dehydrogenase Glucose oxidase

Enantioselectivity Coefficient pKamp

Reference

a

Dominguez et al. (2001)

1.45

Motonaka et al. (1998)

a

Kullick et al. (1994)92

b 2.95 3.09

Kullick et al. (1994)92 Stefan et al. (2003)91 Stefan et al. (2003)93

3.82


a a a a b a b

Aboul-Enein et al. (1999)5 Stefan et al. (1998)6 Aboul-Enein et al. (1999)5 Aboul-Enein et al. (1999)5 Stefan et at. (1998)6 Aboul-Enein et al. (1999)5 Hasebe et al. (1998)

b b b

Stred’ansky et al. (1999) Pasco et al. (1999) Krikstopaitis et al. (2000)

a, no response for the opposite enantiomer; b, not available.

systems are ones with pair of enzymes specific for each enantiomer.89–91 Multi-enzyme biosensors were developed for determination of various drugs using amino acid oxidases. In a different model, two biosensors have been simultaneously used. One sensor is sensing, with similar sensitivity, both D and L species, while the second sensor is sensitive to only one of the enantiomers.92,93 Similar to PEME, two main types of matrices are proposed for enantioselective biosensor design: a polymeric matrix3,4 and a matrix based on carbon paste.16,94 The most reliable membrane design is that based on carbon paste. In PVC-based types, immunodyne membranes are utilized for enzyme immobilization. Each side of the membrane was wetted with 10 mL of enzyme solution at a concentration of 50 mg/mL in a certain buffer. The coupling reaction is allowed to last for 2 min, and then the enzyme membranes, as disks of 8 mm diameter, are washed in 1 mol/L potassium chloride solution for 10 min. They are stored in the buffer at 48C. In carbon paste-based biosensors, graphite powder is heated at 7008C for 15 h in a muffle

furnace and cooled to ambient temperature in desiccators. Hundred microliters of enzyme solution (1 mg enzyme/mL, made in a certain buffer with a certain pH) is then added to 100 mg of the activated graphite powder. The mixture is allowed to react at 48C for 2 h before drying under vacuum for 4.5 h to remove water. An aliquot of 40 mL of Nujol (or paraffin) oil per 100 mg of graphite powder is then added to dry enzymemodified graphite to prepare the paste. Plain graphite–Nujol paste was filled into the sensor body with carbon paste that contains the enzyme filled in the top. Electric contact was made by inserting a silver wire in the carbon paste. The electrode tips were gently rubbed on fine paper to produce a flat surface. When not in use, the biosensor was stored in a dry state at 48C. Recently, a biosensor design was reported where, with the use of nonenantioselective enzyme, the enantioselectivity has been gained by the modification of sensing electrode surface with appropriate conducting polymer, which additionally serves as support for immobilization of enzyme.95

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VALIDATION CRITERIA OF ELECTROCHEMICAL SENSORS AND BIOSENSORS

Given the relatively poor chemical and physical stability of bio-molecules, artificial receptors became of increasing importance as a possible alternative to natural systems.97 The most promising materials in the field of artificial molecular recognition systems are MIPs. Molecular imprinting allows for the creation of specific, tailor-made recognition sites for a target molecule in synthetic polymers. This is achieved if the target molecule is present during polymerization, thus acting as a molecular template. Prior to polymerization, monomers carrying certain functional groups are allowed to form a complex with the target molecule via covalent or noncovalent interactions, and are subsequently copolymerized with crosslinking monomers (Fig. 5). Following polymerization, the functional groups are held in position by the highly cross-linked polymeric network. Subsequent removal of the template by solvent

extraction or chemical cleavage reveals binding sites which are complementary in size and shape to the target molecule. In that way, a molecular memory is introduced in the polymer. It is well known from both biology and chemistry that molecules tend to stick to receptors or surfaces with complementary shape, that is, the ‘‘lock and key’’ theory of enzymes. Thus, molecular imprinting results in polymers capable of molecular recognition and catalysis for target analytes with similar affinity and specificity to antibodies. Chirally imprinted polymers exhibit several attractive features: the elution order of this material is predefined; the enantiomer originally used as template is always more strongly retained; no sophisticated design and laborious synthetic protocols have to be elaborated to generate enantioselective interaction site, and the required components are readily available and inexpensive bulk chemicals. The polymers themselves show beneficial properties such as remarkable stability against mechanical stress, elevated temperature, high pressure, acids and bases, metal ions, and wide range of solvents in combination of long shelf life. There are also several disadvantages with imprinted polymers. Generally, only about 10–15% of the loaded template results in efficient binding site formation and therefore, in relative low loading capacity. Imprinted polymers also suffer from binding site heterogeneity and thus, give rise to extreme peak tailing for the imprinted molecule in chromatography.98 One of the most popular methods of producing MIPs is bulk polymerization,99 in which functional monomers are bound either covalently or noncovalently to a print molecule or template. The correct positioning of these functional groups allows them to converge on the template molecule in a reciprocal fashion. The resulting prepolymer complex is copolymerized with an excess of crosslinking monomer in the presence of an equal volume of inert solvent and a free radical initiator. The template is then removed by extraction or hydrolysis, leaving sites complementary in size and shape to the template molecule. This method usually applied to the preparation of stationary phases for HPLC,100 and can be used to prepare imprinted polymeric membranes101 or polymercoated electrodes for electrochemical sensing.102 Another technique is the in-situ molecular imprinting which is a technique for preparation of imprinted polymers where they are subsequently utilized, that is, onto the surface of a


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The general validation criteria of these classes of sensors have been outlined by Stefan et al.96 These criteria can be summarized as follows: (1) The best chiral selector must be selected, or the enzyme that favors only the reaction of one enantiomer must be selected. (2) The best matrix for the electroactive material must be used. (3) For in-vivo screening analysis, the biocompatibility of materials must be correlated with the response characteristics of the sensors. (4) For PEME, the minimum slope accepted is 50 mV/decade of concentration, with a low limit of detection, minimum two concentration decades for working concentration range, and maximum selectivity over byproducts, and compression and degradation compounds. (5) High enantioselectivity. (6) A minimum of 99.00% for the recovery test of the enantiomer of interest when its concentration is up to 99.99 times lower than that of the other enantiomer.

MOLECULARLY IMPRINTED POLYMERS (MIPs)


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Figure 5. Schematic representation of the noncovalent molecular imprinting principle. The template is CBZ-L-tyrosine and the functional monomer methacrylic acid.

transducer, such as an electrochemical sensor. As a result, imprinted polymers prepared by an in-situ technique do not require any subsequent treatment of the resultant material, except washing to extract the template, and can be used directly in their applications. The in-situ method has been the most reported method for preparation of potentiometric sensors. Different strategies have been utilized for immobilizing the MIPs on transducer surface such as in-situ polymerization, production of thin films of MIP by surface coating, entrapment of MIP particles into gels or membranes, and production of MIP-based composites. In terms of signal transducing various methods of measurement can be used, including spectroscopic techniques (colorimetry, fluorescence, infrared evanescence wave, surface plasmon resonance) and masssensitive methods (quartz crystal microbalance (QCM), surface-acoustic waver oscillator). Among electrochemical methods, these are used: ellipso-

metry, capacitance, conductometry, amperometry, voltammetry, potentiometry, and ionselective field effect transistors.103 MIPs for over 20 classes of compounds have been reported.100,104 The list includes sugars, amino acids, peptides and proteins, therapeutic drugs, steroids, aromatic hydrocarbons, dyes, phosphonate esters, and pesticides. The larger part of applications of MIPs is their use as solidphase adsorbents for HPLC.104 In addition, the use of MIPs in other chemical applications has also expanded greatly to include the development of analytical assays and sensors, CE, TLC, solidphase extraction, and immunoassay-type binding assays,105 and the production of polymers with special functions, such as drug-release matrices.106 Several MIP-based enantioselective sensors have been designed by using a QCM.107–110 A sensor discriminating between S- and Rpropranolol enantiomers in acidified acetonirile

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solution was obtained by copolymerization methacrylatye and S-propranolol on the surface of gold electrode.107 Liao et al. reported stereo-specific MIP sensor for L-histidine based on acrylamide.109 A strong discrimination of response to other amino acids was also shown for these sensors. Percival et al. introduced an enantioselective MIP-based QCM sensor with a noncovalent imprint of 110 L-methanol D-menthol. In potentiometric sensing of chiral amino acids, a surface imprinting technique was used by Zhou et al.111 The technique involved binding octadecylsilane layer to an indium-tin oxide glass plate electrode in the presence of analyte molecule and then washing out the chiral component. The created functionalized surface works as a channel gate opening only to target isomer used as template. The developed sensor was selective to L-N-carbobenzoxy-aspartic acid and showed chiral discrimination to enantiomers of glutamic and aspartic acids. Various electrodeposited conducting and nonconducting polymers has been utilized to modify working electrodes in amperometry and voltammetry in order to use them in enantioselective uptake studies. A poly-pyrrole film doped with L-glutamate, deposited on glassy carbon and Pt electrode showed an enantioselective uptake of L-glutamic acid cation with ratio of L/D isomers >10.112 The enantioselectivity of over-oxidized poly-pyrrole colloids imprinted with L-lactate was reported to different amino acids by Okuno et al.113 In a recent effort by Piletsky et al.,114 new MIPs were developed for either of the enantiomers of phenylalanine using functional monomers bis-acryloyl b-CD and 2-acryloylamido-2-methyl1-propanesulfonic acid (AMPSA). The roles of the hydrophobic interacting CD and the electrostatic interacting sulfonic acid monomers were examined.

of chiral compounds. Another promising alternative is MIPs which have several attractive features: the elution order of this material is predefined; the enantiomer originally used as template is always more strongly retained; no sophisticated design and laborious synthetic protocols have to be elaborated to generate enantioselective interaction site, and the required components are readily available and inexpensive bulk chemicals. Nevertheless, the optimization and adaptation of existing transducer technologies, with respect to the requirements of enantioselective sensor systems, may be a challenging task.

REFERENCES

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81.

82.

83.

84.

85.

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87.

88.

89.

90.

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DOI 10.1002/jps