Environmental Fate of Chiral Pharmaceuticals: Determination, Degradation and Toxicity

Home , Chiral drugs, Chiral switch, Enantiopure drug

Chapter 1 Environmental Fate of Chiral Pharmaceuticals: Determination, Degradation and Toxicity

Ana R. Ribeiro , Paula M. L. Castro , and Maria Elizabeth Tiritan

Contents 1.1 Introduction – Basic Concepts of Chirality ...... 5 1.2 Chiral Pharmaceuticals ...... 8 1.3 Analytical Methods for Quantifi cation/Identifi cation of Chiral Pharmaceuticals in the Environment ...... 13 1.3.1 Chromatography ...... 13 1.3.1.1 Indirect Methods ...... 13 1.3.1.2 Direct Methods ...... 14 1.3.2 Gas Chromatography (GC) ...... 15 1.3.3 High Performance Liquid Chromatography (HPLC) – Liquid Chromatography/Mass Spectrometry (LC/MS) ...... 15 1.3.3.1 Capillary Electro-Chromatography (CEC) ...... 16 1.3.3.2 Other Techniques ...... 23 1.3.4 Electrochemical Sensors and Biosensors...... 24 1.4 Biotic and Abiotic Degradation and Removal Processes of Chiral Pharmaceuticals in the Environment ...... 24 1.4.1 Biodegradation of Non Steroid Anti-Infl ammatory Drugs (NSAIDs), Beta-Blockers and Antidepressants as Individual Enantiomers ...... 25 1.5 Toxicity of Emergent Chiral Pharmaceuticals in Aquatic Organisms ...... 30 1.6 Conclusions ...... 36 References ...... 37

A. R. Ribeiro Centro de Investigação em Ciências da Saúde (CICS) , Instituto Superior de Ciências da Saúde-Norte , Rua Central de Gandra, 1317 , 4585-116 , Gandra PRD , Portugal Centro de Biotecnologia e Química Fina (CBQF), Escola Superior de Biotecnologia , Universidade Católica Portuguesa , Porto , Portugal P. M. L. Castro Centro de Biotecnologia e Química Fina (CBQF), Escola Superior de Biotecnologia , Universidade Católica Portuguesa , Porto , Portugal

E. Lichtfouse et al. (eds.), Environmental Chemistry for a Sustainable World: 3 Volume 2: Remediation of Air and Water Pollution , DOI 10.1007/978-94-007-2439-6_1, © Springer Science+Business Media B.V. 2012 4 A.R. Ribeiro et al.

Abstract Pollution of the aquatic environment by pharmaceuticals is of major concern. Indeed pharmaceutical pollutants have several undesirable effects for many organisms, such as endocrine disruption and bacterium resistance. They are resis- tant to several degradation processes, making their removal diffi cult and slow. Pharmaceuticals reach the environment due to their ineffi cient removal by waste water treatment plants (WWTP), and by improper disposal of unused medicines. In aquatic environments pharmaceuticals reach concentrations at trace levels of ngL −1 – m gL−1 range. Many pharmaceutical pollutants are chiral. They occur in nature as a single enantiomer or as mixtures of the two enantiomers, which have different spatial confi guration and can thus be metabolized selectively. In spite of similar physical and chemical properties, enantiomers have different interactions with enzymes, receptors or other chiral molecules, leading to different biological response. Therefore they can affect living organisms in a different manner. The fate and effects of enantiomers of chiral pharmaceuticals in the environment are still largely unknown. Biodegradation and toxicity can be enantioselective, in contrast to abiotic degradation. Thus accurate methods to measure enantiomeric fractions in the environment are crucial to better understand the biodegradation process and to estimate toxicity of chiral pharmaceuticals. We review (1) general properties of chiral compounds, (2) current knowledge on chiral pharmaceuticals in the environment, (3) chiral analytical methods to deter- mine the enantiomers composition in environmental matrices, (4) degradation and removal processes of chiral pharmaceuticals in the environment and (5) their toxicity to aquatic organisms. The major analytical methods discussed are gas chromatography (GC), high performance liquid chromatography (HPLC), electrochemical sensors and biosensors. These chiral methods are crucial for the correct quantifi cation of the enantiomers regarding that if an enantiomer with more or less toxic effects is preferentially degraded, the assessed exposure based on measurements of achiral methodologies would overestimate or underestimate ecotoxicity. The degradation and biodegradation is discussed using few examples of important therapeutic classes usually detected in the aquatic environment. Few examples of ecotoxicity studies are also given on the occurrence of enantiomers and their fate in the environment which differs with regard to undesirable effects and to biochemical processes.

M. E. Tiritan ( *) Centro de Investigação em Ciências da Saúde (CICS) , Instituto Superior de Ciências da Saúde-Norte , Rua Central de Gandra, 1317 , 4585-116 , Gandra PRD , Portugal Centro de Química Medicinal da Universidade de Porto (CEQUIMED-UP) , Porto , Portugal Instituto Superior de Ciências da Saúde-Norte , Rua Central de Gandra, 1317 , 4585-116 , Gandra PRD , Portugal e-mail: [email protected]; [email protected] 1 Environmental Fate of Chiral Pharmaceuticals: Determination, Degradation... 5

Keywords Chiral chromatography • Enantioselective • Biodegradation • Enantiomeric fraction • Enantioseparation • Racemates • Enantiomers • Chiral switching • Chiral analysis • Waste water treatment plant • Beta-blockers • Antidepressants • Non steroid anti-inﬂ ammatory drugs • Enantiospeciﬁ c toxicity • Chiral stationary phases • Fluoxetine • Propranolol • Atenolol • Ibuprofen • Naproxen • Chiral ecotoxicity

1.1 Introduction – Basic Concepts of Chirality

Chiral compounds are substances with a similar chemical structure that, in general, confers them the same physical and chemical properties like melting point, solubility and reactivity. However they differ in the deviation of polarized light due to the different spatial confi guration originated by planes, axis or centers of asymmetry given two nonsuperposable left-handed and right-handed mirror images compounds, called enantiomers. The planar chirality refers to a planar unit connected to an adja- cent part of the structure by a bond which results in restricted torsion avoiding the symmetry plane as in the monosubstituted paracyclophane (Fig. 1.1a ). When a set of ligands is held around an axis originates a spatial arrangement which is nonsuperposable on its mirror image known as axial chirality. Such cases include ortho - substituted biphenyls and substituted allenes, a group of stereoisomers called atropoisomers which have a restricted rotation about a single bond, being the abso- lute confi guration called R or S preceded by an a (aR or aS ) to distinguish them by other optical active compounds (Fig. 1.1b, c, d ) (Moss 1996 ; Santos et al. 2007 ) . The center chirality consists in a stereogenic center holding a set of four different substituents in a spatial arrangement which also origins two nonsuperposable left- handed and right-handed mirror image compounds (Fig. 1.2 ). The most common type of center chirality is a stereogenic carbon (Fig. 1.3 ), but other atoms such as sulphur or phosphorus can also be a stereogenic center (Fig. 1.4 ). Enantiomers can be identifi ed by the manner to rotate the polarized light, to the right (clockwise) are called dextrorotatory, (d) or (+), and to the left (counter clockwise) are denominated levorotatory, (l) or (−). Concerning their chemical confi guration relative to the spatial orientation of the substituent of the chiral center, enantiomers can be (R ) from the Latin rectus or (S ) from the Latin sinister. The equimolar mixture of two enantiomers is a racemate or a racemic mixture and does not rotate the polarized light (Eliel and Wilen 1994 ) . Despite the similar thermodynamic properties in achiral medium, enantiomers normally have a different behaviour when the environment is also chiral. Amino acids, carbohydrates, deoxyribose and ribose are chiral compounds which are the unity of important natural molecules such as proteins, glycoproteins, DNA and RNA, respectively (Müller and Kohler 2004 ; Hühnerfuss and Shah 2009 ) . As such, in biological processes, enzymes, receptors and other binding-molecules have the capacity to recognize enantiomers in a different way. The model that demonstrate the recognition of an enantiomer by a receptor due to their complementary confi guration is illustrated in Fig. 1.5 demonstrating that the 6 A.R. Ribeiro et al.

aR b

R1 R1

R2 R2

monosubstituted [2.2]paracyclophane ortho -substituted biphenyl c d R1 R3 R1 R4

CC CC C C R2 R4 R2 R3

(Sa )-allene (Ra )-allene

Fig. 1.1 ( a ) Structure of monosubstituted [2.2] paracyclophane possessing planar chirality; ( b ) Structure of ortho-substituted biphenyl with axial chirality; (c , d ) Structures of (aS ) and ( aR )- allenes showing its axial chirality, supposing that the sequence order is R4>R3>R2>R1, originating the ranking 1 for R4, 2 for R3, 3 for R2 and 4 for R1(Eliel and Wilen 1994 )

Fig. 1.2 Stereogenic atom (X) with four different substituents (R1, R2, R3, R4) originating the nonsuperposable structures

other enantiomer does not match the same receptor and thus do not have the same activity in this receptor. However it can be a ligand for other receptor, leading to another activity, side effects, or even toxic effects. When enantiomers of one pharmaceutical compound have the same type of activity but differ in the potency, eutomer is the enantiomer whit higher potency whereas distomer is the one that have such activity reduced, being the ratio of the potencies termed eudismic ratio (Caldwell et al. 1988 ; Cox 1994 ). 1 Environmental Fate of Chiral Pharmaceuticals: Determination, Degradation... 7

NH 2 a O N O H OH F b c O F HO O F H

N N H

Fig. 1.3 Structures of three pharmaceuticals with a carbon stereogenic center. (a ) atenolol; ( b ) propranolol; (c ) ﬂ uoxetine

a b O O P S H2N S

Fig. 1.4 ( a ) Structure of modafinil with a sulphur stereogenic center; (b ) Structure of 2,3-dimethyl-7-phenyl-5-(phenylsulfanyl)-7-(R/S )-phosphabicyclo[2.2.1]hept-2-ene with a phosphorus stereogenic center

Fig. 1.5 Schematic process of the complementary recognition of one enantiomer (a ) rather than the other (b ) 8 A.R. Ribeiro et al.

1.2 Chiral Pharmaceuticals

Pharmaceuticals are chemicals used for diagnosis, treatment, alteration or prevention of disease, health condition, or structure/function of the human body, including veterinary drugs. They act by interaction with the binding site of the drug receptor which is a macromolecule such as enzymes, nucleic acids or membrane-bounded proteins, promoting the pharmacological action by the formation of a drug-receptor complex (Stringer 2006 ) . Their action in humans is well described, but their mechanism and cumulative effects are mostly unknown in non-target organisms (Daughton and Ternes 1999 ) . Chiral pharmaceuticals are becoming of environmental concern because of the different pharmacokinetic, pharmacodynamics, toxicological and ecotoxicological properties occurring between enantiomers which may differently affect aquatic organisms. Concerning to pharmacokinetics, absorption, distribution (e.g. transport by passive or active way, in which the protein binding is important), metabolism and excretion can be different for each enantiomer since they represent biological processes in which binding proteins, membrane proteins, enzymes and other chiral molecules have an important role. Likewise, the pharmacodynamics involves the interaction with receptors like membrane proteins or enzymes which can be different for each enantiomer. Because chiral pharmaceuticals have normally different pharmacokinetic and pharmacodynamics properties, they have different dissociation constant from the binding site and different attachment to it leading to different biological response in quality or quantity (Campo et al. 2009 ) . In a review concerning the chirality in pharmaceuticals, Lima (1997 ) related few examples of chiral pharmaceuticals that can be used as racemates, as in the following cases: (a) The pharmacologic activity of both enantiomers is the same (e.g., prometazine, antihistaminic). This effect is due to the equal pharmacokinetic and pharmacodynamics properties; (b) One enantiomer is biologically more active than the other (e.g., propranolol, b -blocker). In this case both of enantiomers can reach the receptor, binding to it at the binding site with different dissociation constant, leading to a stronger attachment of one enantiomer; (c) One of the enantiomers antagonizes the side effects of the other (e.g., indacrinone). In others cases there are drugs that should be commercialized only as a single enantiomer, mostly due to the association of the enantiomers to different receptors, leading to different responses where (Mannschreck et al. 2007 ) : (a) One enantiomer is biologically active and the other has no activity (e.g., a -metildopa); (b) One enantiomer is biologically active and the other is antagonist (e.g., picenadol) (Franz et al. 1990 ) ; 1 Environmental Fate of Chiral Pharmaceuticals: Determination, Degradation... 9

(c) The pharmacologic activity of enantiomers is different (e.g., propoxyphene: analgesic and antitussive effects); (d) One enantiomer has side effects (e.g., L-Dopa). Table 1.1 shows few chiral pharmaceuticals that can be used as racemates and/or enantiopure formulations, depending on the effects of each enantiomer. The recent advances in stereoselective synthesis and chiral analysis led to an increase of the single enantiomers as drugs available in the market (Hutt 1998 ) . The re-evaluation of the license of the enantiomeric pure drugs which were produced in a racemic mixture, called as chiral switching process, also collaborated to increase the use of single enantiomer (Cordato et al. 2003 ; Hutt and Valentová 2003 ; Caner et al. 2004 ) . However the chiral switching is related to economical, legislation and patent aspects, apart from the evident pharmacologic and toxicological details. Some pharmaceutical companies promote the patenting of new enantiopure pharmaceuticals at the expiring of the patent of the racemic drug (Somogyi et al. 2004 ) . This aspect is highlighted by these authors especially when companies start the marketing approval of the new enantiopure drug when the racemic drug patent is expiring avoiding the release of generics from other companies (Barreiro et al. 1997 ; Somogyi et al. 2004 ; Mannschreck et al. 2007 ) . Besides the advantages of enantiopure pharmaceuticals, like lower therapeutic doses, more safety margin, less interindividual variability, less drug interactions and fewer side effects, chiral switching can lead to unexpected toxicity (Baker et al. 2002 ) . As such, the therapeutic advantage of an enantiopure drug must be clinically proven to justify the cost increase of the new treatment instead of the use of racemates. The single enantiomer and the relative racemate should be compared concerning to pharmacokinetics and toxicological studies (Hutt and Valentová 2003 ; Somogyi et al. 2004 ; Orlando et al. 2007 ) . Fluoxetine is a noted example of a failed chiral switching. Both enantiomers of fl uoxetine are potent inhibitors of the serotonin reuptake pump but the enantiomers of the metabolite norfl uoxetine have differences in this activity, being (R )-norfl uoxetine an inactive metabolite and (S )-norfl uoxetine the active metabolite (Henry et al. 2005 ) . The affi nity of ( R )-fl uoxetine for human

5-HT2A and 5-HT2C receptor subtypes is high, unlike (S )-fl uoxetine, being (R )- fl uoxetine an antagonist for these receptors leading to an increase of extracellular catecholamines, such as serotonine, dopamine and norepinephrine. (S )-norfl uoxetine has a similar binding to the transporter and is recognized as an active metabolite whereas ( R )-norfl uoxetine is an inactive metabolite (Koch et al. 2002 ) . Besides, ( S )- norfl uoxetine is a potent inhibitor of its metabolic enzyme and contributes signifi - cantly to the half-life of the racemate, being ( R )-norfl uoxetine less active regarding this enzyme (Stevens and Wrighton 1993 ) . The chiral switching would be the way to develop a new license market of (R )-fl uoxetine with the same indications as racemic fl uoxetine, with a better half-life, a better selectivity for important receptor subtypes in depression mechanism and less drug-drug interactions, being a good improvement for clinical strategies. However, the development of the (R )-isomer of fl uoxetine was abandoned after an unexpected cardiac side effect, at higher doses, identifi ed at Stage II clinical trials (McConathy and Owens 2003 ) . The company 10 A.R. Ribeiro et al.

Table 1.1 Examples of chiral pharmaceuticals used as racemates or as single enantiomers Qualitative activity Drug Equal Different Mode of action Description References Promethazine X Equal pharmaco- Promethazine Chen et al. logic potency (1992 ) Propranolol X Different Propranolol: Pavlinov et al. pharmacologic (−)-propranolol (1990 ) potency has a pharmacological activity 100 times superior to (+)-propranolol Warfarin X Warfarin: anticoagu- Choonara et al. lant, with greater (1986 ) anticoagulant potency of (S)-warfarin Methadone X Methadone: Huq (2007 ) (R)-methadone has higher affi nity for the m -opioid receptor and longer plasma elimination half-life Bupivacaine X Both enantiomers Bupivacaine: S Huang et al. have pharma- (−)-bupivacaine (1998 ) cological has the same activity but one neural blocking of them has characteristics, but less toxicity has a higher and less side margin of safety effects Fluoxetine X Enantiomers have Fluoxetine: Steiner et al. different (R)-fl uoxetine is an (1998 ) pharmacologi- antidepressant and cal activity (S)-fl uoxetine was tested for migraines prophylaxis Propoxyphene X Propoxyphene: Cooper and (+)-propoxyphene Anders is analgesic and (1974 ) (−)-propoxyphene is antitussive Indacrinone X One enantiomer Indacrinone: Jain et al. antagonize the (S)-indacrinone is (1984 ) side effects of natriuretic and the other (R)-indacrinone is uricosuric (continued) 1 Environmental Fate of Chiral Pharmaceuticals: Determination, Degradation... 11

Table 1.1 (continued) Qualitative activity Drug Equal Different Mode of action Description References Ibuprofen X One enantiomer is Ibuprofen: its Mayer and pharmacologi- anti-inﬂ ammatory Testa cally active and activity is almost (1997 ) the other doesn’t due to have activity (S)-ibuprofen Cetirizine X Cetirizine: its Mannschreck antiallergic et al. (2007 ) activity is due to (R)-cetirizine Dopa X One enantiomer is Dopa: L-dopa is used Hutt and pharmacologi- in Parkinson’s Valentová cally active and disease and ( 2003 ) the other has D-dopa has side side effects effects like nausea, anorexia, involuntary movements and granulocytopenia Thalidomide X One enantiomer is Thalidomide: Smith ( 2009 ) pharmacologi- (R)-thalidomide cally active and was used for the other is insomnia and toxic nausea therapy; (S)-thalidomide is teratogenic Picenadol X One enantiomer is Picenadol: (+)-picen- Franz et al. pharmacologi- adol is an opioid (1990 ) cally active and agonist and the other is (-)-picenadol is a antagonist weak agonist/ antagonist This table resumes the beneﬁ ts and disadvantages depending on the pharmacological or side effect

had hoped that (R )-fl uoxetine would replace fl uoxetine, which was facing imminent expiration of its patent protection. Thus there are many pharmaceutical drugs that are both commercialized at racemic and enantiopure forms, as described in Table 1.2 (Tucker 2000 ; Hutt and Valentová 2003 ; Orlando et al. 2007 ) . There are also some pharmaceuticals, such as Non Steroid Anti-Infl ammatory Drugs (NSAIDs), which suffer chiral inversion, e.g., propionic acid derivatives. These drugs act by inhibition of cyclo-oxygenase (COX) and consequently the synthesis of prostaglandins and thromboxanes. Except for naproxen (Fig. 1.6 ) they are 12 A.R. Ribeiro et al.

Table 1.2 Examples of new enantiopure pharmaceuticals, which racemates were not withdrawn from the market Racemate Therapeutic class Enantiopure pharmaceutical Trade name Citalopram Antidepressant ( S)-citalopram (escitalopram) Lexapro, Cipralex Ibuprofen Anti infl ammatory ( S)-ibuprofen (dexibuprofen) Seractil Ketoprofen Anti infl ammatory ( S)-ketoprofen (dexketoprofen) Sympal, Ketesse Bupivacaine Anaesthetic ( S)-bupivacaine (levobupivacaine) Chirocaine Cetirizine Antihistaminic ( R)-cetirizine (levocetirizine) Xyzal Omeprazole Proton pump inhibitor ( S)-omeprazole (esomeprazole) Nexium Ofl oxacin Antimicrobial ( S)-ofl oxacin (levofl oxacin) Levaquin,Tavanic Salbutamol b 2-adrenergic receptor ( S)-salbutamol (levalbuterol) Xopenex agonist

a b OH OH

R S O O

Fig. 1.6 Structure of (R )-naproxen (a ) and (S )–naproxen ( b )

aO b O O O R S OH OH

Fig. 1.7 Structure of (R )-ibuprofen (a ) and (S )–ibuprofen (b ) commercialized as racemates in most countries. Ibuprofen (Fig. 1.7 ) is a common example of a NSAIDs used for pain, infl ammation, fever and arthritis, which is normally sold as racemate although S -(+)-ibuprofen is practically the responsible for the pharmacologic action. Moreover this drug, as other profens, suffers unidirec- tional conversion to the pharmacologic active S -(+)-enantiomer in vivo which diffi cult to obtain the enantiopure (S )-ibuprofen with a good percentage of conversion justifying the use of racemates. The disadvantage of the racemic ibuprofen is that ( S )-ibuprofen has less gastric side effects than the racemic mixture; is more hydro- soluble leading to a more rapid action; the chiral inversion can originate interindividual variability in analgesia; and the accumulation of the intermediate ( R )-ibuprofen-CoA in the chiral inversion, which can react with triglycerides leading to the accumulation at fatty tissue in the organism (Lima 1997 ; Tucker 2000 ; Bonabello et al. 2003 ; Carvalho et al. 2006 ; Chávez-Flores and Salvador 2009 ) . Another therapeutic option is the use of chiral drugs with an enantiomeric ratio different from 1 to improve the benefi ts of each enantiomer, when applicable. Enantiomeric Fraction (EF) is the proportion of the concentration of one enantiomer 1 Environmental Fate of Chiral Pharmaceuticals: Determination, Degradation... 13 to the total concentration, expressing the relative concentration of an enantiomer’s compound. Racemate exhibits an EF of 0.5 while an enantiomerically pure compound has a value of 0 or 1. Thus, a mixture of two enantiomers in a proportion different to 1:1 has an EF between 0 and 1 and different to 0.5. As an example, the advantage of the administration of a mixture of S 0.75: R 0.25 bupivacaíne rather than the racemate was reported, with the same anaesthetic properties and with less toxicity (Gonçalves et al. 2003 ) . In this review we will focus on three pharmaceutical classes: Beta-blockers, Antidepressants and Non Steroid Anti-Infl ammatory Drugs (NSAIDs), due to their persistence in the environment and respective ecological effects (Ternes 1998 ; Trenholm et al. 2006 ; Vanderford and Snyder 2006 ; Pérez and Barceló 2008 ; Fernández et al. 2010 ; Santos et al. 2010 ) .

1.3 Analytical Methods for Quantiﬁ cation/Identiﬁ cation of Chiral Pharmaceuticals in the Environment

1.3.1 Chromatography

The basis of analytical methods for analyses of chiral compounds is the formation of diastereoisomers between the enantiomers and other optical pure compound which provides different retention times, leading to the separation of the enantiomers. The diastereoisomers, which are non-mirror image compounds, can be separated by chromatography due to their different physical and chemical properties (Carvalho et al. 2006 ). In chromatography methods, indirect and direct experimental approaches can be used (Bojarski 2002 ; Hühnerfuss and Shah 2009 ) .

1.3.1.1 Indirect Methods

Indirect methods require the derivatization of the racemic mixture with an optical pure reagent, leading to the formation of diastereoisomers. Since diastereoisomers have different physical and chemical properties, they can be resolved by an achiral stationary phase. After the separation in an achiral column, enantiomers can be recovered by inversing the derivatization reaction. This approach is possible if the racemate has at least one suitable functional group to allow the reaction. As advantages, indirect methods can be performed in conventional chromatographic columns as RP-C18 , it is possible to introduce chromophores or ﬂ uorophores and also it is easy to predict the elution order. However, the complex and time-consuming derivatization step with different reaction rates for each enantiomer, the poor availability of optical pure chiral reagents, the possibility of racemization and the difﬁ culty to apply to environmental samples turns this method weak for environmental analysis (Bojarski 2002 ; Carvalho et al. 2006 ; Hühnerfuss and Shah 2009 ) . 14 A.R. Ribeiro et al.

1.3.1.2 Direct Methods

Direct methods use a chiral selector as an additive in the mobile phase (Chiral Mobile Phase Additive) or on the stationary phase (Chiral Stationary Phase or CSP). In both cases, the chiral selector has more affi nity with one of the enantiomers. Chiral Mobile Phase Additive is a more complex and expensive method because it requires higher amounts of chiral selector to prepare the mobile phase, which is not recovered and thus it is wasted in a large scale. This kind of direct method can also interfere with the detection mode (Lämmerhofer 2010 ) . Chiral Stationary Phase (CSP) consists in a chiral selector adsorbed or covalently bonded to a solid support, which forms transitory diastereoisomeric complexes with the two enantiomers with different stability (Francotte 2001 ; Lämmerhofer 2010) . The more stable complex will elute with larger retention time. There are many different type of commercial chiral columns: Pirkle type or donor-acceptor CSP, crown ethers CSP, ligand-exchange CSP, polysaccharide derivatives CSP, cyclodextrin CSP, protein CSP, macrocyclic glycopeptides antibiotics CSP and others such as CSPs based on synthetic polymers (Pirkle and Pochapsky 1989 ; Haginaka 2008 ; Lämmerhofer 2010 ) . They are performed in open or tubular columns, open tubular capillaries and thin layer plates (Wistuba and Schurig 2000 ; Wistuba 2010 ) . The major advantage of this method is the ability of the analyte to remain unmodifi ed. Despite the numerous of commercial CSP, an universal column is not available, thus making the trial versus error a challenge. The choice of the CSP is based on the experience and on the available lit- erature, being often empirical by trial and error evaluation. However some authors start this with polysaccharides or macrocyclic antibiotics because of their broad application and the possibility to operate in normal, reverse or polar organic mode (Perrin et al. 2002a, b ; Andersson et al. 2003 ; Cass et al. 2003a ; Sousa et al. 2004 ; Ates et al. 2008 ; Pirzada et al. 2010 ) . Thus, the method developing time is reduced and the prob- ability of success is enhanced. The more usual separation mode is normal-phase, however this tendency is turning to reversed-phase and polar organic mode, being all applicable to the most used polysaccharides or macrocyclic antibiotics CSPs (Cass and Batigalhia 2003 ; Cass et al. 2003b ; Montanari et al. 2006 ) . Andersson et al. (2003 ) performed a screening of polysaccharides or macrocyclic antibiotics CSPs to compounds used in pharmaceutical industry. They reported that when using polysaccharides CSPs in normal-phase, the alcohol used as modifi er affects selectivity, elution order and retention time. The additives used for separation of basic compounds in normal-phase mode must be basic to reduce tailing, whereas for separation of acidic compounds the additive must be acid to reduce the excessive retention of these compounds. Neutral compounds are unaffected by these additives so they can be processed with any of them. Thus, besides the type of column and the alcohol chosen, the additive used is of great importance for the success of the resolution of enantiomers. Concerning to polar organic mode, better resolution is achieved with neutral and weak acidic and basic compounds. Using macrocyclic antibiotics CSPs, the number of compounds enantioresolved is lower than using polysaccharides CSPs, although they complement the enantioselectivity and can resolve important classes of pharmaceutical compounds like b -blockers, broncho- dilators and some local anaesthetics (Andersson et al. 2003 ) . 1 Environmental Fate of Chiral Pharmaceuticals: Determination, Degradation... 15

Molecularly Imprinted Polymers are a synthetic alternative in which the target compound is used in the polymerization process as a template, to produce a speciﬁ c receptor to one enantiomer which is known to be more retained. The removal of the template by a solvent or a chemical reaction provides a complementary site in the receptor to the target molecule, providing a Molecularly Imprinted Polymer which can be used as CSP avoiding the choice based on the trial and error method. Commonly these Molecularly Imprinted Polymers are incorporated in conventional HPLC columns. These materials are easy to obtain, highly selective and stable, and the elution order is predictable. The template is the more retained enantiomer being the last to eluate. The drawback of Molecularly Imprinted Polymers is the peak tailing produced due to the heterogeneous binding site. Molecularly Imprinted Polymers can be coupled to HPLC, Capillary Electro-Chromatography, Thin Layer Chromatography and Analytical Sensors (Wistuba and Schurig 2000 ; Sancho and Minguillón 2009 ) .

1.3.2 Gas Chromatography (GC)

Gas Chromatography (GC) can be performed in the indirect and direct mode, having the advantages of high efﬁ ciency, sensitivity, speed of separation, and ability to separate analytes from impurities. The indirect mode requires the derivatization of the chiral compound with an optical pure reagent, resulting in diastereoisomers that are then separated on an achiral stationary phase. The direct mode is performed with a CSP. GC has the great advantage of not needing optimization of mobile phase concerning solvents, pH, modiﬁ ers and gradients (Eljarrat et al. 2008 ) . However, there are only a few chiral columns that can be used in GC, which limits the application of this technique. The most used columns are cyclodextrin-based and have demonstrated a great applicability in organochlorine pesticides (Wong and Garrison 2000 ; Eljarrat et al. 2008 ) . Another useful CSP are based on metal complexes such as metal-b -diketonate polymers (Rykowska and Wasiak 2009 ) . One disadvantage of GC is the need of derivatization, in some cases, to increase the volatility, to prevent the peak tailing and thus to improve detection limits by the peak shaping (Zhang et al. 2009 ) . The high temperature used in GC is a drawback when the analytes are not volatile and when the chiral compound can suffer racemization or decomposi- tion (Schurig 2001 ; Jiang and Schurig 2008 ) . Thus GC has several limitations to environmental analyses of chiral pharmaceuticals (Huggett et al. 2003 ; Lamas et al. 2004 ; Jones et al. 2007 ) .

1.3.3 High Performance Liquid Chromatography (HPLC)-Liquid Chromatography/Mass Spectrometry (LC/MS)

High Performance Liquid Chromatography (HPLC) has become a technique of routine analysis in replacement of GC because of the numerous combinations of the 16 A.R. Ribeiro et al. available columns with the potential mobile phases, and the detection methods that can be coupled, such as Mass Spectrometry (MS) or optical detectors when the analytes are UV transparent (Aboul-Enein and Ali 2004 ; Görög 2007 ) . The high variety of chiral commercial columns, like Pirkle, crown ethers, ligand exchangers, cyclodextrins, polysaccharides, proteins, macrocyclic glycopeptides antibiotics, and others, usually design for HPLC analysis and the different elution mode makes this technique a powerful tool for chiral analysis (Perrin et al. 2002a, b ; Andersson et al. 2003 ; Zhang et al. 2010 ) . HPLC with different type of detection has been used for the determination of the EF of many chiral pharmaceuticals in a variety of matrix, including environmental samples (Francotte 2009 ; Kasprzyk-Hordern et al. 2010 ) . Few examples are shown in Table 1.3 . Liquid Chromatography/Mass Spectrometry (LC/MS) and LC–tandem MS (LC/ MS/MS) have played a crucial role in environmental analysis (Niessen 2003 ; Kot- Wasik et al. 2007 ; Pérez and Barceló 2008 ) mainly due to their versatility, sensitivity and selectivity (González et al. 2007 ) . Recent trends in environmental Mass Spectrometry methods have been emerging with special focus on hybrid mass spectrometers such as Quadrupole-Time Of Flight (Qq-TOF) (Farré et al. 2008 ) and Quadrupole-Linear Ion Trap (Qq-LIT) (Díaz-Cruz et al. 2008 ) . However, Triple Quadrupole (QqQ) mass analysers are the most used analytical technology in the environmental analyses (Gros et al. 2006 ) . Few publications have also employed Ion Trap (IT) mass spectrometers for environmental determinations (Feitosa-Felizzola et al. 2007 ; Madureira et al. 2009 ; Barreiro et al. 2010 ) . Despite the largely application to environmental analyses the ion suppression of the interest compounds peaks with the components of the matrix may consist in a limitation of the method. So, the MS/MS detectors are a powerful tool that must be used in environmental analysis because of their high selectivity (Pérez and Barceló 2008 ; Madureira et al. 2009 ) . EF is an important tool in biodegradation, toxicological studies and wastewater monitoring (Fono and Sedlak 2005 ; Hashim et al. 2010 ) . It was demonstrated that the determination of EF can be affected by the peak integration method (Asher et al. 2009 ) . They found that the Deconvolution Method, a method which considers two independent Gaussian-based mathematic functions, is more accurate especially when peaks overlap (poor resolution or asymmetry). Instead, the commonly used method, called Valley Drop Method (VDM), showed signiﬁ cant bias in EF determination. Thus, for environmental analysis the peak integration method must be evalu- ated and Deconvolution Method is preferred when poor resolution or tailing occurs.

1.3.3.1 Capillary Electro-Chromatography (CEC)

Capillary Electro-Chromatography (CEC) is a hybrid separation technique of HPLC and Capillary Electrophoresis (CE) since it is based on the partition of solutes between two phases (HPLC) and on the different mobility of the same solutes towards an electric fi eld (CE) and has the advantages of both CE and HPLC. Likewise HPLC it can be applied also to neutral compounds and has high Table 1.3 Analytical methods of separation of several enantiomers in different matrices Enantioselective Elution Chiral stationary Matrix Drug method mode phase Mobile phase Detection limit application Observations Refs Omeprazole 2D-LC/UV or Reverse-phase RAM BSA C8 and Phosphate buffer (pH 5.0 m gL−1 (UV-vis); Waste and It was developed a Barreiro et al. IT-MS/MS amylose 6.8; 0.1 M NaCl) 0.025 m gL−1 estuarine two-dimensional ( 2010 ) tris-(3,5-dimeth- (IT-MS/MS) water liquid chromatog- ylphenylcarbam- samples raphy method ate) of coated which allows the onto APS- use of 1 mL of Nucleosil sample Lansoprazole 2D-HPLC/UV Reverse-phase RAM BSA C8 and (35:65, V/V) 0.025 m g mL−1 Human plasma It was performed an Gomes et al. amylose tris ACN:water automatized (2010) (3,5-dimethoxy- method which phenylcarbam- allows the sample ate) coated onto cleanup and APS-Nucleosil quantifi cation of the analyte in the sample 10 b -blockers HPLC/UV Normal-phase Cellucoat n-heptane/EtOH/ 3.0–297.0 m g mL−1 Screening study Good enantioresolu- Ali et al. DEA tion of ten (2009a ) b -blockers in a single column Fluoxetine and HPLC/FD Reverse-phase Chiralcel OD (75:25, V/V) 5.0 ng mL −1 Human plasma A sensitive method Silva et al. norfl uoxetine potassium was developed to ( 2009 ) hexafl uorophos- further analysis of phate 7.5 mM plasma samples, and sodium following the phosphate therapy with 0.25 M solution antidepressants (pH 3.0)/ACN (continued) Table 1.3 (continued) Enantioselective Elution Chiral stationary Matrix Drug method mode phase Mobile phase Detection limit application Observations Refs Venlafaxine HPLC/UV Normal-phase Chiralpak AD and 0.5%TFA or DEA in n.a Semi-preparative The quality of Caccamese (antidepressant) IA n-hexane/2- HPLC for separation is et al. and 11 analogs propanol or in vitro affected by the pH (2009 ) EtOH pharmaco- and alcoholic logical modifi er of mobile essays phase Methadone LC/MS Reverse-phase Chiralcel OJ (65:35, V/V) 1 ng mL −1 Human plasma It was used an Ansermot ACN/0.02% TEA isotope-labelled et al. in water internal standard (2009 ) to avoid matrix effects Dechloroethylifosfamide LC/MS/MS Reverse-phase Chiralpak-AGP Gradient of 10 mM 1 ng mL−1 Human plasma To consider the Aleksa et al. metabolites of ammonium application of the (2009 ) Ifosfamide acetate in water less toxic (pH 7.00) and enantiomer 30 mM (pH 4.00) 62 pharmaceuticals HPLC/UV Polar-organic Chiralcel AD-RH, (100:0.1:0,1, V/V/V) n.a Screening study ACN/DEA/TFA is the Ates et al. phase OD-RH, AS-RH ACN or MeOH/ fi rst choice of ( 2008 ) and OJ-R DEA/TFA solvent system followed by MeOH/DEA/TFA 62 pharmaceuticals HPLC/UV Polar-organic Sepapak-2 and (100:0.1:0.1, V/V/V) n.a. Screening study The CSP preference is Ates et al. phase Sepapak-3 ACN or MeOH Sepapak- ( 2008 ) or EtOH/DEA/ 2>Chiralcel TFA OD-RH>AS- RH>OJ-R Ketoprofen HPLC/UV Reverse-phase Diamonsil C18 (71:29, V/V) 0.03 m g mL −1 (LQ) Rat plasma It was observed a Wang et al. MeOH/0.01 M (in vitro stereoselective ( 2007 )

KH 2PO 4 (pH 4.5) release test) release of ketoprofen, being faster for (S)-ketoprofen Enantioselective Elution Chiral stationary Matrix Drug method mode phase Mobile phase Detection limit application Observations Refs b -blockers, selective LC/MS/MS Reverse-phase Chirobiotic V (90:10, V/V) 0.2–7.5 ngL −1 WWTP infl uents Able to quantify MacLeod serotonine MeOH/20 mM and effl uents b -blockers at et al. −1 re-uptake inhibitors NH 4OAc in ngL levels in ( 2007 ) and salbutamol water, 0.1% aqueous samples formic acid (pH with little sample 4) pre-treatment Bufurarol HPLC/UV Polar ionic Chirobiotic V (100:0.015:0.010, 2 ngL−1 Plasma samples Good recovery which Hefnawy et al. phase V/V/V) MeOH/ and allows therapeutic ( 2007 ) acetic acid/TEA pharmaceu- drug monitoring tical formulations Fluoxetine and HPLC/FD Normal-phase Extrasil CN/Apex 75% tetramethyl 0.2–0.25 ng mL−1 Plasma and This method allows Unceta et al. norfl uoxetine Silica ammonium cerebral the assessment of ( 2007 ) chloride in ACN; cortex the relationships (40:60, V/V) between the tetrahydrofurane/ concentration of isooctane individual enantiomers. Fluoxetine HPLC/UV Normal- Chiralcel AD, OD, Hexane/isopropanol/ n.a Screening study The better resolution Zhou et al. phase// OJ, Cyclobond I DEA or was obtained with ( 2007 ) Polar- 2000 DM and MeOH/0.2% Chiralcel AD, OD organic Kromasil triethylamine and Cyclobond. phase CHI-TBB acetic acid (Cyclobond) b -blockers LC/MS/MS Reverse-phase Chirobiotic V (90:10, V/V) 3–17 ngL−1 WWTP infl uents Enough resolution to Nikolai et al. MeOH/0.1% (effl uent); and effl uents quantify ( 2006 ) TEAA in water, 17–110 ngL−1 b -blockers at ng/L acetic acid (pH 4) (infl uent) levels in aqueous samples (continued) Table 1.3 (continued) Enantioselective Elution Chiral stationary Matrix Drug method mode phase Mobile phase Detection limit application Observations Refs b -blockers (atenolol, HPLC/UV Normal-phase Chiralcel OD Hexane/EtOH or n.a. Biological Rapid and effi cient Singh et al. betaxolol, 2-propanol/DEA samples and enantioseparation ( 2006 ) metoprolol, and, or acetic pharmaceu- which provide the nadolol) acid tical simultaneous formulations determination of the b -blockers b -blockers and profens HPLC/UV Reverse-phase Chirobiotic V or V2 MeOH/0.1–1.0% n.a Screening study Chirobiotic V had Bosáková triethylamine higher retention et al. acetate buffer and enantioresolu- (2005 ) tion for the majority of analytes b -blockers and profens HPLC/UV Polar-organic Chirobiotic V or V2 MeOH/0.005-0.1% n.a Screening study Polar-organic (Bosáková phase acetic acid/0.005- separation mode et al. 0.1% TEA showed better 2005 ) enantioseparation b -blockers HPLC/UV Reverse-phase Synthesized resin (60:40, V/V) ACN/ n.a Screening study New stationary phase Agrawal and m-[(+)-a -methyl sodium based on a chiral Patel benzyl acetate-acetic selector attached (2005 ) carboxamide] acid buffer (pH to polymeric XAD-4 4.1) support achieved a better separation with shorter retention times Bupivacaine HPLC/UV Normal-phase Kromasil CHI-TBB (98:2:0.3:0.05, V/V) n.a To develop The column has a Da Silva hexane/2-propa- continuous great enantioreso- Júnior nol/acetic acid/ chromato- lution for et al. TEA graphic bupivacaine ( 2005 ) SMB unit Enantioselective Elution Chiral stationary Matrix Drug method mode phase Mobile phase Detection limit application Observations Refs Fluoxetine and HPLC/UV Reverse-phase Chiralcel OD-R (65:35, V/V) 10 ng mL −1 (LQ) Human plasma A simple, accurate Gatti et al. norfl uoxetine potassium and precise ( 2003 ) hexafl uorophos- method of phate 100 mM/ separation of four ACN (pH 3.0) enantiomers in less than 25 min 53 compounds used in HPLC/ Normal-phase Chiralcel AD, OD, (15:85:0.1, V/V) n.a Screening study The four columns Andersson pharmaceutical UV-DAD AS and OJ EtOH or showed et al. industry 2-propanol/ enantioselectivity ( 2003 ) isohexane/TFA for 85% of the or DEA (for tested compounds. acidic or basic The additive was and neutral DEA for basic compounds, compounds and respectively) TFA for acidic Polar-organic (50:50, V/V) MeOH compounds phase EtOH (for basic and neutral compounds) 53 compounds used in HPLC/ Polar-organic Chirobiotic R, V (100:0.02:0.01, V/V) n.a Screening study The three columns Andersson pharmaceutical UV-DAD phase and T MeOH/acetic showed et al. industry acid/TEA (for enantioselectivity ( 2003 ) basic and acidic for 65% of the compounds) tested compounds Reverse-phase (25:75, V/V) MeOH/ TEA (0.1%, pH 6) (for neutral and acidic compounds) (continued) Table 1.3 (continued) Enantioselective Elution Chiral stationary Matrix Drug method mode phase Mobile phase Detection limit application Observations Refs 8 b -blockers HPLC/UV and Reverse-phase Kromasil C8 Gradient of 0.03% 0.7– 1.3 nM corneal The gradient HPLC Ranta et al. FD TFA in water and permeability associated to UV ( 2002 ) 0.03% TFA in studies and FD detection (50:50, V/V) allowed the water/ACN determination of all b -blockers 36 pharmaceuticals HPLC/ Normal-phase Chiralcel AD, OD n-hexane/2-propanol n.a Screening study The three columns Perrin et al. UV-DAD and OJ or n-hexane/ showed (2002b ) EtOH complementary and enantioselectivity for the tested compounds Fluvastatin LC/FD Reverse-phase Chiralcel OD-R (40:60, V/V) ACN/ 1 nM (LQ) Blood plasma The aim was develop Toreson and phosphate buffer an on-line Eriksson pH 2.5 automated method (1997 ) to determinate fl uvastatin and its enantiomers Note that these methods are widely used in pharmaceutical, biomedical and environmental applications ACN acetonitrile, EtOH ethanol, MeOH methanol, DEA diethylamine, TEA triethylamine, TFA trifl uoracetic acid, LQ limit of quantifi cation , WWTP Waste Water Treatment Plants, n.a . not applicable 1 Environmental Fate of Chiral Pharmaceuticals: Determination, Degradation... 23 selectivity because of the wide range of mobile and stationary phases available. As CE it has high peak effi ciency without the necessity for pump systems to provide high pression (Sancho and Minguillón 2009 ) . So, it makes this an easy technique with a short time of analyses and a low consumption of sample and electrolyte, providing a high potential in the pharmaceutical, biomedical and environmental analysis (Li et al. 2010 ) . Enantiomers can be separated within a wall coated open tubular capillary, being the chiral selectors bound to the capillary wall. In the case of packed capillaries, they are obtained by packing chiral selectors in a gel. Otherwise, packed capillaries can be obtained by polymerization in situ providing monolithic phases that can be classifi ed in two major categories, the silica-based and the organic-polimer-based (polyacrylate, polyacrylamide or polystyrene) (Scriba 2003 ; Gübitz and Schmid 2004 ) . Alternatively, the chiral selectors are directly added into the separation electrolyte. The chiral selectors used in Capillary Electrophoresis include crown ethers, cyclodextrins, polysaccharides, proteins, and macrocyclic glycopeptides antibiotics, the same selectors used in HPLC as reviewed by Scriba (2003).

1.3.3.2 Other Techniques

There are other chromatographic related techniques that are used in a less extent. Micellar Electrokinetic Capillary Chromatography (MEKC) is a technique which consists in the addition of a surfactant molecule (above its critical micellar concentration) in Capillary Electrophoresis, leading to the formation of a micellar pseudo- stationary phase in the solution. Thus, the chromatographic principle is present. The surfactant forms micelle occurring partition mechanisms between the micellar pseudo-stationary phase and the mobile phase. Enantioresolution can be achieved using chiral surfactants or using chiral agents that are added to an achiral micellar buffer, such as combination of micelles and cyclodextrins or modifi ed cyclodextrins. To improve the enantioresolution it can be also combined chiral surfactant and cyclodextrins. A recent approach is the use of polymeric surfactants, a group of high-molecular-mass molecules. Despite the advantages of the so called molecular micelles, such as lower concentrations that can be used, more stability and rigidity, the latter allows slower mass transfer of the analytes between the pseudo-stationary phase and the mobile phase, leading to a lower resolution compared to the conventional micelles (Ha et al. 2006). Super Critical Fluid Chromatography (SFC) is a hybrid technique of GC and LC and combines the better characteristics of each. Because of the equal density, dis- solving capacity, the intermediate viscosity and diffusion coeffi cient of its mobile phase, it has the advantages of providing higher fl ow rates and faster separation than LC and a lower dispersion. The mobile phase is compared with those used in GC and HPLC due to the ability to carry compounds and to dissolve them, respectively. Open tubular columns can be used with Flame Ionisation Detection (FIA), Electron Capture, nitrogen phosphorus, and sulphur chemiluminescence. Otherwise packed columns are usually coupled to an UV detector (Taylor 2009 ) . This technique is an 24 A.R. Ribeiro et al. alternative to HPLC when the enantioresolution is partial by normal phase or is not achieved by reverse phase, providing the lower consumption of solvents and the use of non-toxic and non-explosive solvents. However, there are some limitations like high cost of the equipment, complexity of the hardware and the weak experience in this technique (Maftouh et al. 2005 ) . Thin Layer Chromatography (TLC) is preferentially used in indirect mode but it is limited because of its low resolution and weak power to detect low concentrations like those found in the environment. TLC can be used as complementary to HPLC since it is less expensive and allows the optimization of the separation parameters in an inferior time and with less costs (Bhushan and Martens 1997 ) , so the application on environmental analysis is very limited.

1.3.4 Electrochemical Sensors and Biosensors

Electrochemical Sensors and Biosensors provide an enantioselective analysis using an electrochemical cell coupled to a chiral receptor (Potentiometric Enantioselective Membrane Electrodes, PEME), an enzyme (Chiral Amperometric Biosensors) or an antibody (Enantioselective Immunosensors), in which the sample ﬂ ows without separation steps. The principle of PEME is based on the thermodynamics of the reaction between each enantiomer and the chiral selector, with different stability in the complexes formed with each enantiomer, leading to different reaction energies (Izake 2007 ) . These techniques have advantages like direct determination in the matrix, high precision, fastness and the possibility of on-line detection with Flow Injection Analysis and Sequential Injection Analysis systems (Izake 2007 ) . It was developed a method for detection of (S )- and (R )-captopril using two amperometric biosensors based on L- and D-amino acid oxidase with an on-line SIA system (Stefan et al. 2000a ) . The same researchers developed a PEME based on maltodex- trin for the assay of (S) -captopril and an amperometric biosensor for the assay of (R )-captopril with an on-line SIA system (Stefan et al. 2000b ) . Both of them provide more than 30 assays per hour.

1.4 Biotic and Abiotic Degradation and Removal Processes of Chiral Pharmaceuticals in the Environment

Removal of pharmaceuticals in surface waters can be due to photo-transformation, biodegradation, hydrolysis and partition to sediment (Liu et al. 2009 ) . Pharmaceu- ticals are generally highly polar compounds since they are produced in a way which promotes their transport and excretion in the organism. Thus, their removal in Waste Water Treatment Plants (WWTP) is mostly restricted to biodegradation and to abiotic processes as oxidation and sorption. Air-stripping and photo-transformation are also abiotic processes but normally are insigniﬁ cant in the removal http://www.springer.com/series/11480