Applications of LC-MS to quantitation and evaluation of the environmental fate of chiral drugs and...

Trends Trends in Analytical Chemistry, Vol. 27, No. 10, 2008

Applications of LC-MSto quantitation and evaluationof the environmental fate of chiraldrugs and their metabolitesSandra Perez, Damia Barcelo

This review provides a brief overview on chirality as a structural characteristic of pharmaceuticals. This property has received

very little attention in the field of environmental analysis, despite the fact that many drugs are marketed as racemates. The review

covers the different methodologies commercially available on chiral liquid chromatography (LC) columns to provide good

resolving power. It also reviews recent results obtained in chiral analysis of drugs and their metabolites with an array of modern

mass-spectrometric analyzers coupled to an LC system that provides high selectivity and sensitivity. It further covers the

occurrence and the fate of chiral drugs in the environment.

ª 2008 Elsevier Ltd. All rights reserved.

Keywords: Chirality; Chiral drug; Emerging pollutant; Environmental analysis; Environmental fate; LC-MS2; Mass spectrometric analyzer; Metabolite;

Pharmaceutical degradation; Source of contamination

Sandra Perez, Damia Barcelo*

Department of Environmental

Chemistry, IDAEA-CSIC c/ Jordi

Girona, 18-26, 08034

Barcelona, Spain

Damia Barcelo*

Department of Environmental

Chemistry, IDAEA-CSIC c/ Jordi

Girona, 18-26, 08034

Barcelona, Spain

Catalan Institute for Water

Research (ICRA), Parc Cientıfic i

Tecnologic de la Universitat de

Girona, Edifici Jaume

Casademont, Porta A, Planta 1 -

Despatx 13C/ Pic de Peguera,

15E-17003 Girona, Spain

*Corresponding author.

Tel.: +34 934 006 100x435;

Fax: +34 932 045 904;

E-mail: [email protected]

836

1. Introduction

A number of environmental pollutants ofanthropogenic origin are chiral (i.e. theyexist in stereoisomeric forms that are non-superimposable on their mirror images,the so-called enantiomers). Althoughenantiomers have identical chemico-physical properties (with the exception oftheir interaction with polarized light), theyusually differ in their biological propertiesbecause of their stereoselective interactionwith enzymes, receptors or other naturallyoccurring molecules. Biological effects(e.g., toxicity, mutagenicity, carcinoge-nicity and endocrine-disrupting activity)are generally stereoselective, as are bio-transformation processes of enantiomerstaking place in living organisms and theenvironment [1].

There have been a vast number ofstudies on the fate of organic micropollu-tants in the environment published, but,in the large majority of cases, the role ofchirality in their behavior was not takeninto account, so knowledge of their

0165-9936/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2008.09.003

environmental whereabouts remainsincomplete. It is only recently that chiral-ity of environmental contaminants hasbeen addressed, with most efforts beingdedicated to investigating persistent or-ganic pollutants (POPs) [e.g., PCBs andDDT derivatives, pesticides (phenoxy-acidherbicides and organophosphorous insec-ticides) and plasticizers (phthalates)] [2].

However, little is known about theenvironmental fate of emerging contami-nants that are chiral (but are not coveredby existing regulations of water qualityand have not been studied previously).Emerging contaminants are considered topose a potential threat to environmentalecosystems and human health and safety.Specifically, this category of compoundsincludes pharmaceuticals, drugs of abuse,personal-care products, steroids and hor-mones, surfactants, perfluorinated com-pounds, flame retardants, and industrialand gasoline additives, as well as theirtransformation products [3]. The watersolubility of some of these synthetic or-ganic compounds makes them mobile in

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Trends in Analytical Chemistry, Vol. 27, No. 10, 2008 Trends

the aquatic environment, so they are potential candi-dates for infiltrating into the ground, and then firstreaching groundwater supplies and eventually drinkingwater.

With respect to pharmaceuticals, the main concernrelates to their continual entry into water bodies aspollutants originating from use of drugs in farming,aquaculture and human health, in addition to improperdisposal of expired medication. Although their biologicaleffects are well characterized in organisms, whose healththey are designed to improve, due to their inherentbiological activity, the exposure of living organisms inenvironmental systems to drug residues can lead to ad-verse effects [4].

Many chiral drugs were marketed as racemates foreconomic reasons [5], while newly developed drugs aremarketed as single enantiomers for their pharmacoki-netic properties, patent protection and side effects of theundesirable enantiomer (this kind of development beingknown as a racemic switch). Currently, approximately50% of marketed drugs are chiral, and of these approx-imately 50% are mixtures of enantiomers rather thansingle enantiomers.

The enantiomers of a racemic drug differ in pharma-codynamics and/or pharmacokinetics. Since the US Foodand Drug Administration (FDA) issued guidelines for thedevelopment of stereoisomeric drugs in 1992 [6], bio-logical and pharmaceutical sciences have focused muchon the chiral nature of drug molecules. Numerous ana-lytical techniques have been applied, among them liquidchromatography (LC), which in recent years has becomeone of the main separation techniques for chiral drugs.Mass spectrometry (MS) now plays an important role innumerous enantiomeric determinations of pharmaceu-ticals and their metabolites, especially tandem MS (MS2)that provides a fast, sensitive and selective data. MS2

spectra can also give structural information aboutmetabolites.

In the human body, drugs can be transformed into oneor more metabolites that, for the sake of excretability,generally exhibit higher polarity than the parent com-pound. The study of the metabolism of chiral drugs inthe human body is of great importance because of theirstereoselective interaction with optically-active macro-molecules [2]. Once these compounds are excreted fromthe human body via urine or feces and then dischargedinto sewage, further enantioselective biotransformationcan take place in wastewater-treatment plants (WWTPs)and in the environment.

This review summarizes the present implications of thechiral nature of the pharmaceuticals for the human bodyand the environment. It also briefly covers advances inLC-MS for analyzing chiral pharmaceuticals and theirmetabolites in biological samples. We review the use ofan unusual polar organic phase mode for high-perfor-mance LC (HPLC) as well as normal phase with the

atmospheric pressure photoionization (APPI) interfacefor the determination of drugs and their metabolites inclinical studies. We also report on selected applications ofchiral separation for the determination of the occur-rence, fate and transformation of chiral drugs in theenvironment.

2. Advances in LC-MS for enantioselective analysisof drugs

2.1. Separation techniquesEnantioselective gas chromatography (GC) with deriva-tization and capillary electrophoresis (CE) have beenused extensively in the enantioselective analysis of drugs[7]. Fewer applications of enantioselective HPLC havebeen published because of the difficulty of performingenantiomeric separations of drugs and their metabolitesin the past.

Since advances in enantiomeric LC separation in1980s, the situation has changed significantly with theavailability of commercial chiral stationary phases(CSPs). The enantiomeric separation always impliesinteraction with a pure chiral compound in reversed-phase or normal-phase mode in chromatography, andcan be divided into two major types of work: indirect anddirect.

In the indirect chromatographic method, a chiralderivatizing reagent converts the two enantiomers in amixture of diastereomeric derivatives differing in physi-cochemical properties and separated on that principle.Although such indirect methods have been used exten-sively in the past, the direct methods are the methods ofchoice because of their simplicity.

In the direct resolution of enantiomers, the separa-tion is provided by an optically-active moiety, termed achiral selector, and it may be incorporated into thestationary phase, or be present as a mobile-phaseadditive, in which case an achiral stationary phase canbe used. However, the trend is to use CSPs becausemany additives are costly, the derivatization can betime consuming and the detection mode may limit thechoice of additives [8].

Many CSPs have been developed, but only a fewdominate the market (e.g., polymer-based, Pirkle type,protein bonded and macrocyclic based). Although Pirkle-type CSPs are more selective and well characterized,polysaccharide derivatives (one of the polymer-basedclasses) are currently the most popular chiral selectorsfor enantioseparation of various compounds due to theirversatility, durability and loading capacity [9]. They areeffective under not only normal-phase conditions, butalso reversed-phase conditions using the appropriatemobile phases [10].

Protein-bonded CSPs have become popular due to thecharacter of the chiral selector that can be changed by a

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simple change in the mobile-phase composition (e.g., thenature and the concentration of uncharged modifier orpH), allowing a wide range of enantiomers to be sepa-rated [11,12]. However, they are not very efficient andgenerally give broad sample peaks with fewer than 3500theoretical plates [13]. The more common protein-bonded phases include bovine serum albumin, humanserum albumin, a1-acid glycoprotein, ovomucoid, anda-chymotripsin.

The macrocyclic-based CSPs (primarly vancomycin,teicoplanin and ristocetin A) are commonly used forchiral separations in HPLC. Whereas the macrocyclicglycopeptide and aromatic-derivatized cyclodextrins arehighly effective in the normal-phase mode, some linearderivatized carbohydrate CSPs have been conditioned towork in reversed-phase mode. Most of the chiral recog-nition elements incorporated into the CSPs are non-target specific in nature, making reliable prediction ofthe separability and order of elution of a pair ofenantiomers unfeasible.

Molecular imprinted polymers (MIPs) offer the oppor-tunity to modify CSPs with predefined chiral recognitionproperties by using the analytes of interest as binding-site-forming templates [14]. However, the chromato-graphic use of MIP-type CSPs has been hampered by thedifficulties associated with engineering suitable chro-matographic formats and the inherent mass-transfercharacteristics of imprinted polymers.

The field of chiral chromatography is constantlydeveloping with truly new technologies entering themarket to bring solutions for enantiomeric separation[15]. However, the use of nanotechnology to structurechiral cavities has disappeared [16], but the use of zir-conia as a versatile substrate for CSP development hasemerged [7].

2.2. Detection systemsThe major applications of enantioselective HPLC analysisof drugs and their metabolites are in the field of drugdevelopment, since knowledge of the pharmacokinetic,absorption, distribution, metabolism and excretioncharacteristics of each enantiomeric pharmaceutical isessential in that field. Two decades ago, most drugs witha chiral center were commercialized as racemates. Al-though chirality is a critical factor in in vivo systems, itwas often ignored due to the difficulty of separating anddetecting the enantiomers.

In the past, stereoisomer HPLC analysis was performedusing two main detectors: UV and fluorescence detection(FLD) using derivatization agents. Among the differentpossible detection techniques that can be coupled to LC,MS has become popular for the analysis of pharmaceu-tical compounds due to its sensitivity, speed and speci-ficity. The development of atmospheric pressureionization (API) interfaces [e.g., electrospray ionization(ESI) and atmospheric pressure chemical ionization

838 http://www.elsevier.com/locate/trac

(APCI)] contributed to the increasing utilization of massdetection.

Enantiomers can be separated using different LCtechniques: normal phase, polar organic solvent andreversed phase. Analytical LC-MS methods for theanalysis of drugs and their metabolites are currentlybased on the combination of reversed-phase HPLC withESI or APCI MS. However, in chiral analysis, the use ofnormal phase is often required and ESI and APCI arehardly applicable for this kind of separation [17]. ESI andAPCI are incompatible with normal-phase conditionswhen flammable mobile solvents (mixtures of alkanesand alcohols) are used at a high flow rate, due to con-cern about the explosion hazards associated with APCI(corona discharge) and ESI (high voltage discharge)[18].

There have been three approaches taken to overcomethis problem:(1) lower the APCI-probe temperature at the expense

of sensitivity [10];(2) add a high content of aqueous polar organic make-

up-flow by post-column addition to reduce alkanes;or,

(3) use an APPI interface.If the chiral column permits work with the reversed-

phase mode, the three interfaces can be used. Usually,ESI requires the use of volatile mobile-phase modifiers(e.g., formic acid, ammonium formate or ammoniumtrifluoroacetate) to enhance sensitivity. However, theseadditives introduce a memory effect to the CSPs, which isattributed to some undefined alteration in the tertiarystructure of the polymer, and shortens the life of thechiral column, thereby adversely affecting the enantio-separation [19]. This problem can be avoided by usingpolar organic solvent chromatography (POSC) [20].

Although the advanced LC-MS techniques can obtainlow-noise chromatograms, an adequate sample-prepa-ration step is crucial in stereospecific analysis in order toobtain cleaner extracts that are less likely to causematrix effects [21]. In addition, labeled internal stan-dards can have a beneficial effect, especially with respectto signal reproducibility. As enantiomers have the samemass spectra, chromatographic baseline separation isessential in order to obtain reliable results.

3. Enantioselective LC-MS analysis of drugs andtheir metabolites

3.1. Stereoselectivity of enantiomers in humansAfter drugs are administered, the body changes them toform metabolites. Depending on the pharmaceutical,metabolites can make up a large fraction in the massbalance of an administered drug upon excretion. How-ever, some drugs are not metabolized much and aretherefore excreted largely unchanged.


Enantiomers of chiral drugs generally differ in theirpharmacological properties (the most renowned examplebeing thalidomide, whose S-enantiomer proved terato-genic). Many physiological processes show a high degree ofstereoselectivity, including the metabolism of xenobiotics.Drug-metabolizing enzymes may therefore discriminatebetween the enantiomers. In addition, prochiral drugsmay give rise to chiral metabolites. Some biotransforma-tion pathways in the human body are regioselective andenantioselective, so the two enantiomers of a drug canhave distinct efficacy and toxicity profiles. One enantiomermay influence desirable physiological, pharmacological,pharmacodynamic and pharmacokinetic properties, whilethe other often behave very differently, with differentinteractions with living organisms as well as differentactivities in chemical and biotechnological processes. Thestereospecific determination of chiral drugs and theirmetabolites is therefore important.

Separation and detection of enantiomers of chiraldrugs is currently performed with LC-MS in clinicalstudies, and there was a considerable increase in thenumber of published papers in 2007 dealing with thedetermination of chiral drugs and their metabolites inhuman samples. Table 1 shows some examples of ste-reospecific analysis of drugs and their metabolites inhuman samples using LC-MS.

3.1.1. Bupropion. A sensitive, stereoselective methodwas used for the determination of bupropion, a chiraldrug marketed as a racemic mixture and used clinicallyas anti-depressant, and its major metabolites (R,R)-hydroxybupropion and (S,S)-hydroxybupropion in hu-man plasma and urine samples. It used a protein-basedcolumn, a1-acid glycoprotein, for the enantiomericseparation and (+ESI)LC-MS2 [22]. Table 1 shows thedetails of the LC-MS2 method.

Validation of the method was subject to a number ofdifferent considerations: accuracy; stability of thesamples in the matrix; stability of the enantiomers inurine samples after the hydrolysis of the glucuronicconjugates; racemization; recovery; ion suppression; andcarry-over in the MS instrument. Accuracy was >98%for all analytes and all concentrations. Hydrolysis of thehydroxybupropion conjugates with b-glucuronidase at37�C and 60�C during 2 h or 24 h was studied todetermine the conditions for maximum hydrolysis andthe minimum racemization of the analytes. Hydrox-ybupropion concentrations in urine tripled with hydro-lysis at 37�C for 2 h; at 60�C, its concentration did notincrease, but this temperature caused substantial race-mization of hydroxybupropion and bupropion(bupropion racemization occurred after only 2 h at37�C) [22].

Potential racemization of the analytes was expressedin terms of enantiomeric excess (ee). When urine andplasma samples were stored at –20�C, ee was stable for 7

days. However, at 10�C, the analytes began to racemizewithin 24 h.

Recovery was >70% in plasma and >55% in urinesamples due to the hydrolysis of the glucuronide conju-gates.

The signal loss due to ion suppression was enantio-selective and negligible for all analytes except for (S)-bupropion in plasma, where the response was 75% ofthat without matrix effect and was corrected with deu-terated internal standards [22].

Carry-over was less than 0.01 ng/L for bupropionenantiomers and 0.8 ng/L for the hydroxylated enanti-omers.

The chromatographic resolution for the analytes was3.0 for bupropion and 7.3 for hydroxybupropion, andthe predominant enantiomers in both urine and plasmawere (R)-bupropion and (R)-hydroxybupropion.

3.1.2. Citalopram. The enantioselective analysis ofcitalopram, a chiral drug, and its metabolite, demethyl-citalopram, in human and rat plasma used chiral(+ESI)LC-MS2 [23]. Citalopram is an anti-depressant,available as a racemic mixture or an (S)-pure enantio-mer. The authors compared the resolution and the sen-sitivity using different chiral columns, namely ChiralcelOD-R and OD-H, Chiralpack, Chirex, a1-acid glycopro-tein and Chirobiotic V.

Direct separation of citalopram and its metabolite wereobtained on Chiralcel OD-R and Chirobiotic columns V.However, the sensitivity of ion monitoring with the MS2

detector was better for Chiralcel than for Chirobiotic V.The resolution for the cellulose tris(3,5-dimethylphe-

nylcarbamate), Chiralcel OD-R column, was 0.88 forcitalopram and 1.0 for demethylcitalopram (Table 1).

Other parameters that the authors considered in thepublication were limits of quantification (LOQs) thatwere 0.1 ng/mL for the two analytes, 50 times lowerthan the LOQs of 5 ng/mL presented by Kosel et al. [24]in the study citalopram and its demethylated metabolitesin plasma with a stereoselective method using a Chiro-biotic column and LC-DAD.

Moreover, the method developed [23] was precise andaccurate showing coefficients of variation of less than15% and recoveries of 70% for both enantiomers.

Evaluation of the stability of the samples after threefreeze-thaw cycles with the Student t-test revealed nodegradation of the samples after 12 h.

With this method, citalopram was detected in humanand rat plasma samples in a higher proportion of the(R)-citalopram compared to (S)-citalopram. Although(S)-demethylcitalopram was in a higher proportion inhumans than (R)-demethylcitalopram, in rats, thecontrary obtained [i.e. (R) > (S)] [23].

3.1.3. Ifosfamide. A sensitive, specific (+ESI)LC-MSmethod was developed and validated for the


Table 1. Stereospecific analysis of pharmaceuticals and their metabolites in human and animal samples by LC-MS

Compound/metabolites HPLC conditions: column and mobile phase Resolution API interface and MS mode Matrix/Predominant enantiomers Ref.

Bupropion (BUP) a1-acid glycoprotein bBUP:3.0 (+)ESI Human plasma and urine [22]

Hydroxybupropion (OH-BUP) 20 mM ammonium formate (pH 5.7): methanol,

linear gradient; T: 28�C (aRP)

OH-BUP: 7.3 MS2 (API 4000 QTRAP) (R)-BUP> (R)-BUP

(R,R)-OH-BUP> (S,S)-OH-BUP

Citalopram (CITA) Chiralcel OD-R CITA: 0.88 (+)ESI Human and rat plasma [23]

Demethylcitalopram (DCITA) Acetonitrile:methanol:water (30:30:40 v/v/v)+0.05

diethylamine, isocratic; T: 25�C (RP)

DCITA: 1.0 MS2 (QqQ) (R)-CITA> (R)-CITA

(R)-DCITA> (R)-DCITA(Human)

(R)-DCITA> (R)-DCITA (Human)

Ifosfamide (IF) Chirabiotic T IF: 1.20 (+)ESI Human plasma [25]

2-N-dechloroethylifosfamide (2-DCl-IF) 2-propanol:methanol (60:40 v/v), isocratic; (cPOSC) 2-N-IF: 1.17 MS (R)-IF> (R)-IF

3-N-dechloroethylifosfamide 3-N-IF: 1.20 (R)-2-DCl-I= (R)-2-DCl-IF

(3-DCl-IF) (R)-3-DCl-I> (R)-3-DCl-IF

Ketamine (Ket) a1-acid glycoprotein Ket: 1.17 (+)ESI Human plasma [27]

Norketamine (Norket) 10 mM ammonium acetate (pH 7.6): 2-propanol

(6:94), isocratic; T: 25�C (RP)

Norket: 1.58 MS (R)-Ket P (R)-Ket

(R)-Norket P (R)-Norket

Methadone (Met) a1-acid glycoprotein (AGP) 10 mM ammonium

acetate (pH 7.0):acetonitrile; (RP)

Met: 1.30 (+)ESI Human plasma [29,30]dEDDP EDDP: 1.17 MS (R)-Met P (R)-Met

(R)-EDDP> (R)-EDDP

Mirtazapine (MRT) Chiralpak AD-RH MRT: 2.0 (+)ESI Human plasma [33]

8-hydroxymirtazapine (8-OHM) acetonitrile-methanol-ethanol + 0.2% diethylamine

(98:1:1, v/v/v); T: 23�C (POSC)

8-OHM: 1.11 MS2 (QqQ) (R)-MRT= (R)-MRT

Demethylmirtazapine (DMR) DMR: 4.53 (R)-8-OHM= (R)-8-OHM

(R)-DMR= (R)-DMR

Omeprazole (OME) ReproSil Chiral-CA gnr (+)eAPPI Human plasma [35]

5-hydroxyomeprazole (HOME) 2-propanol-acetic acid-diethylamine (100:4:1, v/v/v):

hexane; T: 20�C (fNP)

MS2 (QqQ) (R)-OME P (R)-OME

Omeprazole sulfone (OMES) (R)-OME> (R)-OME

Propafenone (PPF) a1-acid glycoprotein PPF: 2.4 (+)ESI Human plasma [40]

5-hydroxypropafenone (5-OHP) 10 mM ammonium acetate (pH 5.96): 1-propanol

(100:9, v/v); 10 mM ammonium acetate (pH 4.1): 2-

propanol (100:0.9, v/v); T: 20�C (RP)

5-OHP: 2.3 MS2 (Ion trap LCQ) (R)-PPF> (R)-PPF

(R)-5-OHP= (R)- 5-OHP

Phenprocoumon (PPC) Chira Grom nr (+)ESI (R)-PPC P (R)-PPC [41]

4 0-hydroxyphenprocoumon (4 0-OH-PPC) 5 mM ammonium acetate (pH 3.9): methanol; (RP) (Baseline separation) MS2 (QqQ)

6-hydroxyphenprocoumon (6-OH-PPC)

7-hydroxyphenprocoumon (7-OH-PPC)

Tramadol (TRA)o Chiralpak AD nr (+)ESI (R)-TRA> (R)-TRA [42]

O-desmethyltramadol (ODT) Isohexane:ethanol:diethyl-amine (97:6:0.2, v/v) ; T:

25�C (RP)

MS2 (QqQ) (R)-ODT P (R)-ODT

aRP, Reverse phase; bResolution = 1.18 (tb-ta)/(W1/2a+W1/2b); cPOSC, Polar organic solvent chromatography; dEDDP, 2-ethylene-1,5-dimethyl-3,3-diphenyl-pyrrolidine; eAPPI, Atmosphericpressure photoionization; fNP, Normal phase; gnr, Not reported.

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enantioselective determination of ifosfamide, a chiralmolecule, which is used as an anticancer agent andadministrated as racemic mixture, and its two metab-olites (2-N-dechloroethylifosfamide and 3-N-dechloroe-thylifosfamide) [25]. The chromatographic separation ofthe enantiomers used a macrocyclic-based CSP column,Chirabiotic, containing macrocyclic antibiotic teicopla-nin (Table 1).

The enantioselective separations were achieved usinga mobile phase comprising 2-propanol:methanol (60:40v/v). The use of POSC allowed enantioselective separa-tion of (R,S)-3-N-dechloroethylifosfamide.

The same group previously reported an enatioselectiveLC-MS method using Chirabiotic T as a chiral column[26]. Using reversed-phase mode and ESI or APCI, (R,S)-3-N-dechloroethylifosfamide was not enantioselectivelyresolved under the chromatographic conditions used inthis study [25].

For ifosfamide, 2-N-dechloroethylifosfamide and 3-N-dechloroethylifosfamide, the resolutions calculated usingPOSC were 1.20, 1.17 and 1.20, respectively. The lowerlimit of detection (LOD) was 5.0 ng/mL, the inter-dayand intra-day precisions were in the ranges 3.63–15.5%RSD and 10.1–14.3% RSD, respectively, and the accu-racy in the range 89.2–101.5% of the nominal values.

3.1.4. Ketamine. Ketamine, an analgesic agent and achiral molecule commercially available as a racemicmixture, undergoes extensive metabolism to norketa-mine involving N-demethylation [27]. A sensitiveenantioselective methodology using a CSP based uponimmobilized a1-acid glycoprotein and (+ESI)LC-MS wasdeveloped and validated. Buffer concentration, the typeand concentration of organic modifiers and the pH of themobile phase usually affect enantioselective separations[28]. For enantioseparation in a1-acid glycoprotein ofketamine and its metabolite, the type of buffer, organicmodifier and buffer pH were optimized as follows: 10 mMof ammonium acetate; 2-propanol (6:94, v/v); and, pH7.6 [27].

The resolution calculated as a was 1.17 and 1.58 forthe parent compound and its metabolites. It is worthnoting that a small change in the organic modifier from10% of 2-propanol to 6% altered the retention times ofthe analytes and the resolution between the enantio-mers. The intra-day and inter-day RSDs were less than8.0% and the lower LOQs were 1 ng/L for both com-pounds versus the previously reported lower LOQs of5 ng/ml for ketamine and 10 ng/ml for norketamineusing LC-DAD [27].

This validated method was applied to the analysis ofhuman plasma of 60 patients, obtaining levels of con-centration of (S)-ketamine, (R)-ketamine, (S)-norketa-mine and (R)-norketamine of 27.1 ng/mL, 28.9 ng/mL,3.4 ng/mL and 2.5 ng/mL, respectively.

3.1.5. Methadone. The enantiomers of methadone werealso separated on a1-acid glycoprotein and detected inLC-(+ESI)–MS [29,30]. Methadone is a synthetic opiatethat is metabolized in the human body to 2-ethylene-1,5-dimethyl-3,3-diphenyl-pyrrolidine. This assay hadgreater sensitivity (LOQ for the metabolite was 0.1 ng/mL per enantiomer and the LOD was 0.1 ng/mL perenantiomer) than the previously reported methods usingLC-DAD (LOQ for the metabolite was 8 ng/mL [31] or35 ng/mL [32] per enantiomer).

3.1.6. Mirtazapine. Mirtazapine is an antidepressantdrug that is available as a racemic mixture. In humans,mirtazapine is extensively metabolized to 8-hydroxy-mirtazapine and demethylmirtazapine (DMR). Themethod for the stereoselective determination of mirt-azapine and its metabolites in plasma with LC-MS2 wasvalidated [33].

The separation of the target analytes was optimized intwo columns, Chiralpak AD and Chiralpak AD-RH,under polar organic phase conditions using acetonitrile-methanol-ethanol (98:1:1, v/v/v), because previousresults obtained by the same group had shown poorresolution of these compounds under normal-phase orreversed-phase conditions.

Although the use of diethylamine as an additive in themobile phase is discouraged when working in +ESI, itwas added to the mobile phase in order to reduce theinteraction of the basic drugs with the silanol groups ofthe silica support. With the previous conditions, theresolution for the analytes was more than 1.11 (SeeTable 1) and the LOQs were 1.25 ng/mL for all analytes.

In a previous study from the same authors [34], usingChiralpack AD and LC-DAD, the LOQs were higher thanthose reported, which were in the range 6.25–625 ng/ml [33].

3.1.7. Omeprazole. A validated method was used for thesensitive determination of omeprazole, an anti-ulcerdrug, and two of its metabolites, the chiral 5-hydroxy-omeprazole and the achiral omeprazole sulfone, using(+APPI)LC-MS2 [35]. For enantioresolution, a ReproSilChiral-CA column in normal-phase mode was chosen.The separation of the omeprazole enantiomers waschallenging, and prior work on the stereospecific sepa-ration of omeprazole and 5-hydroxyomeprazole sug-gested that they could be separated on a stationaryphase designed for carboxylic acids in normal-phasemode [36]. The authors used normal-phase mode withhexane and alcohols in the mobile phase. The combi-nation of alcohols and hexane is normally incompatiblewith ESI or APCI, due to concerns about potentialexplosion hazards and poor ionizability [18]. To achieveproper ionization in the ion source of the mass spec-trometer and to avoid the explosion hazard of normal-



phase eluent, a post-column make-up liquid containingreverse phase solvents and/or buffers can be added [37].However, the authors [35] applied the APPI mode thatcan overcome the limitations, achieving low LODs(0.2 ng/mL and 1 ng/mL for omeprazole and 5-hydroxyomeprazole, respectively) and low LOQs (5 ng/mL and 2.5 ng/mL for omeprazole and 5-hydroxy-omeprazole, respectively) [31].

By contrast, using a reversed-phase HPLC method forthe stereoselective separation of omeprazole and itsmetabolites, Kanazawa et al. [38] reported LOQs of50 ng/mL.

3.1.8. Propafenone. Two different mobile phases [10mM ammonium acetate buffer (pH5.96):1-propanol(100:9,v/v) and 10 mM ammonium acetate buffer(pH4.1):2-propanol (100:0.9,v/v)] in reversed-phasemode had to be used for the enantioseparation of pro-pafenone, an antiarrhythmic agent, used clinically as aracemic mixture, and its metabolite, 5-hydroxypropafe-none [39]. For the separation and detection, an a1-acidglycoprotein and (+ESI)LC-MS2 were used to achieveLOQs of 20 ng/mL for the entire analytical method. Theresolution for propafenone was 2.4 and for its metabolite2.3.

They also determined by indirect method after enzy-matic hydrolysis, the glucuronides and sulfate conju-gates.

3.1.9. Phenprocoumon. A stereoselective method deter-mined phenprocoumon, a vitamin K antagonist, and itsmetabolites based on (+ESI)LC-MS2 [40]. Two newmetabolites using chiral column Chira Grom weredetermined.

3.1.10. Tramadol. A synthetic analogue of codeine,tramadol, and its metabolite, O-desmethyltramadol, inhuman plasma were determined with a simple, sensitiveand selective (+APCI)LC-MS2 method [41]. The authorsused a Chiralpack AD column in normal-phase modecoupled to the APCI interface with the APCI-vaporizertemperature set at 250�C. Because APCI ionization occursin the gas phase, a lower probe temperature may result inincomplete desolvation and vaporization of column efflu-ents, therefore giving rise to lower sensitivity. Despite thelow temperature, the LOQ was 0.5 ng/mL.

The authors reported that they could use APCI inter-face safely because a relief valve protected the ionizationsource against overpressure, and, when a small amountof polar modifier combined with the apolar majority ofthe mobile phase, ignition of an explosion was highlyunlikely [41].

3.2. Stereoselectivity in the environmentAfter their excretion, human drugs and their metab-olites reach WWTPs. The biological treatment of


sewage in WWTPs plays a crucial role in reducing theload of pharmaceuticals and their human metabolitesbefore they are finally discharged into the aquaticenvironment.

As in the human body, stereoselective processes canalso govern microbial degradation of chiral compoundsin WWTPs [42]. Overall, the enantiomeric ratio of chiraldrugs may have been altered after they leave the humanbody and before they end up in the aquatic environment.

Once in the aquatic environment, chiral drugs andtheir metabolites can be transported and distributed inrivers and streams and be subject to further degradationby both abiotic and biotic processes. Whereas in theabiotic processes the fate of enantiomers is generally notaffected by the stereochemistry, biological processes maydiscriminate between enantiomers of chiral compoundsand result in the enrichment of one of the enantiomerscompared with the other.

Recent monitoring studies demonstrated that, byemploying enantioselective analysis, differences in ster-eoselective biodegradation rates in WWTPs could beexploited in terms of distinguishing drug (metabolite)contamination in the environment originating fromdischarge of untreated sewage compared to that releasedfrom WWTPs [42]. After sewage treatment, the chiralpharmaceutical compounds used in human medicinecan enter the aquatic environment as a mixture ofenantiomers. In the environment, one form can be ac-tively degraded by microorganisms and the other formcan be more resistant to degradation. Early research inenantioselective degradation of pesticides indicated thatthe environmental behavior of chiral compounds is notstraightforward, so it is not always possible to predictenantiospecific transformations [43]. Microbial popula-tions in the environmental matrices can change, andeven reverse, the enantiomeric ratios. However, some-times the abiotic degradation processes are sufficientlyrapid for both enantiomers, so that enantioselectivedegradation is not so important [3].

Chiral analysis can give information about theoccurrence, the fate and the transformation of drugs andtheir metabolites in the environment. Since enantiomersof a chiral compound can interact differentially withother chiral molecules in the environment (e.g., enzymesin living systems) and may have different biological andtoxicological effects, chirality must be taken into accountfor understanding their environmental fates and theirrisk assessments [44].

There have been few studies published on the enan-tioselective analysis of drugs in the environment. In part,this is due to the limited separation power of chiral HPLCcolumns that commonly yield broad peaks, which, withcomplex environmental samples, are susceptible to co-eluting interferences.

Only two methods for stereoisomer quantification ofchiral b-blockers in environmental matrices using LC-MS


have been reported (Table 2). However, no metabolitedetermination was reported.

A stereospecific method was used for the determina-tion of the b-blockers, atenolol, metoprolol and propan-olol, in influents and effluents from WWTP using aChirobiotic V vancomycin-based chiral column and LC-MS2 [44]. The resolutions calculated for atenolol,metoprolol and propanolol were 0.8, 0.7 and 0.9,respectively (Fig. 1).

Using the same column, but with a mobile-phase pH of5.0, better resolution for atenolol and propanolol couldbe obtained (1.1 and 1.5, respectively) [45].

For method validation, LODs (sum of enantiomers)and recoveries were determined. LODs of target analyteswere in the ranges 3–17 ng/L (effluent samples) and 17–110 ng/L (influent samples). Analyte recoveries fromspiked wastewater were in the range 67–106% ineffluent and influent samples.

The authors also calculated the enantiomer fractions(EFs) of all three analytes [EF = E1/(E1 + E2)] in influentand effluent samples from two Canadian WWTPs [44].In one WWTP, the EFs were close to 0.5 in the influentsamples for three target analytes (Table 2).

However, the EFs for atenolol and propanolol in theeffluent samples from the same WWTPs were non-racemic in August and racemic for atenolol andmetropolol in November, indicating that stereoselectiveprocesses in the WWTPs had altered the enantiomercomposition and apparently varied between seasons.

In the second WWTP, metropolol was stereoselectivelyeliminated to give a racemate, showing that the

Table 2. Stereospecific analysis of pharmaceuticals in environmental sam

Compound

(metabolites)

HPLC conditions:

column and mobile

phase

Resolution

Atenolol (ATL),

metoprolol (MTL),

propanolol (PRL)

Chirobiotic V(90:10)

Methanol/water +

0.1%

triethylammonium

acetic acid (pH 4); T:

nr, (aRP)

aATL: 0.8MTL: 0.7PRL:

0.9

Atenolol (ATL),

metoprolol

(MTL),nadolol (NDL),

pindolol (PNL),

propanolol (PRL), sotalol

(STL), citalopram

(CITA),fluoxetine

(FLUX)salbutamol (SBL)

Chirobiotic V(90:10)

Methanol/water +

20 mM ammonium

acetate (pH 4); T: nr,

(aRP)

ATL: 1.15MTL:

1.10NDL: 0.70PNL:

0.99PRL: 1.32STL:

1.34CITA:FLUX:SBL:

aResolution = (tb�ta)/0.5x(Wa+Wb).

enantioselectivity within biological wastewater treat-ment was reversed compared to the first WWTP [44].

The resolution of the target analytes reported in theprevious study was improved with a modified mobilephase comprising methanol/water (90:10) and 20 mMammonium acetate (pH 4) [46] (Table 2). The develop-ment of a reversed-phase enantioselective method usingChirobiotic V and LC-MS2 was described for the deter-mination of six b-blockers (atenolol, metoprolol, nadolol,pindolol, propanolol and sotalol) two selective serotoninre-uptake inhibitors (citalopram and fluoxetine) and b2-antagonist salbutamol in influents and effluents fromWWTPs [46]. Apart from getting better resolution, thenew mobile phase improved the LODs to 0.2–7.5 ng/Lfrom 2–17 ng/L. It is known that the mobile phase thatcontains triethylammonium acetate suppresses ESI inpositive-ion mode. The authors had enantiomeric purestandards for only three compounds: atenolol, propan-olol and fluoxetine. The EFs for the remaining analyteswere calculated on basis of enrichment of the first-elutedenantiomer. Propanolol was racemic in the influent, butenriched in (S)-propanolol in effluent. Metoprolol wasracemic in both influent and effluent and atenolol ininfluent was enriched in (R)-atenolol while it was race-mic in the effluent [46].

Enantioselective analysis is a dynamic field that canbring new information about the dominant source ofpollution in the environment as well as estimation of thenatural attenuation in a big system like a river. Threepapers have been published using chiral GC-MS forenvironmental fate of pharmaceuticals, but no other

ples by LC-MS

API interface Matrix/ Predominant enantiomers Ref.

(+)ESIMS2(API 4000

QTRAP)

Wastewater [44]

Influent WWTP1(R)-ATL = (S)-

ATL(R)-MTL P (S)-MTL(R)-PRL =

(S)-PRL

Effluent WWTP1(R)-ATL P (S)-

ATL(R)-MTL = (S)-MTL(R)-PRL >

(S)-PRL

Influent WWTP2(R)-ATL > (S)-

ATL(R)-MTL < (S)-MTL

Effluent WWTP2(R)-ATL = (S)-

ATL(R)-MTL = (S)-MTL(R)-PRL >

(S)-PRL

(+)ESIMS2(API 4000

QTRAP)

Wastewater [46]

Influent WWTP(R)-ATL > (S)-

ATL(R)-MTL = (S)-MTL(R)-PRL =

(S)-PRL(R)-FLUX > > (S)-FLUX

Effluent WWTP1(R)-ATL = (S)-

ATL(R)-MTL = (S)-MTL(R)-PRL <

(S)-PRL(R)-FLUX > (S)-FLUX


Figure 1. HPLC-ESI-QqQ-MS chromatogram corresponding to the separation of the racemates of atenolol, metoprolol and propanolol on aChirobiotic V column (Adapted from [44]).


papers have been published using LC-MS. We thereforeinclude these GC-MS papers because the studies are ofgreat interest for the review.

3.2.1. Distinguishing the source of contamination in theenvironment. This can be done with a chiral pharma-ceutical (e.g., propanol) that has been proposed as atracer of untreated sewage [47]. When an overflowoccurs in the WWTP, untreated wastewater is a sourceof wastewater-derived contaminants in surface watersand is often ignored because of the difficulty of discrim-inating it from wastewater influent [47].

Observing the changes of the enantiomeric ratios ofpropanolol, Fono et al. could distinguish the contami-nation of surface waters from raw-sewage contaminantsor treated-sewage contaminants. The method includedderivatization to convert the two enantiomers ofpropanolol to diastereomers and separating them inGC-MS2. Propanolol is prescribed as a chiral racemicdrug. In raw sewage, propanolol exists in the racemicform; however, (R)-propanolol is preferentially degradedin WWTPs. The EF of propanolol decreased from racemicto values significantly below racemic during biologicalwastewater treatment in each of the seven WWTPs thatwere sampled in this study.

The authors also simulated the biodegradability of theenantiomers of propanolol in batch reactors involving


with secondary wastewater and river water. They foundthat biodegradability was enantiospecific in wastewater,but, in the river-water reactor, propanolol did not de-grade, showing racemic EF. These results suggested that,when propanolol is detected in surface waters, an EF of0.5 indicates that its source is raw sewage and an EF of0.42 or less indicates that its source is treated sewage[47].

Another paper assessed the sources of contaminationof chiral pharmaceutical ibuprofen, commonly detectedin WWTPs and surface waters with GC-MS [48]. Theauthors studied the elimination of ibuprofen in WWTPsand the occurrence in surface waters, also evaluatingthe changes of enantiomeric ratios (ERs) in those sam-ples. The ERs, calculated using ER = E1/E2, changedbetween influent and effluent samples from (S/R) being5.5–8 to 0.9–2, indicating that (S)-ibuprofen is some-what faster degraded than (R)-ibuprofen. It has beenshown that, in humans and other mammals, inactiveform (R)-ibuprofen undergoes extensive chiral inversionto (S)-ibuprofen and this confirms the predominantpresence of (S)-ibuprofen in influent samples. In surfacewaters, ERs are up to 2.0, suggesting extensive degra-dation of ibuprofen during lengthy residence in theaquatic environment. A possible exception was acatchment area with long residence time and low pop-ulation density, where the ER was 4.2, which may point


to some input of untreated or insufficiently treatedwastewater [45].

3.2.2. In-stream attenuation of pharmaceuticals in longrivers. To assess the importance of in-stream attenua-tion of pharmaceuticals in long rivers, Fono et al. com-pared the decreases in concentration of fourpharmaceuticals (i.e. gemfibrozil, ibuprofen, metoprololand naproxen), but only for metoprolol were the changesof EF assessed [49]. The concentrations of the fouranalytes was determined with prior derivatization withGC-MS2 and decreased by 75–90% as the water traveleddownstream. Metoprolol, a racemic drug, was employedas tracer for assessing biodegradation.

In previous studies assessing the changes of EF ofmetoprolol in a WWTP, Macleod et al. [46] showed thatno shift in the EF was observed through the WWTP.Assuming that, the average EF for metoprolol wasdetermined in the river, and it decreased with distancedownstream from 0.44 at the first sampling point to0.31 at the last sampling point. As a result, the changein the EF, photolysis and sorption were not the processesresponsible for the attenuating the pharmaceuticals,because abiotic processes should not affect the EF ofmetoprolol. The changes in the EF of metoprolol there-fore provided strong evidence that biodegradation oc-curred in the river [49].

4. Conclusions

The increasing number of papers published on chiralseparation of drugs and their metabolites demonstratesthat enantiomeric separations are of interest toresearchers and pharmaceutical companies. Severalchiral columns have been developed for these pur-poses. In 2007, there were reports on several enan-tioselective methods for the determination of drugsincluding their metabolites in clinical studies withLC-MS.

Important factors have to be taken into account whena stereoselective method is being developed:� choice of a suitable chiral column is crucial for a given

chiral separation problem and is by no means an easytask because achieving enantioresolution is oftenpurely empirical; and,

� choice of an appropriate mobile phase is critical be-cause small differences in organic concentration inthe mobile phase and its pH strongly affect retentiontimes or separation of the enantiomers (resolution).Regarding the detection systems, using DAD, the

selectivity for chiral drugs and their metabolites is notsufficient for clinical and environmental studies. LC-MS2

generally results in more selective and sensitive deter-minations (see LOQs of chiral drugs and their metabolitesin Table 1).

Although excellent methods have been developed forthe stereoselective determination of chiral drugs andtheir metabolites, environmental studies have histori-cally ignored stereospecific analysis of the environmentalpollutants and failed to evaluate which enantiomerspersist in the environment, so stereospecific analysis of-fers new approaches for investigating the occurrenceand the fate of chiral drugs in the environment.

AcknowledgementsThe work presented in this article was supported by theSpanish Ministerio de Educacion y Ciencia, CEMAGUA(CGL2007-64551/HID). This work reflects only the au-thors� views and the European Community is not liablefor any use that may be made of the information con-tained therein.

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