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This article gives an up-to-date commentary on chiral liquid chromatography coupled with mass spectrometry for the determination of pharmacologically active chiral compounds (cPACs) (including illicit drugs) in environmental matrices. Several applications are discussed to demonstrate the benefits of performing environmental analysis of cPACs at the enantiomeric level. Finally, future perspectives in this rapidly developing field of research are outlined.
Determination of pharmacologically active chiral compounds (cPACs) at the enantiomeric level in environmental matrices is essential. Such an approach yields vital information for improved wastewater-based epidemiology, development of more accurate environmental risk assessment, and improved understanding of cPAC fate in wastewaters and the environment. This article gives an up-to-date commentary on chiral liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) to determine cPACs, including illicit drugs, in environmental matrices. Several applications are presented to demonstrate the benefits of performing environmental analysis of cPACs at the enantiomeric level. Finally, future perspectives in this rapidly developing field of research are outlined.
Stereochemistry plays an important role in the life of plants and animals, but it is also vital in the agricultural, pharmaceutical, and chemical industries. Stereoisomers are compounds that differ only in the spatial arrangement of their constituent atoms or groups. They can be classified into two groups: enantiomers and diastereomers. Enantiomers are stereoisomers which are non-superimposable mirror images of one another and are described as chiral isomers (Figure 1). The fundamental difference between enantiomers and diastereomers is that enantiomers of the same chiral molecule, as opposed to diastereomers, have identical intramolecular distances between non-bonded atoms, the same energy content, and therefore the same physico-chemical properties. As a result they are very difficult to separate. Because of this difficulty, chiral pharmacologically active compounds (cPACs) are often sold as a 50:50 mixture of the two possible enantiomers (a so-called "racemic mixture" or "racemate"). However, it is well known that biological activity is often associated with only one enantiomer.
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Chiral analysis relies on the separation of the analyte of interest in a chiral environment (for example, a chiral derivatizing agent or a chiral stationary phase), which enables differentiation of enantiomers through the formation of distinguishable diastereomers via multipoint interactions (1). The chiral recognition mechanism is usually explained by the "three point interaction model" (Figure 2). In this a minimum of three simultaneous interactions between the chiral selector and at least one of the enantiomers, with at least one of these interactions being stereochemically dependent, is required (3). The physico-chemical properties of these diastereomers differ, enabling their separation using indirect or direct methods. In the indirect approach, the enantiomers are derivatized with an enantiomerically pure reagent followed by their separation in an achiral environment. In the direct approach, the enantiomer interacts with a chiral compound that can be either bound to an immobile support or as a mobile-phase additive.
Despite the importance of chiral analysis in the environment, most studies do not explicitly account for individual stereoisomers. This leads to inaccurate conclusions because they incorrectly assume that enantiomers have identical environmental behaviour (4). For example, toxic effects of fluoxetine and propranolol to aquatic biota are enantiomer dependent (5,6).
Figure 1: Structure of ketoprofen enantiomers.
The enantiomeric fraction (EF) of a cPAC is the relative concentration of each enantiomer and a value of 0.5 denotes a racemic mixture. The EF can change significantly following intake by human metabolism and excretion, during biological wastewater treatment, and when present in the aquatic environment. Biological processes may discriminate between enantiomers of cPACs, which can lead to stereoselective enrichment or depletion of one enantiomer. Enantiomers can also undergo chiral inversion to form an enantiomer of potentially higher toxicity (7).
Figure 2: The "three point interaction model". Adapted and reproduced with permission from Trends in Environmental Analytical Chemistry 1, Sian E. Evans and Barbara Kasprzyk-Hordern, Applications of Chiral Chromatography Coupled with Mass Spectrometry in the Analysis of Chiral Pharmaceuticals in the Environment, e34–e51 (2014) © Elsevier.
Currently, the direct analysis approach is the method of choice for enantioseparations. The most important selectors are chiral stationary phases (CSPs) based on cyclodextrins, polysaccharides, macrocyclic antibiotics, synthetic chiral macrocycles (crown ethers, other synthetic macrocycles), chiral synthetic polymers, chiral imprinted polymers, protein-based CSPs, and ligand- or ion-exchange CSPs (8,9). CSPs based on polysaccharides, proteins, and macrocyclic antibiotics are the most used in enantioselective liquid chromatography tandem mass spectrometry (LC–MS–MS) separation methods for environmental analysis. Despite the large number of available CSPs, it is not possible to find a universal stationary phase for a range of different cPACs. This is a result of the different mechanisms involved and the high selectivity of chiral selectors required to achieve chiral recognition. For polysaccharide-based CSPs (made by various derivatives of cellulose and amylose molecules), H-bonding, π-π, dipole, and steric interactions are the main mechanisms involved, while for protein-based (α-acid glycoprotein, cellobiohydrolase, or albumin) and antibiotic-based (vancomycin, teicoplanin, or ristotecin A) CSPs, H-bonding, hydrophobic and ionic interactions, and inclusion complexes are the main interactions that determine chiral separation (Figure 3).
Figure 3: (a) Potential binding sites and mechanisms in teicoplanin and (b) cellulose tris(3,5-dimethylphenylcarbamate). Adapted and reproduced with permission from Trends in Environmental Analytical Chemistry 1, Sian E. Evans and Barbara Kasprzyk-Hordern, Applications of Chiral Chromatography Coupled with Mass Spectrometry in the Analysis of Chiral Pharmaceuticals in the Environment, e34–e51 (2014) © Elsevier.
Chiral analysis can provide useful information about the occurrence, fate, and transformation of cPACs and their metabolites in the environment because enantiomers of the same cPAC can interact in different ways when exposed to a chiral environment, such as in biological systems (enzymes, proteins). There are four main areas where chiral analysis of PACs in the environment is studied:
Fate of cPACs during Wastewater Treatment: Following consumption by the human population, cPACs reach wastewater treatment plants (WWTPs) in a modified form (for example, as metabolites) or with their EF altered. During wastewater treatment they are subjected to biotic processes that lead to further changes in enantiomeric composition. This stereoselective degradation needs attention because the discharged effluent might be enriched with one of the enantiomers and current risk assessment will not provide a realistic view. To date, there are relatively few studies that have reported enantiomeric concentrations of cPACs in influent and effluent wastewaters and in sewage sludge (7,12–18).
Wastewater-Based Epidemiology: Wastewater-based epidemiology (WBE) is a new approach to estimate the consumption of illicit drugs by a community by determining their concentration in influent wastewater (19). The incorporation of enantiomer-specific analysis in this approach can help address current difficulties such as: distinguishing between legal and non-legal use of drugs; verification of the method of synthesis of illicit drugs; route of administration; identification of whether drug residue results from consumption of an illicit drug or metabolism of other drugs; and monitoring trends in drug abuse (20).
cPACs as Chemical Markers of Water Contamination with Wastewater: A small number of studies have revealed the potential for cPACs to be used as effective indicators of human sewage contamination in water courses, differentiating between sources of untreated effluents and treated effluents discharged from WWTPs. As Evans et al. (21) stated, such a marker must be consistent in samples where wastewater is found and must consistently and significantly change its EF during its residency in WWTPs. This must be in a way that is not characteristic with attenuation in the environment and propranolol was identified as a possible marker (12).
Environmental Risk Assessment and Fate in the Environment: Stereoselectivity requires attention because current environmental risk assessment does not account for individual enantiomers and, considering that enantiomeric specific toxicity can occur, the risk posed may be underestimated. Once present in the environment, changes in the EF could be used to differentiate biotransformation from other removal processes. Unlike abiotic mechanisms (that is, sorption, photochemical transformation, air-water, and soil-water exchange), which are believed to be the same for both enantiomers, biotic mechanisms can be enantioselective. However, the absence of enantioselectivity does not explicitly mean achiral degradation because not all biotic processes show preference for the transformation of one enantiomer over the other.
Sampling Strategy: Although often overlooked, sampling is a critical aspect of any environmental monitoring procedure. An excellent critical evaluation of different sampling modes has been conducted by Ort et al. (22). The collection of grab/spot samples has obvious limitations because it can only give information for a specific point in time. Findings therefore may not be representative as cPAC concentration and wastewater or river flow can vary throughout the day. Alternatively, automated samplers are used to collect a composite sample that can be either time or volume proportional over a 24 h period. Volume proportional sampling is considered to be the most representative mode of sampling because it accounts for variations in flow. However, the stability of cPACs in composite samplers is unknown for a variety of matrices. Significant degradation (>15%) has been observed for some PACs in wastewater over a 12 h period despite storage at 4 °C (23). This is even more important for chiral analysis because stereoselective degradation occurs for several cPACs (24). Consequently, EF may change substantially from the time of sample collection to sample processing. To overcome this, biological activity can be inhibited by acidification or the addition of sodium azide to the collection bottles within the samplers. This approach has yet to be validated for chiral analysis.
Sample Preparation: Sample preparation procedures usually applied in the analysis of cPACs are achiral (that is, they should not show any stereoselectivity towards chiral analytes). The preparation of environmental matrices (typically 50 mL to 500 mL) for aqueous phase analysis is often conducted offline and involves centrifugation and/or vacuum filtration through glass fibre filters, solid-phase extraction (SPE), elution and evaporation in a suitable solvent, reconstitution in mobile phase, and filtration through pre-LC–MS filter membranes. Such a large number of steps during sample preparation increase the likelihood of incorporating error into the final measurement (25). Therefore the use of deuterated surrogates is essential to correct for such errors and ensure accuracy of the final measurement. Despite the use of deuterated surrogates, recovery of target PACs must be maximized because very low method quantitation limits are required for environmental monitoring. The loss of PACs to glassware surfaces (specifically during evaporation of SPE extracts) can impact analyte recovery significantly. For example, using silanized SPE vials gave recoveries six times higher than non-silanized vials for some PACs (25). Even with this knowledge, silanized glassware is often not used in reported analytical methods.
Coupling Chiral Chromatography with Mass Spectrometry: Mobile phase composition is essential for chiral chromatography, not only for achieving enantiomeric separation while using chiral stationary phases, but also to enable compatibility with mass spectrometry (MS) (21). Coupling chiral chromatography to MS is crucial for environmental analysis where complex heterogeneous matrices are encountered. The high selectivity and sensitivity of MS detection enables the quantification of cPACs present at low levels (ng to µg/L) in environmental matrices. However, the majority of established analytical methods for chiral analysis use UV detection, often for quality control purposes within the pharmaceutical industry. These mobile phases often contain non-volatile buffers or additives not compatible with MS. There is therefore a need to develop more chiral methods that can be coupled to MS for environmental analysis. Furthermore, there is a lack of chiral ultrahigh-performance liquid chromatography (UHPLC) columns available. Most chiral columns used in environmental analysis are only capable of high performance liquid chromatography (HPLC) performance and are limited to maximum operating pressures of 2000 psi. The introduction of chiral UHPLC columns would enable improved separations in shorter analysis times; this is currently the rate limiting step of current analytical methods.
A further issue of coupling chiral chromatography or any LC system with MS for environmental analysis is matrix suppression, particularly with electrospray ionization (ESI) (26). The majority of methods in the literature report the use of polymeric-based SPE cartridges for sample preparation because of their suitability for PACs which possess a wide range of chemistries (21). However, this type of sorbent is non-selective and unwanted materials are co-extracted with target chemicals. This can result in >50% signal suppression of analyte signal strength in some cases (7). Furthermore, matrix suppression is known to vary between enantiomers of the same cPAC, reinforcing the importance of using racemic mixtures of deuterated surrogates in analytical methods. López-Serna et al. (27) reported that several compounds (propranolol, metoprolol, and timolol) are subject to stereoselective matrix effects. To demonstrate, EF for propranolol-d7 was 0.50 in a standard solution, 0.58 in surface water, and 0.66 in wastewater influent.
Most enantiomeric profiling of cPACs in environmental matrices has focused on beta-blockers, antidepressants and ephedrines. Such methods tend to use columns containing vancomycin or cellobiohydrolase stationary phases as chiral selectors. Lopez-Serna et al. (27) successfully separated 14 cPACs including metabolites at the enantiomer level. These belonged to several therapeutic classes including beta-blockers, antidepressants and anti-inflammatories. This was achieved using a column containing a vancomycin-based stationary phase (250 mm × 2.1 mm, 5-µm) operated under isocratic conditions at a flow rate of 0.1 mL/min. The mobile phase consisted of 4 mM ammonium acetate in methanol containing 0.005% formic acid. The total run time was 66 min. The addition of ammonium acetate as a mobile phase buffer was essential because it improves ionization but, more importantly, it increased the resolution of enantiomers during LC separation (27). A column containing a vancomycin-based stationary phase is versatile as it can also be used in either normal- or reversed-phase modes. On the other hand, a column with a cellobiohydrolase stationary phase is more restrictive, only enabling operation in reversed-phase mode. Nevertheless, it has successfully resolved enantiomers of amphetamines, ephedrines, some antidepressants, and beta-blockers in environmental matrices.
Beta-blockers are ubiquitous in wastewaters and they are the best studied of the cPACs at the enantiomeric level. Many studies have found atenolol to be enriched with the S(–)-enantiomer in raw wastewater (18,28,32,34). However, a recent study by Vazquez-Roig et al. (32) found a racemic EF that coincided with a higher estimated weekly population usage (1.3 g d-1 1000-1 inhabitants vs. 1 g d-1 1000-1 inhabitants), suggesting the higher concentration may be from disposal of the drug, rather than human usage and metabolism. Stereoselective biodegradation of atenolol has been observed in some WWTPs (13,18,32). It is hypothesized that the degree of atenolol stereoselectivity during wastewater treatment is dependent on the season (34) and WWTP technology (32,34).
Other beta-blockers including metoprolol (12,13,34), propranolol (13,27,29,34), and albuterol (27) behave similarly and undergo enantioselective biodegradation. The antidepressant fluoxetine is also found to exhibit steroeselectivity. In wastewater effluents fluoxetine has been found to be enriched with S(+)-fluoxetine (29). This is a significant observation as S(+)-fluoxetine is more toxic than the corresponding enantiomer to Pimephales promelas (5,6). Interestingly, the stimulant ephedrine has two chiral centres and therefore occurs as natural, 1R,2S(–)-ephedrine and 1S,2S(+)-pseudoephedrine, and synthetic, 1S,2R(+)-ephedrine and 1R,2R(–)-pseudoephedrine. Only the natural diastereomers are usually found in raw wastewater (18,32). During the winter months concentrations of the whole drug were higher and enriched with 1S,2S(+)-pseudoephedrine. This is likely a result of higher usage of over-the-counter medications that contain this enantiomer for the treatment of colds.
Many illicit drugs are chiral. Since human pharmacokinetics show stereoselectivity in many chiral xenobiotics, differences in potency and activity are also found in illicit drugs. For example, R-(–)-MDMA has a more hallucinogenic effect than the S-(+)-form (21), while (+)-LSD is 20 times more psychoactive than (–)-LSD (20). Despite this knowledge and possible differences in ecological impact between enantiomers of the same drug, there are a very limited number of illicit drugs analyzed at the enantiomeric level in environmental matrices.
Figure 4: Extracted influent wastewater showing cPACs separated using a chiral column (METH: methamphetamine; AMPH: amphetamine; MDMA: 3,4-methylenedioxymethamphetamine; MDA: 3,4-Methylenedioxyamphetamine). Adapted and reproduced with permission from Environmental Science & Technology 46, Barbara Kasprzyk-Hordern and David S. Baker, Enantiomeric Profiling of Chiral Drugs in Wastewater and Receiving Waters, 1681–1691 (2012) © American Chemical Society.
The enantiomeric analysis of amphetamine-like compounds in wastewater has been performed previously using a column with a cellobiohydrolase stationary phase (100 mm × 2 mm, 5-µm) operated under isocratic conditions with a mobile phase consisting of 1 mM ammonium acetate/2-propanol 9:1 (Figure 4) (7). Enantioselective separation was achieved by using a pH of 5. This enables ionic bindings between positively charged analytes and the chiral selector (isoelectric point = 3.9).
Consequently, a higher retention for these analytes was observed in response to an increased degree of net charge of the chiral selector. A comparison between a column with a cellobiohydrolase stationary phase and a column containing a vancomycin-based stationary phase (the two most commonly used chiral columns for environmental analysis) for the analysis of several illicit drugs was performed by Bagnall et al. (29). Greater resolution was achieved with a column with a cellobiohydrolase stationary phase than a column containing a vancomycin-based stationary phase (i.e., Rs MDMA CBH 1.9 > Rs MDMA CBV 1.0) because of combinations of ion exchange, hydrogen bonding, and hydrophobic interactions. These methods operate at flow rates of ≤0.1 mL/min and run times of ≥1 h to achieve adequate enantiomeric resolution.
Studies have shown enantioselective degradation of MDMA leading to the enrichment with the R-(–)-enantiomer during wastewater treatment in the UK (23) and Spain (32). The extent of enantioselective degradation was found to vary between different types of chiral drugs and was dependant on the wastewater treatment technology used and the time of year (18). Enantiomer profiling of some illicit drugs and metabolites (MDMA, MDA, amphetamine and methamphetamine) has also been applied in WBE. All illicit synthesis methods produce racemic MDMA (EF = 0.5). However, S-(+)-MDMA undergoes preferential metabolism over R-(–)-MDMA.
This leads to the enrichment of residual MDMA excreted in urine (and then found in wastewater) with the R-(–)-enantiomer (EF>0.5). This characteristic change in EF of MDMA allows the verification of whether drug residues present in wastewater results from its consumption (EF>0.5) or direct disposal of unused drug (EF = 0.5). For example, the results of several sampling campaigns in England (35) and in the Netherlands (37) showed that MDMA is usually present in wastewater as a result of direct consumption (EF>0.5; MDMA enriched with R-(–)-enantiomer). However, excessively high mass loads of MDMA during one sampling campaign in a WWTP in Utrecht (the Netherlands) proved to be racemic, indicating direct disposal of unused MDMA. This coincided with a police raid earlier at a nearby illegal production facility within the catchment area (37).
Chiral chromatography coupled with MS is an emerging tool in environmental analysis of cPACs. Enantiomeric profiling of cPACs in environmental matrices could aid risk assessment to help determine the fate and toxicity of these chiral pollutants. A selection of contemporary applications to detect a range of important cPACs in environmental matrices can be obtained from reference (21).
Unfortunately, because of gaps in knowledge and the lack of robust analytical protocols for the analysis of chiral compounds at the enantiomeric level, false conclusions might be drawn from environmental data. One example concerns enantiomer-dependent signal suppression in the ESI–MS interface, which can lead to false measurements of individual enantiomers if undertaken in the absence of labelled analogues of target analytes.
There are several aspects of stereoselective environmental analysis using chiral LC coupled with MS that require immediate attention. There is an urgent need to: (i) introduce robust protocols for sampling and adequate sample preparation approaches limiting stereoselective microbial degradation and enantiomer-dependent enrichment of analytes; (ii) introduce new chiral stationary materials, which could be applied in UHPLC applications with the aim of increasing the speed of analysis and column efficiency while maintaining high enantioselectivity, as well as increasing the capability to resolve as wide a spectrum of analytes as possible; and (iii) ensure high precision and accuracy of measurements at the enantiomeric level by using labelled analogues of target analytes to limit analytical errors leading to changes in chiral recognition (for example, stereoselective signal suppression in the ESI interface). In the future we will see significant advances in environmental MS in terms of both sensitivity and selectivity. It is important to realize that without tackling the issue of stereochemistry and the effects of chiral pollutants, false, inaccurate, or misleading conclusions will still be drawn from the environmental data.
This work was supported by the UK Engineering and Physical Sciences Research Council [grant number EP/I038608/1 and EP/K503897/1/], the UK Natural Environment Research Council [grant number NE/I000534/1], and the European Union's Seventh Framework Programme for research, technological development, and demonstration [grant agreement 317205, the SEWPROF MC ITN project, 'A new paradigm in drug use and human health risk assessment: Sewage profiling at the community level'] and [grant agreement 629015, the MC IEF project 'Chiral veterinary medicines in the environment']. The support from Wessex Water is also greatly appreciated.
Bruce Petrie is a research officer at the University of Bath. He was awarded his PhD in water engineering at Cranfield University. Dr Petrie moved to Bath in 2013 to work on the major interdisciplinary programme co-funded by Wessex Water and the University of Bath's EPSRC Impact Acceleration Account, titled Holistic Approaches to Sustainable Water Supply.
Maria Dolores Camacho Muñoz has been a Marie Curie Postdoctoral IEF Fellow at the University of Bath since 2014. Originally from Spain she completed her PhD in chemistry at the University of Seville (Spain) in 2013. She also obtained a MSc in advanced studies in chemistry (University of Seville) and an MA in quality management systems (IMF Business School, Spain).
Erika Castrignanò is a Marie Curie Early Stage Researcher (Department of Chemistry, University of Bath, UK). She has a MSc in pharmaceutical chemistry (La Sapienza, Rome, Italy). She has also graduated from the National School on Addiction and the National School of Analytical Methodologies in Mass Spectrometry.
Sian Evans is an EPSRC and University of Bath funded PhD student (Department of Chemistry, University of Bath, UK). She has a MSc in toxicology (Birmingham University, UK). She has also graduated from Plymouth University with a BSc (Hons) in environmental Sciences.
Barbara Kasprzyk-Hordern is a reader in environmental and analytical chemistry in the Department of Chemistry at the University of Bath. She received her PhD in chemistry from Adam Mickiewicz University in 2004. Having completed her PhD she has worked as a lecturer at the Faculty of Chemistry at Adam Mickiewicz University, a Marie Curie Transfer of Knowledge Research Fellow in analytical environmental toxicology at the University of Glamorgan, and a lecturer/senior lecturer in analytical science at the University of Huddersfield and at the University of Bath.
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