Lab-Based Approaches To Support the Screening and Identification of

Chapter 5

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Lab-Based Approaches To Support the Screening and Identification of Transformation Products by LC-HRMS Bettina Seiwert, Cindy Weidauer, Kristin Hirte, and Thorsten Reemtsma* Department of Analytical Chemistry, Helmholtz Centre for Environmental Research - UFZ, Permoserstrasse 15, 04318 Leipzig, Germany *E-mail: [email protected].

The screening for transformation products (TPs) of contaminants in complex environmental samples is a difficult task. Lab-based systems that simulate environmental transformations are helpful for both the detection and the identification of TPs formed or occurring in the environment. Oxidation, reduction, hydrolysis, photolysis and conjugation may be performed in lab experiments and the product mixtures analyzed by LC-HRMS with all-ion fragmentation or data-dependent MS/MS experiments. The full mass spectrometric data are stored. It is not necessarily recommended to elucidate all TPs of the lab-based approaches, but to focus on those peaks that occur also in the environmental sample. This approach allows detection and tentative identification of TPs in the environmental sample and to ascribe it to the right parent compound. As elevated concentrations may be used in the lab experiment more meaningful fragment spectra are produced, which facilitates the structure elucidation.

Introduction The detection of transformation products (TPs) of contaminants in environmental samples, their assignment to parent compounds and their identification is cumbersome. Targeted approaches by multimethod that focus on TPs as for pesticide TPs are rare due to the limited number of available © 2016 American Chemical Society Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


standards of these TPs (1). For pharmaceuticals that often enter the environment by wastewater, standards of the main TPs are often commercially available, but for pesticides and industrial chemicals this it is not necessarily the case. Thus suspect screening and non-target screening are the only possible techniques to detect their TPs. However as stated by Zedda et al. only a limited number of compounds have been identified or tentatively identified so far in environmental samples by non-target screening (2). Non-target screening approaches are often used to compare different treatments, environmental samples of different regions or time points by multivariate statistics. This approach is powerful and works well to show that there are differences and it may lead to potential targets of interest (3). Generally, however, even the selection of peaks of interest by non-target approaches is time consuming and it depends on the ionization efficiency of the compound of interest and the matrix effects of the respective sample whether a transformation product is detected with a sufficient intensity and thus selected or overlooked. Adduct formation sometimes prevents the elucidation of molecular formulas. The identification of TPs, however, in such a real world sample remains cumbersome. Without sufficient a priori information (such as the possible parent compound of a TP or the reaction conditions that causes its transformation) structure elucidation often turns out to be impossible. High resolution mass spectrometry provides information on the exact molecular mass and, supported by the isotopic pattern, information on the presence of certain elements. The preferred ionization mode and the likelihood of adduct formation together with the fragmentation pattern supply information on the absence or presence of functional groups. But if there are not enough characteristic fragment ions such efforts do not lead to the final proposal of a molecular structure. Additionally, structural isomers may not be distinguishable. For example carbamazepine epoxide and hydroxy-carbamazepine (both carbamazepine + O (m/z 253.0977)) lead to the same fragment ions (m/z 236.0712 (C15H10NO2); m/z 210.0919 (C14H12NO)) as these are stable ions. Even more worrying the TP trans-dihydroxy-dihydro-carbamazepine experiences an in-source loss of water that leads to a primary ion of the same mass as carbamazepine plus O rather than to a molecular ion. Only a careful search for adducts with sodium or potassium will prevent misinterpretation. The relative intensities of fragment ions are not necessarily comparable between mass spectrometers of different vendors. This may limit the benefit of mass spectral libraries like MassBank (4). If the sample amount is limited and the analytes or their corresponding TPs become quite polar so that large volume injection is not possible, then the chance to detect characteristic fragment ions decreases. Mass spectral databases that link parent compounds with known TPs would help. Such information will already guide the user to further TPs and depending on whether or not the set of TPs is detected the tentative identification will be strengthened or put into question. One attempt for such a database in the context of water contaminants is the DAIOS database, which provides links from parent 68

Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


compounds to their expected TPs. However this database stores retention times and selected fragment ions, but no full mass spectra (5, 6). As far as microbial transformation is concerned one possibility is to use software-based approaches (EAWAG-BBD Pathway Prediction System (http://eawag-bbd.ethz.ch/predict/index.html)), which predict possible TPs based on known transformation processes for structural moieties of the parent compound. Another option are mass spectrometry vendor based-approaches like UNIFI from Waters to generate possible molecular formulas and simply screen for their exact masses. But the suspect screening for software predicted TPs usually results in a large number of false positive findings, especially when the control sample is not adequate. The problem of detecting and identifying TPs is especially challenging when substances are investigated that contain only the atoms C, H, N, O. In complex samples like influents or effluents of wastewater treatment plants (WWTPs), mixtures of chemicals and their TPs are present and linking a TP to a certain parent compound is cumbersome due to the possibility of structural isomers. One example is tramadol ([M+H]+, C16H26NO2, m/z 264.1958, MassBank Record: AU111702), that can occur in the effluent of a WWTP, but desvenlafaxine (MassBank Record: EA105303) with the same molecular formula can be present as well. According to the mass spectral library MassBank, a water loss leads to the same fragment ion (m/z 246.1856) for both compounds and there are only minor further fragment ions expected for tramadol and desvenlafaxin (4). In such situations information on the retention time of TPs can be decisive. A direct correlation between and a molecule’s polarity (expressed as log P or log D) and its retention time is not necessarily found. Moreover, the error in predicting log P is usually too large to allow a useful retention time prediction. This is even worse on the basis of log D prediction, as the prediction of the required pKa-values increases the error further (7). One example where this approach failed are structural isomers, as 2- and 3-hydroxy-carbamazepine. They have the same predicted log D value (log D 2.4, pH 7.4, ChemAxon) but elute at very different retention times, whereas the log D value of Oxcarbazepine (log D 1.87, pH 7.4) differs, but this compound nearly coelutes with 3-hydroxy-carbamazepine after separation on a reversed phase column (8, 9). The final confirmation depends on the availability and the agreement of the mass spectrometric data and the retention time with reference compounds. Another option is to verify the structural proposal by NMR spectroscopy like shown by Kaiser et al., but this often requires a preconcentration and clean-up as NMR needs elevated analyte concentrations and a high purity (10).

The Principle of Using Lab-Based Approaches Because of these difficulties in detecting and identifying TPs directly from LC-HRMS data of environmental samples, it can be very useful to combine it with a simulation of transformation reactions in the laboratory. Data exploitation then changes from a non-target screening to a suspect screening. 69 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


The approach is illustrated in Figure 1. The lab experiment leads to a new class of suspects, which we called here smart suspects. Their structure is not confirmed, but by having the same retention time and the same fragment ions as those in the environmental sample the initial information content is higher than for “normal” suspects; the origin (parent) is confirmed and the conditions of formation are known. For “normal” suspects only proposed structures and– depending on its origin (software-based or based on literature data) expected fragment ions are available.

Figure 1. Scheme of the identification approach by lab experiments.

The parent compound is subjected to one or more different transformation reactions and the reaction solution subsequently analyzed by LC-HRMS under standard conditions using all-ion fragmentation or data-dependent fragmentation. One option is to elucidate all formed TPs and to store the information in a libraries of tentatively identified TPs (4). Another option is to perform the tentative identification only for those peaks , that match with peaks in the environmental sample, with respect to retention time, exact mass, associated adducts and isotopic pattern. By that the identification of possible peaks of interest in a real-world sample is already shifted from a non-target or statistical approach to a suspected approach. This last approach is much less time consuming. Information on other TPs is retained in the data set and can be utilized in future screenings for TPs of the respective parent compound. Structure elucidation is then best performed using the fragment ion data of the lab sample, which are usually less noisy, and by searching for possible isomers and comparing their fragmentation and retention times. 70



The lab experiment can be performed at elevated concentration, so that more intense product ion spectra are obtained. Furthermore it is possible to develop and validate enrichment or clean-up methods for the TPs in the sample of interest. This also allows to produce sufficient material of a TP for further elucidation by NMR and for quantification after certain clean up steps. Thus small peaks in real samples without enough information on fragment ions can be elucidated. Additionally by having a mixture of possible TPs of the compound of interest one can optimize the LC-MS method, including the ionization mode and the addition of organic or inorganic modifiers. The influence of different chromatographic conditions on the retention time (pH, organic solvent, modifier, or column) may help in further structure elucidation. If the sample amount is limited lab-based experiments help to generate TPs for further experiments. In one particular example the chromatographic separation at two different pH-values (pH 3 and pH 5) and with different solvent (methanol or acteonitril) helped to elucidate the structure of one of the TPs (11). Fragment ion spectra do not only provide information on the structure of the particular TPs. Rather, characteristic fragment ions found in the spectra may be used for a screening in the whole data set for yet undetected and structurally related TPs. With this approach one additional TP was detected from CBZ (11). This illustrates that laboratory experiments are very useful to support TP detection and identification, even though they may not always provide us with the full set of TPs formed in the environment.

Environmentally Relevant Processes Environmentally relevant processes are reduction, oxidation and hydrolysis, which may happen abiotically or by biotic catalysis, as well as direct and indirect photolysis. Additionally conjugation processes may occur in higher organisms, that may further mask possible TPs. Laboratory methods to simulate these transformations should be fast and performed in a reproducible manner, so that they are largely independent from the laboratory in which they are performed. Figure 2 provides an overview on the environmental transformation processes and suitable lab methods to simulate them. In the environment the above-mentioned processes may not necessarily happen individually. Rather one initial transformation may lead to more reactive species that are then prone to further transformations. One advantage of lab-based methods in general is that it is easy to perform an additional experiment, to prolong the reaction times or to select harsher reaction conditions to generate additional knowledge on the stability of TPs or to form subsequent TPs. Hydrolysis Hydrolysis is one key reaction in the environment as water is a ubiquitous reactant. It often leads to smaller and more polar TPs, which preferably enter the aqueous phase and, if stable, remain in the water cycle. Otherwise this process 71



frequently serves as an initial step to enable consecutive reactions. There are different functional groups that are prone to hydrolysis like carboxylic acid esters and amides, their cyclic analogs lactons and lactams, thioesters, carbamates, ureas and sulfonylureas, thio- and dithiocarbamates, halogenated aliphatic hydrocarbons, epoxides, phosphates, phosphonates as well as thiophosphates, imides and sulfonamides. Hydrolysis itself is mainly affected by pH and temperature, but often only the kinetics are studied and less attention is given to the pH-dependence of the hydrolytic degradation pathways and the hydrolysis products. If only one hydrolysable group is present the hydrolysis product is easily predicted. If more than one such group is present in a parent compound or if there is the possibility of follow-up reactions, e.g. to form cyclic structures that are more stable - these pathways may lead to structurally diverse hydrolysis products that are not predictable by software-based approaches. As was shown by Hirte et al. for amoxicillin, a simple experimental setup with three different buffer systems (pH 3, pH 7 and pH 11) led to a detailed understanding what may happen (12). Amoxicillin (AMX) is a widespread β-lactam-antibiotic and, together with some of its TPs (AMX penicilloic acid, AMX penilloic acid, AMX 2’,5’-diketopiperazine, and 3-(4-hydroxyphenyl)pyrazinol), a known environmental contaminant.

Figure 2. Overview of the possible processes in the environment and lab-based methods to simulate them.

A comparison of lab samples with samples from WWTPs confirmed that AMX penicilloic acid was the major TP in the WWTP influent compared to the other three TPs in the effluent. Additional matches were not detected in the 72 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


effluent and related surface water samples. But the lab-based experiment resulted in additional information on the stability of these detected TPs in the environment. The first three TPs of the above mentioned were not stable but transformed into 23 yet unknown hydrolysis products within three to four weeks. Consequently the large number and slow formation kinetics which will lead to comparatively low environmental concentrations of these later TPs of AMX may explain why they were not detected in the environment so far, even after applying an adapted SPE method to screen for them. This example illustrates the importance of several aspects – the information about possible peaks (with retention times, exact masses and expected fragment ions) that are connected to a compound and additionally about the variability of TPs and their stability that may be investigated by using the lab-based approach. This provides information on whether TPs formed in the lab experiment may be expected to occur in the environment. Oxidation The most important degradation pathways of xenobiotics in the environment, such as biodegradation or photolysis involve redox reactions. Oxidation reactions may also proceed abiotically, catalyzed by metal or metaloxides, for example with Fe(II) or Mn(III/IV) oxide present in suspended particles in surface water or in soils. Electrochemistry (EC) off-line or on-line coupled to mass spectrometry is a well-accepted method to study phase I and phase II metabolism of drugs (13). It has been shown in many publications that EC is a powerful tool as it is able to simulate a large number of transformation reactions, such as the hydroxylation of activated aromatics, benzylic hydroxylation, N-dealkylation of amines, dealkylation of ethers and thioethers, S-and P-oxidation, oxidation of alcohols to aldehydes and dehydration (14). Transformation by EC has been compared to liver microsom for several pharmaceuticals and other substances (15). One advantage of EC transformation is that it proceeds without the addition of any reagents. Therefore, the reaction solutions can be analyzed directly without any need for clean-up, contrary to when liver homogenate was used to simulate biotransformation by higher organisms. Such clean-ups may lead to the loss of TPs, which then remain un-recognized. Often, however, the diversity of TPs formed by EC is larger than that found with microsomes. These surplus TPs can be useful, too, as they may help elucidate other transformation processes taking place in WWTPs or upon ozonation (16). EC is equally helpful for the determination and identification of TPs generated by microbial processes. Correspondingly, EC has been used to support screening for TPs of the recalcitrant pharmaceutical carbamazepine (CBZ) formed by the white-rot fungus Pleurotus ostreatus (16). EC with LC-HRMS facilitates detection and identification of TPs since the TP mixture is not superimposed by biogenic metabolites and elevated substrate concentrations can be used. Ten TPs formed in the microbial process were detected by comparing the data (m/z versus retention time) of the fungally treated CBZ with the EC treated CBZ solution (16). In such cases it is not recommended to elucidate every peak in the electrochemically generated mixture. Rather only those signals have to be studied closely that occur also in the environmental sample under investigation. However 73



the information on additional TPs generated by EC supports the structure elucidation. Furthermore, transformation pathways are best elucidated by applying EC to intermediate TPs and follow their transformation (17). While in biological studies, this requires a complete set of new exposure experiments, which may last for days to weeks, EC oxidation to examine a certain pathway is performed in minutes. In the presented example this was proven for the oxidation of acridine to acridone (11). Instead of EC, other oxidizing agents such as the fenton reaction, KMnO4, ferrate (Fe(VI)) or MnO2 may be used to study the oxidation of anthropogenic substances in the lab (18–21). However, the advantage of the electrochemical cell is its variability in terms of the applied oxidation potential and that it is a pure reagentless method, which may be coupled online with mass spectrometry. As shown by van Leeuwen et al. the observation of short-lived intermediates is possible that may help to elucidate follow-up reactions by understanding the transformation pathways (22). Furthermore by using different redox potentials the product spectra and more important the relative intensities of the TPs are influenced without requiring longer reaction times. Another parameter that may be varied is the working electrode. It is known that a boron-doped-diamond electrode (BDD) and the glassy carbon electrode are leading to different TPs, due to the possibility of the BDD electrode to generate hydroxyl radicals. Reduction Reductive transformation processes can be of importance in sediments as well as in the subsurface. To simulate reductive transformation either catalytic hydrogenation (Pt/H2) or reduction with an electrochemical cell may be used. However, apart from dehalogenation of iodinated analytes and reductive metabolism of nitro aromatic xenobiotics there are no examples in literature, where the electrochemical cell has been used to simulate reductive transformation (23, 24). This may be due to the fact that the reductive potentials (down to -3 V for a BDD electrode and down to -2 V for a glassy carbon electrode, both measured against Pd/H2) are insufficient to dehalogenate chlorinated compounds. Recently the BDD electrode was used to transform brominated flame retardants. Tetrabromobisphenol A (TBBPA) showed a single, a twofold and threefold debromination. However the direct comparison with anaerobic bacteria that yield bisphenol A as a final product failed, because two TPs were missing (see Figure 3) (25). Moreover, a cleavage product (dihydroxydibromo benzene) formed by electrochemical conversion was not formed by the strictly anaerobic bacteria. The catalytic hydrogenation (Pt/H2) is a clean system allowing the identification of TPs formed under strongly reducing conditions and thus shows extreme conditions. A surface reaction takes place, where the H–H-bond of the sorbed H2 molecules is weakened and a syn-addition to C–C double bonds takes place. By this approach König et al. formed a total of nine reduction products of CBZ, where an increasing degree of hydrogenation correlated with an increase 74



in the retention time (26). The presence of two independent reduction pathways was proven by separate experiments with 10,11-dihydro-CBZ ((2H)-CBZ) as a precursor. (2H)-CBZ could not be further reduced, indicating that the stepwise reduction of CBZ to the fully saturated alicyclic (16H)-CBZ proceeds along another pathway. However, in natural samples from bank filtration sites no reduction product except 10,11-dihydro-CBZ ((2H)-CBZ) could be detected so far (26).

Figure 3. Comparison of the mass chromatograms of the reductive dehalogenation of Tetrabromobisphenol A by strictly anaerobic bacteria (bottom) and the products derived by an reduction in a electrochemical cell (top).

Combined Reduction/Oxidation As reductive and oxidative zones may be found in close proximity to each other in the subsurface, it can be of interest to study the combination of reductive and oxidative transformation also in the lab. For example, in our lab a solution of CBZ was reduced to form (2H)-CBZ and subsequently oxidized by the electrochemical cell at a redox potential of 1.5 V (measured against Pd/H2). An oxidative transformation of (2H)-CBZ along a pathway quite different from that known for CBZ was observed. All TPs of (2H)-CBZ had lost the carbamoyl moiety. As a first step hydroxylation of one of the two aromatic rings occurred probably in para-position to the nitrogen, which was proven by the presence of 75 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

the corresponding quinone imine. This quinone imine is suspected to be quite reactive (Michael acceptor), which probably prevents their observation in the environment. In this case the lab experiment did not lead to an identification of TPs found in the environment but provided an explanation why no further reaction products of (2H)-CBZ have yet been found in the environment. It also explains why a removal of dihydro-CBZ is observed in WWTPs but so far no TPs were detected that correlated with this removal.


Photolysis For compounds that are not biodegradable, photolysis is one reaction that may be important to initiate degradation in surface waters. Photolysis reactions are divided into direct and indirect photolysis. In the latter process singulet oxygen (1O2), hydroxyl radicals (.OH), peroxy radicals (.OOR) or photo-excited organic matter can be involved. For the investigation of possible photolysis products it is extremely important to use a light source that adequately simulates the sunlight spectrum in terms of intensities and wavelengths. Otherwise quite different TPs may be formed (27). Thus a xenon-lamp is the best choice. Commercially available are the suntestXPS and the QSun test chamber as a test-box that additionally keep the temperature stable during photolysis and enable the easy implementation of filters to simulate the wavelength distribution of natural sunlight. As proven by Sevilla-Moran et al. even the kinetic parameters obtained with this setup are in good comparison to degradation by sunlight thus the same is valid for the product distribution (28). With slight modification, even direct coupling to an LC-HRMS is possible (29). Furthermore, the pH during photolysis may influence the product distribution and thus the comparability with environmental samples. As shown for lamotrigine (pKa 5.7) the photolysis of a solution at pH 3 and pH 7 results in different product distribution (30). Dechlorination without hydroxylation (TP220, see Figure 4) occurs only if the neutral species is present. Additives can influence the product distribution and help to elucidate the reaction mechanism of the photolysis: acetone acts as a triplet photosensitizer, non-polar solvents like acetonitrile favour mechanisms involving radical intermediates, isopropanol or bicarbonate are radical scavengers, nitrate enhances the involvement of OH radicals, degasing by nitrogen avoids the involvement of oxygen species (either as 1O2 or 3O2), while addition of sodium azide can verify the involvement of 1O2 as it generates 1O2 (31–33). The knowledge on the formation pathway may support the elaboration of structure proposals. One such example is shown in Figure 4. Three lamotrigine phototransformation products are selected – two are formed by a radical driven mechanism and one is considerably increased by the addition of the radical scavenger isopropanol. Thus the dechlorination leading to TP220 and the isomerisation leading to TP256 take place via a radical intermediate, whereas it was proven that no radical hydroxylation leads to TP259. Together with the fragment information the three structures were proposed. 76 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 4. Selected phototransformation products of lamotrigine and the influence of the solvent on their formation that helps to elucidate their structure. The stability of primary TPs against further redox reactions and, thus, their stability in the environment can be tested by using the initial reaction mixture for a subsequent oxidation or reduction experiment. In Figure 5 this is shown for the three photolysis products of lamotrigine (structurs shown in Figure 4) at EC potentials of +2 V (oxidation) and -2 V (reduction). In this case TP220 was proven to be sensitive to oxidation.

Figure 5. Stability test of TPs of the photolysis of lamotrigine, by oxidation and reduction in the electrochemical cell. 77 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Furthermore the investigation of derivatives or TPs together with the parent compound helps to elucidate structures. An illustrative example is shown in Figure 6, where the photolysis of carbamazepine was investigated. Two peaks (tR=4.9 min and tR=5.2 min) are observed in the extracted ion chromatogram for m/z 251.081. The sum formula leads to three different possible structures. One of them is BQM, an ozonation product of CBZ. Its structure was already proven by NMR (34). By comparing the retention time, peak shape and fragment ions with a ozonated sample of CBZ the identity of the peak with tR =5.2 min in the photolysis sample of CBZ was proven to be BQM (structure b in Figure 6). Possible structures for the second TP with m/z 251 (tR 4.97 min) are structure a and c shown in Figure 6. An additional photolysis experiment with Oxcarbazepine (Ox-CBZ) in which the same TP at tR = 4.9 min was formed rules out structure c). Therefore this peak at 4.9 min in Figure 6 should correspond to structure a).

Figure 6. Extracted ion chromatogram of m/z 251.081 for the photolysis of CBZ, Ox-CBZ and ozonolysis of CBZ, the possible structures of TP251(a,b,c) and the observed fragment spectra are included.

Biodegradation Batch Experiments Simulation of biodegradation under well-defined conditions in a batch experiment at elevated concentrations of the analyte of interest is an additional approach that is often applied (3, 6, 35). In principle any microbial community (fungi or bacteria) including mixed cultures (e.g. sewage sludge) may be used. To check for the biodegradability there are three OECD tests available: The closed bottle test (CBT, OECD 301 D), which simulates surface water, the manometric respirometry test (MRT, OECD 301 F) and the Zahn-Wellens test (ZWT, OECD 302 B) that simulates biodegradation in wastewater treatment (3). These tests are widely used and also applied to investigate the formation of TPs, e.g. for different 78 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


pharmaceuticals or benzotriazoles (6, 36, 37). Each experiment consisted of active bioreactors and several controls. However, such experiment require days to weeks. Spiked and non-spiked samples are run in parallel and analyzed by LC-HRMS to determine peaks of interest. Using time-resolved measurements, the increasing concentration of the formed TPs helps to elucidate peaks of interest and possible pathways and to close the mass balance like shown for lamotrigine (38). Furthermore a combination with sand-filled columns is possible to simulate in the lab what happens in bank filtration sites (39, 40). As presented above for photolysis, the experiments may be performed with different commercially available TPs to prove degradation pathways. Brezina et al. showed that the degradability of 2-OH-carbamazepine and 3-OH-carbamazepine on sand filters differed strongly (39) An anaerobic batch degradation test led to interesting TPs of carbamazepine by reduction, which had not been reported before (26). The molecular formula (C16H19N2) and the fragment ions suggest that here the carbamoyl moiety of (2H)-CBZ, the primary reduction product, was further reduced to an amine and then methylated. This example illustrates that such an experiment even covers follow up reactions. However, all these experiments, when analyzed by LC-HRMS, lead to relatively complex data sets. As not only the concentration of the selected parent compound and its TPs but also of secondary metabolites produced by the bacterial community may change over time, the selection of signals of interest in such data sets is intricate. This is especially the case if the parent compound does not exhibit a unique isotopic pattern or mass defect. Consequently, exploitation of such data sets takes considerably more time than required for the less complex and cleaner chemical transformations mentioned above. Moreover, as for the other approaches the outcome has to be proven in the real environment. In-Vitro and in-Vivo Methods to Simulate Phase II Metabolism Phase-II metabolites are of interest, when humans, animals or plants are involved. Conventional methods to study the metabolism and prepare mixtures of potentially formed TPs are in-vivo methods using animals like rats. Animal testing, however, has to be reduced and replaced. Liver-based in-vitro technologies are often used in such instances. However, in human liver microsomes enzymes like N-acetyltransferase (NAT), Glutathione-S-transferase (GST) and cytosolic cofactors are missing. This limits the metabolic reactions and reduces the ‘metabolic competence’ (41). Consequently the number of TPs is limited, which is undesirable when libraries of possible TPs are the aim of the investigation. As an alternative the zebrafish embryo (ZFE) with its recently elucidated extensive metabolism may be used for metabolism studies (42). The ZFE is small, develops fast and it is not considered an animal test until 96 hours post fertilization (TG OECD 236). The ZFE is highly metabolically active already in early life stages, i.e. within the first four days of development. A broad variety of phase I and phase II TPs have been shown to be formed, which corresponds to gene expression studies (43). The detected TPs are potentially formed by humans as well and may, therefore, also be found in wastewater. In case of clofibric acid it 79



was shown that compared to other animals additional TPs were formed by the ZFE (42). Their cyclic core structure suggests formation from di-hydroxy clofibric acid via decarboxylation. This TP, however, was not found in environmental samples so far. In the case of metoprolol the comparison of the TPs of ZFE and the influent and effluent of a WWTP led to the identification of an additional TP –ketometoprolol. This TP is not known from humans or other animals, but has been detected before as a transformation product produced in WWTP. As ketometropol is not available as a reference compound the ZFE offers a way to “produce” it in the lab and, also, to verify its formation from metoprolol as parent compound. Another important potential of the ZFE is its possibility to perform phase II reactions as outlined by Brox et al. (42). An illustrative example to show the transferability of phase II metabolism from one species to another is shown in Figure 7. An acetylated carbamazepine was detected in cucumber by comparison with the ZFE extract.

Figure 7. TP252 of CBZ detected in ZFE and a cucumber extract by comparison, the signal intensity of the fragment spectra allowed the structure elucidation only for the ZFE extract.

As far as metabolism in plants is concerned, plant cell experiments may be used instead of whole plants, as shown by Macherius et al. (44). Such plant cells can show the same TP pattern as real plants but the establishment of plant specific cell cultures can be cumbersome. 80 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Conclusion The selected examples show that lab experiments combined with LC-HRMS analyses using all ion fragmentation can be a very useful combination to detect and to identify transformation products in environment samples. The lab experiments are generally easy to perform. Examples for hydrolysis, oxidation, reduction, photolysis and conjugation, as well as combined processes were provided for illustration. Thre are two options how to process and to utilize the LC-HRMS data of the lab experiments: either all possible TPs are processed, their structure elucidated as far as possible and the data with structure proposals stored in a lab-based or open-source mass spectral database for (later) use. Or the whole data set is stored but only those signals are actually processed that are also found in the sample of interest. The latter approach saves time but keeps all data for a later comparison with other samples. Therefore it is essential that standard conditions for the LCHRMS analysis of the lab experiments and the environmental samples have been established in a lab. Then, the lab experiments are a powerful tool and source of LC-HRMS data to detect and to tentatively identify transformation products in environmental samples, even when reference standards are not (commercially) available. Furthermore such lab experiments can clarify whether TPs are really expected to occur in the environment or whether follow up reactions likely take place.

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