HRMS Approaches for Evaluating Transformations ... - ACS Publications

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HRMS Approaches for Evaluating Transformations of Pharmaceuticals in the Aquatic Environment Michael Hannemann,1 Bozo Zonja,1 Damià Barceló,1,2 and Sandra Pérez*,1 1Water

and Soil Quality Research Group, Department of Environmental Chemistry (IDAEA), Spanish National Research Council (CSIC), c/ Jordi Girona, 18-26, 08034 Barcelona, Spain 2Catalan Institute of Water Research, c/Emili Grahit, 101, Edifici H2O, Parc Científic i Tecnològic de la Universitat de Girona, E-17003 Girona, Spain *E-mail: [email protected]. Phone: ++34-93 400 6100 ext 5310. Fax: ++34-93 204 5904.

Pharmaceuticals and their related transformation products (TPs) are distributed into the aquatic environment. UPLC & HPLC combined with HRMS techniques are able to detect these emerging contaminants in environmental samples. However, due to the lack of commercially available standards for detection and quantification of TPs, suspect screening is rapidly gaining popularity in the scientific community of HRMS users. This chapter reports on the application of suspect screening to detect pharmaceutical compounds and their TPs in environmental samples and also on its new application for the evaluation of the fate of selected pharmaceuticals in the aquatic environment. The authors include two examples using suspect screening published in the literature: the evaluation of the biodegradation of lamotrigine and the photolysis of ICMs. Qualitative methods for the investigation of the fate of drugs are described for manual workflows as well as for more sophisticated approaches using suspect screening; further it provides advantage of recent software developments in automated data analysis. Hence, suspect screening is

© 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.


straightforward, and if the transformation pathways of the parent compounds are unknown, the combination of lab-scale experiments and new approaches based on HRMS like the analysis of MS/MS fragmentations, mass defect, and isotopic pattern are useful methods that help researcher achieve their objectives. While detection and quantification are gaining adequate results, it is important to include newly identified TPs in environmental fate studies of pharmaceuticals and, if they are associated with a high environmental risk, to consider them for inclusion in future water quality guidelines.

Introduction Pharmaceuticals are a group of chemical substances that have medicinal properties. In 2015 the industry generated approximately one trillion US-$ revenue worldwide, with the biggest markets being located in North America and Europe (1). The occurrence of pharmaceutical compounds in the environment is ubiquitous (2–5). In the aquatic environment they can be transported and distributed in rivers and streams and are subject to degradation by both abiotic and biotic processes. These processes form transformation products (TPs) which can be more mobile and polar than the parent compound and may exert adverse effects on aquatic organisms (6–8). In case of bioactive compounds their pharmacological effect can be retained when the pharmacophore of the molecule remains intact after its transformation. The most frequent detection of transformation products of emerging contaminants (ECs) occurs in surface water and wastewater samples and they are formed mainly by biodegradation and photolysis processes (9–11). In order to assess the biodegradability of a compound at lab-scale there are several standardized test like e.g. the OECD guidelines (12). However, they rely on applying small doses of wastewater or activated sludge biomass to degrade a compound, which may result in a considerable increase in the degradation rate. On the other hand, many degradation studies reported in the literature use mixed liquor from aeration tanks of wastewater treatment plants (WWTPs). As an alternative, batch reactors can be filled with activated sludge and diluted with either ultrapure water, groundwater of wastewater effluent (13). This gives the additional benefit of performing the degradation in more related conditions as it happens in the WWTP, but the dilution would reduce the effect of sorption (13). So, the typical concentration of the activated sludge is about 4 - 5 gas/L of total suspended solids (TSS) and can be lowered down to 0.5 - 1 gas/L TSS without compromising the reaction kinetics. As the biological transformation is evaluated, appropriate control reactors (without biological activity) have to be run in parallel in order to determine the relevance of abiotic processes such as chemical hydrolysis or light-induced degradation. These control reactors are typically filled with activated sludge, which has either been autoclaved or supplemented with inhibitors like formaldehyde or azide. In some cases, WWTP effluent can also be used. For reliable results it is also important to control and maintain the pH, temperature and reactor 26

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.


volume constant over the course of the degradation period. Biodegradation is the main degradation pathway of pharmaceuticals in WWTPs. However, when they are discharged to surface waters photodegradation is considered a key process governing the whereabouts of organic micro-pollutants (14). In order to evaluate the photodegradability of a compound experiments with spiked surface water with exposure either simulated solar radiation or natural sunlight are performed (15). The samples are irradiated with appropriate dark-control experiments to account for possible hydrolytic reactions. The performance of these processes for the degradation of pharmaceuticals can be evaluated using liquid chromatography high resolution mass spectrometry (LC-HRMS). Two HRMS platforms, namely time-of-flight instruments (ToF) and Orbitrap are now the most powerful tools for the determination of pharmaceuticals and their TPs because the large majority of them are frequently detected at very low concentrations in complex matrices and there is a lack of available commercially standards. Three main analytical approaches are now applied for environmental analysis using HRMS: target analysis, suspect screening and non-target analysis. This book chapter deals with suspect screening analysis applied to environmental analysis and evaluation of transformation processes of pharmaceuticals.

Suspect Screening Approach Currently the method of choice for the analysis of pharmaceuticals in environmental samples is a target analysis. This term refers to the analysis of a predefined set of known substances in an environmental sample which covers at best 1-2% of all the contaminants present therein (9, 16). It relies on the availability of reference standards for quantification, which substantially increases the cost, and poses particular problems when a wanted target compound is not commercially available or is difficult to obtain by chemical synthesis. By contrast, if the given chromatographic method is likely to separate all compounds, HRMS instruments are capable of screening and detecting a virtually unlimited number of compounds, provided that they ionize under the given experimental conditions and their m/z values are within the mass range of the full-scan MS data set (4, 17). One of such techniques is the so-called suspect screening, for which databases (instead of reference standards) are used to tentatively identify and confirm the presence of known analytes. A suspect list has to be created including known parental chemical structures and their TPs, their elemental compositions and their exact monoisotopic masses. Then, all m/z values are searched in the environmental samples and confirmed on the basis of mass accuracy, retention time (RT), isotopic pattern determination, and structure confirmation using MS/MS experiments, Figure 1. Additional benefit is that spectral libraries can be shared by many laboratories or can be accessed via online repositories like MassBank. However, suspect screening is not making a difference between ecotoxicological effects. It is further necessary to evaluate ecotoxicological effects of TPs due to the cocktail mixture of synthetic contaminants present inside the aquatic environmental compartments (18). New approaches like effected directed analysis (EDA), the use of multivariate statistics, and environmental 27


risk assessments are leading to more focused studies based on environmental detection (16). Needless to say that any TPs originating from the degradation of pharmaceuticals are a priori also detectable without having the reference standard at the laboratory (19).

Applying Suspect Screening for Environmental Samples


Suspect Screening for Detection of Pharmaceuticals and Their Transformation Products Ultra/high performance LC (UPLC or HPLC) separates well polar compounds with functional groups, such as acids, phenols and amines; further UPLC combined with HRMS is especially useful for thermolabile, polar compounds. Table 1 shows state to the art UPLC/HRMS suspect screening methods for anthropogenic organic micropollutants such as pesticides, pharmaceuticals, drugs of abuse, sweeteners, surfactants, flame retardants, benzothiazoles and benzotriazoles, phthalates, disinfectants, and nowadays due to resistant bacteria, additionally antibiotics; further it summarizes the software which was used for detection purposes. Thereby, suspect screening achieves adequate results for tentatively identified analytes, as all of the examples show positive results. Furthermore, to meet the challenges posed when analyzing a mixture of many known and unknown compounds at low concentrations in complex matrices, a range of different HRMS instruments have been developed in recent years (19). Eichhorn et al. 2012 (20) compared the two most prominent (QExactive vs. QTOF), and while it is still not clear which is the best option for environmental samples, both are important resources for suspect and non-target screening of environmental water samples. This is mainly due to their selectivity, sensitivity, and the capacity of MS/MS fragmentation. Manual data mining is outsourced and the parameters, like metabolomics, pathways, or chemical reactions have to be set in the beginning of the experiment. However, software is normally reducing the effort and skill needed, but in the case of novel computer aided approaches it is getting more complex. HRMS is possible to detect several thousand compounds in water samples and manual data mining faces the challenge of noise elimination. Analytical methods vary from experiment and research group capability, but detection has to be confirmed by degradation experiments or, at least by literature data about ECs. As described in the introduction, TPs should be considered during monitoring of environmental samples. Overall, toxicological data on effects of target TPs of PPCP’s on ecosystems is rare; in particular, there are no systematic studies on their environmental impact and therefore research on this topic should be encouraged (2). Thereby, suspect screening is a promising approach due to its no longer requirement of analytical standards, and therefore opens possibilities of detecting new ECs; while exact mass filtering gives the possibility to quantify matching measured RT to predicted RT, fragment patterns with MS/MS to MS/MS databases, of suspect compounds. 28 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.

Matrix

Analytical technique

Suspect screening list and source of the compound list

Data mining (name of the software)

Tentatively identified analytes

Confirmed analytes

Reference

Detection and evaluation of the fate Wastewater

SPEUHPLC-ESIQTOF MS

± 500 pesticides, pharmaceuticals, drugs of abuse and transformation products

MassLynx v4.1 ChromaLynx

4 pesticides, 3 pharmaceuticals, 1 drug of abuse, 1 metabolite of drug of abuse

n.a.

(42)

WWTP effluent

4 x SPE-UHPLC–ESI-Q Exactive MS

internal TP-list, 12-13 pharmaceuticals

Mass Frontier (FISh), SIEVE

1 pharmaceutical, 3 human metabolites, and a synthetic impurity

1 pharmaceutical, three human metabolites, and a synthetic impurity

(32)

WWTP effluent

SPEUHPLC-ESITOF MS

147 pharmaceuticals and 54 metabolites

MassLynx v4.1 ChromaLynx XS

25 pharmaceuticals

4 pharmaceuticals

(43)

WWTP effluent

SPE-HPLCESI-LTQOrbitrap MS

1706 LC-MS and ESI amendable compounds produced or used in local industry + 325 chemicals reported to occur in surface water

MZmine v2.9, R-nontarget, MetFrag

13 compounds

1 UV filter, 4 chemical synthesis intermediates, 1 pharmaceutical

(44)

29


Table 1. Examples of Suspect Screening Methods Applied to Different Environmental Water Samples; SPE = Solid Phase Extraction, API = Active Pharmaceutical Ingredients; Usual Software Like Xcalibur (Thermo Fischer Scientific) Is Not Additionally Mentioned. Table modified and adapted from Reference (41), Copyright 2015, Trends Anal. Chem.

Continued on next page.


30


Table 1. (Continued). Examples of Suspect Screening Methods Applied to Different Environmental Water Samples; SPE = Solid Phase Extraction, API = Active Pharmaceutical Ingredients; Usual Software Like Xcalibur (Thermo Fischer Scientific) Is Not Additionally Mentioned Matrix





Confirmed analytes

Reference

WWTP effluent


394 compounds from 15 classes of homologous series (e.g. linear alkyl benzyl sulfonates, sulfophenyl alkyl carboxylic acids, etc.)

RMassBank, R-nontarget, enviMass, MetFusion

69 compounds related to 11 classes of homologous series

n.a.

(45)

WWTP effluent, surface water

UHPLC-ESIQTOF MS

metabolites of 6 pharmaceuticals

LecoChromaTOF, PeakView- IDA Explorer

6 metabolites

2 metabolites

(46)

WWTP effluent

4x SPEHPLC-ESI-Q Exactive MS

867 API on suspect screening list, 119 known target compounds (IMS data)

Thermo Scientific Formulator enviMass v1.2 enviPat R

77 APIs

26 new APIs on target screenining list

(47)

WWTP effluent, surface water

LC–ESI–QTOF 5 pharmaceuticals MS

MassLynx v4.1 MetaboLynx XS

22 transformation products


(48)







Confirmed analytes

Reference

WWTP effluent & influent


17 parent/TP pairs with batch experiment

R’s enviPick, R’s enviMass, R’s OrgMassSpec, R’s Nontarget, R’s MassBank MOLGENMS/MS, ProteoWizard 3.0 MetFrag & MetFusion

13 pairs of TPs (mix between suspect and non-target screening, but new interesting approach)

4 transformation products > 0.5, thereby 1 is 100% confirmation (non-target)

(16)

WWTP effluent

SPE-LC-ESILTQ Orbitrap

146 pollutants, pharmaceuticals (33) & metabolites (113)

ExactFinder 2.5

69 compounds

-

(49)

WWTP effluent & surface water

SPEUHPLCQTOF MS & SPE-LCLTQ-Orbitrap MS

107 pharmaceuticals and illicit drugs

TraceFinder ChromaLynx

28 compounds

18 compounds

(50)

Surface water


1794 predicted or known transformation products of 52 pesticides and pharmaceuticals

MassFrontier, UM-PPS,

19 pesticides and pharmaceuticals


(22)

31

Matrix

Continued on next page.


Matrix





Confirmed analytes

Reference

Surface water

LVI-HPLCESI-QTOF MS

1200 pharmaceuticals and personal care products

PeakView IDA Explorer Analyst TF 1.5 MultiQuant

5 pharmaceuticals

n.a.

(51)

Surface water

SPE-HPLCESI-QOrbitrap MS

140 pesticides and transformation products having log Kow < 5 (water relevant substances) and at least one heteroatom (ESI amendable)

MassFrontier 6.0, MetFrag

19 pesticides and 11 transformation products (not taking into account predicted MS/HRMS fragments)

13 pesticides and 5 transformation products

(52)

Surface water

SPEUHPLC-ESIQTOF MS

1212 pharmaceuticals, 546 pesticides, 378 polyphenols and 233 mycotoxins

Analyst, Peak View 1.0 MultiQuant 2.0 MarkerView Formula Finder

31 pharmaceuticals, 8 pesticides, 1 polyphenol, 2 mycotoxin

n.a.

(53)

32


Table 1. (Continued). Examples of Suspect Screening Methods Applied to Different Environmental Water Samples; SPE = Solid Phase Extraction, API = Active Pharmaceutical Ingredients; Usual Software Like Xcalibur (Thermo Fischer Scientific) Is Not Additionally Mentioned



Suspect Screening Applied To Evaluate the Fate of Pharmaceuticals For the study of transformation processes, the typical scheme consists of the generation of TPs in laboratory settings, followed by their identification by applying an array of analytical techniques, the detection, and ultimately quantification (if possible), of the TPs in the environment (15, 21). Two approaches can be distinguished: a “TPs profiling approach” and a more “sophisticated method approach” using HRMS. The overall goal is identical but due to advances in data analysis software, analytical workflows have changed leading to an improvement of the whole procedure (16, 19, 22–24). TPs profiling is mostly done manually; not only is it time-consuming but also prone to missing TPs of minor intensity in the complex full-scan MS data set. In the sophisticated approach, using suspect screening, largely automated data analysis allows for a more exhaustive screening in much reduced time. The first part of the suspect screening is to create a list. In order to generate a suspect screening list for a given pharmaceutical and its TPs, required for evaluating the transformation of the parent compound in the aquatic environment, different options have been proposed in the literature: mining for known TPs already reported in the literature, identification of novel TPs in controlled lab-scale experiments, and in silico prediction of TPs based on common degradation pathways of compounds like xenobiotics. Different setups have been used for simulating the transformation processes that pharmaceuticals can undergo in the aquatic environment, including the commonly used sunlight simulator and simple custom-made batch-reactors for biodegradation studies (see introduction). In addition, innovative software can be of help for the prediction of transformation pathways such as the University of Minnesota Pathway Prediction System (UM-PPS) and the Meteor Environmental Pathway Prediction System (Lhasa Limited, UK) (25). Following the detection of the TPs in samples from the degradation studies or the in silico prediction of the metabolites, a suspect list can be created and then used to search for TPs in real samples from the aquatic environment. Although this search can be performed manually by successively extracting from the total ion chromatogram (TIC) the ion masses of the suspect analytes with narrow mass windows, an automatic search largely facilitates this process. To this end, several software packages are available in the public domain such as MZmine (26) or XCMS (27) while commercial solutions such as MetWorks or SIEVE (Thermo Fisher Scientific) are also designed for rapid feature detection. Using an alternative protocol HRMS data analysis, MS/MS data can be screened for fragment ions that might be shared by the parent compound and its TPs (MassFrontier, HighChem, Thermo Scientifc). Furthermore, working with HRMS allows to take advantage of mass-defect filters (MDF) which can be an interesting approach for the detection in HRMS data of TPs differing in their structure from the parent compound by only minor modifications. The mass defect is defined as the difference between the exact mass of the molecule and its mass number expressed in atomic mass units and thus can be either positive or negative. Simple transformation reactions such as hydroxylation or demethylation result in only very small changes of the mass defect, and thus applying a MDF help detect such TPs. Processing software 33 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.


of different platforms have implemented MDF algorithms allowing for automated mass defect filtering. Once the list of TPs has been created, the next step is their structural elucidation. However, since it is essential to focus the efforts on the identification of the most relevant TPs, a prioritization step has been proposed before initiating the tedious compound identification process. By this, the complete list of TPs, observed for example in a laboratory experiment, can be reduced to a few key degradates. In general, the identification comprises of several steps. The first step determines the change in elemental composition between the pharmaceutical and the TP taking into account their differences in exact masses. The second step consists of comparing their MS/MS spectra, which aims to differentiate structural moieties having been altered during the degradation process from those remaining unchanged in the TP. As with the feature search and the MDF, instead of time-consuming manual data analysis the comparison of MS/MS spectra can also be accelerated by software-assisted algorithms. For example, Mass Frontier (HighChem) and MetFrag (28) are software used for the prediction of MS/MS fragmentation pathways. Finally, in order to ensure the complete coverage of all TPs formed in controlled laboratory experiments, a mass balance between untreated and treated sample for the target pharmaceutical and its TPs has to be quantitative (Figure 1).

Examples of Suspect Screening for the Evaluation of the Fate of Pharmaceuticals in the Environment Suspect screening, when used, is mostly applied for the evaluation of the fate of a target pharmaceutical in the aquatic environment using the combination of laboratory degradation experiments and suspect screening with HRMS techniques. In this case two types of compounds were chosen for a detailed explanation: lamotrigine (LMG) and X-ray contrast media (ICM). ICMs are one of the most frequently detected compounds in environmental samples. These, highly polar compounds are used in high amounts, up to 200 g for one examination, as imaging agent for organs or blood vessels during medical diagnostic tests (15). Due to their metabolic stability in the human body, they are collected as unmodified parent compounds in WWTP where their can undergo microbial degradation or adsorption only to a certain degree. Thus they eventually break through the facility and are discharged with the treated effluent into surface waters. Accordingly, their concentrations in sewage-impacted rivers are considerable (15, 29, 30). On the other hand, LMG is an anticonvulsant for the treatment of epilepsy, and bipolar disorder and is commonly used in therapy together with carbamazepine. In the U.S. neuro-active pharmaceuticals are estimated to be used by about 8% of the population (31). Unlike iopromide, LMG is highly metabolized in the human body (32–34). Ferrer et al. (34) could demonstrate that LMG and its TPs are almost completely bypassing the treatment process in the WWTP and occur in measurable concentrations in various environmental compartments including wastewater effluents, surface water, groundwater and drinking water samples. 34



Figure 1. Workflow of the application of suspect screening for detection of pharmaceuticals and their TPs as well as for the evaluation of the fate of pharmaceuticals in the aquatic environment.

Biodegradation Studies of Lamotrigine In 2010 Ferrer & Thurman (34) reported the first detection of LMG and its two major metabolites in surface water, groundwater, drinking water, and wastewater. Concentrations of LMG ranged from 488 ng/L in wastewater to 108 ng/L in surface water, with two out of seven drinking water samples having been tested positive. The concentrations of N2-glucuronide LMG were 209 ng/L in wastewater and 195 ng/L in surface waters. In humans LMG undergoes hepatic metabolism to form this prominent N2-glucuronide, along with N2-methyl-LMG. Both were reported to display similar activity as the parent compound (34). The authors used a QToF-MS to acquire full-scan MS data, ran a peak detection algorithm, and automatically assigned the most likely elemental compositions of the detected peaks based on their accurate mass, isotope spacing and relation isotope intensities. They then filtered the resulting list by compounds containing two chlorine atoms, which displayed their characteristic isotope pattern. This eventually provided strong evidence for the presence of LMG and its two human metabolites in the water samples. The authors emphasized that the occurrence of 35



drug glucuronides in WWTP effluents should be more thoroughly investigated. In follow-up article from 2014 the same authors reported on the degradation pathways of LMG under advanced treatment by UV, hydroxyl radicals, and ozone (33). The result of this study showed that LMG responded only slowly to direct photolysis or oxidation by ozone. In wastewater treatment advanced oxidation processes using hydroxyl radicals (HO·) are effective but this degradation process showed low degradation rates. The main transformation pathway was hydroxyl group addition to the benzene ring during the reaction with (HO·), while ozone opened the triazine ring structure and direct photolysis dechlorinated the benzene structure. With the molecular structure containing a triazine ring and a benzene ring a fast degradation is not favored (33). The work of Zonja et al. 2016 (32, 35) has shown the complexity of the fate of LMG in the environment. The authors analyzed influent and effluent samples from WWTPs for a total of twelve LMG-related compounds including human metabolites, impurities, and TPs gained from literature search, further batch experiments, and detection in water samples. Thereby, the batch reactors were spiked with the parent compound LMG and additionally with labeled 13C3-LMG in order to gather evidence that supported the MS-derived identities of the TPs postulated in the degradation of unlabeled LMG. The parent compound, three metabolites and one synthetic impurity (known as OXO-LMG) were quantified in wastewater effluents, while glucuronide oxidation, deconjugation, and biotic transformation were confirmed and are the proposed transformation pathways. The appearance of OXO-LMG was unexpected and additional batch experiments provided evidence that the N2-glucuronide LMG was the actual source, while LMG itself was not further degraded. The relevance of LMG N2-glucuronide TPs in the transformation pathway of LMG in WWTPs was confirmed after conducting mass balance studies for corresponding raw and treated sewage. Both sewages were compared to each other and mass balance calculation was possible. Only when taking all TPs and metabolites into account the mass balance could have been closed (Figure 2). In 2013 Writer and colleagues (31) described the natural attenuation of 14 neuro-active pharmaceuticals and their associated metabolites, including LMG and CBZ. The authors used a newly developed lagrangian sampling method, which followed a stretch of the river as it flows downstream. Thereby, LMG and its metabolites were confirmed to be persistent. The primary mechanism of their removal was interaction with bed sediments and stream biofilms. LMG is in general more persistent than its metabolites and there is a clear need for more investigations on the environmental fate of LMG. While the majority of research on attenuation processes of neuro-active drugs has used controlled laboratory studies, the approach comparing differences between natural and laboratory conditions is a valuable approach. Schollée et al. 2015 (16) analyzed influent and effluent wastewater of a WWTP and those from a lab-scale batch experiment followed by multivariate statistics of HRMS and MS/MS data. The authors compared peaks detected in the influents and effluents with those found in the batch experiments, WWTP influent was spiked with parent compounds and human metabolites, and WWTP effluent with known TPs whose commercial standards were available. This proof-of-concept study was used to see whether it would be possible to link parent compounds with 36



potential TPs formed in the secondary treatment of a WWTP with the non-target applied to spectral symmetry comparison. Later, in a real sample with non-target analysis, they detected a surfactant homologue series with their associated TPs. A reduction of the number of quantified compounds between influent and effluent indicated degradation during the treatment; with principal component analysis (PCA) the separation of influent and effluent compounds was achieved. Thirteen basic modification reactions were used; further filtering of RT reduced the overall number of potential links of parent compounds and TPs in influent and effluent. In order to elucidate the structures behind the pairs, MS/MS information was collected. To further prioritize elucidation efforts on pairs, the spectral similarity between supposed parents and TPs was calculated, as it is postulated that similar structures would yield similar spectra. MS/MS similarity was visualized with head-to tail plots. It was possible that the intensity of the TPs peak was higher than the intensity of the parent compound in the influent samples. Linking the parent and the TP fragmentation ions opened the possibility to search for new TPs in the related sample from WWTP to assign possible structures for the TP. Admittedly, when a parent compound structure is unknown, the identification of the TP structure cannot be accomplished. The procedure is not yet as successful as anticipated but useful for wastewater comparison and considered a new method in its early stages.

Figure 2. Workflow sketch approach for LMG, Bozo et al. 2016 (32) and ICM compounds, Pérez et al. 2006 & 2009, (37), (38); full line is TP profiling approach, dotted line is more sophisticated approach and dashed line is both possibilities combined or at the same time; mass balance is taken into account, but some approaches bypass directly to target analysis. 37 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.


X-ray Contrast Media Photolysis Study In surface waters one of the main processes to transform organic pollutants is photolysis (7, 21, 33, 36). For its evaluation, similar approaches to the ones applied to wastewater samples have been used. The typical approach consists of conducting experiments in lab-scale batch-reactors, analysis of the aqueous samples by full-scan MS in conjunction with targeted or untargeted MS/MS data acquisition, and manual inspection of the MS data. This workflow was successfully applied by Pérez et al. 2009 (37, 38) and is sketched in Figure 2 for TP profiling and further more sophisticated approaches. In two publications the authors described TP profiling for the evaluation of the bio/ photodegradability of iopromide, in waste and surface waters (15, 38). The objective of the first study was to identify the TPs of iopromide when the parent compound was degraded in mixed liquor from a WWTP in a batch-reactor experiment. The generated biodegradation products were structurally characterized by ESI-ion trap-MS (low resolution instrument) in combination with H/D-exchange experiments. The authors detected three TPs, could elucidate their structures based on the MS/MS data, and proposed the metabolic pathway as oxidation of the primary alcohol on the side chains forming carboxylates. Three years later they applied a similar approach to identify photoproducts of iopromide. The acquisition of a high-resolution QToF-MS (10,000 resolving power) allowed the characterization of photoproducts by accurate mass measurements in combination with H/D exchange experiments. The experimental approach consisted of spiking surface water samples with iopromide followed by exposure to artificial sunlight. Four principal photoreactions were detected for the photolysis of iopromide: gradual, and eventually complete, deiodination of the aromatic ring, substitution of the halogen by a hydroxyl group, N-dealkylation of the amide in the hydroxylated side chain, and oxidation of a methylene group in the hydroxylated side chain to the corresponding ketone (38). The knowledge on the photodegradation pathways of the iodine-bearing X-ray contrast agent iopromide was advanced by investigating the formation of photoproducts originating from a series of five structurally related X-ray contrast media. In order to avoid the tedious process of manual data mining, in this instance the photo-TPs were searched while using peak-picking software (SIEVE, Thermo Scientific) allowing to detect differences, i.e. putative photoproducts, in the TICs of treated and control samples. Once chromatographic peak alignment was performed with described software (more sophisticated approach), this yielded a list of 108 photoproducts, which was used to build a compound database (15). With the goal of assessing the environmental relevance of these photoproducts obtained under controlled laboratory conditions, real surface water samples were probed for their presence based on accurate MS/MS data and retention time matching. For confirmation purposes mass errors of up to 5 ppm were accepted. This led to the prioritization of eleven photoTPs based on their high detection frequency in real samples. Their structure elucidation was eventually accomplished by comparison of the characteristic fragmentation patterns with those of the respective parent compounds. Finally, in order to quantify and elucidate the structures of the priority photoTPs in surface water samples, semi-preparative LC of the irradiated laboratory samples obtained 38 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.

reference standards of the transformation products. The median concentration of the parent compounds ranged from 29 ng/L to 6 μg/L (iomeprol) while the photoTPs were found at median concentration of 30 ng/L, with maximum concentrations of 0.4 μg/L for one of the photoTPs of iomeprol (15).


Conclusion and Future Advances This chapter has reviewed HRMS strategies to detect and evaluate the fate of pharmaceuticals in the aquatic environment using suspect screening methods. Each approach has its advantages and disadvantages and should be considered while choosing the method for following experiment. Thereby the sophisticated approach is the more promising one. The disadvantage of TP profiling is the narrow screening window of compounds, and the time effort in preparation. However, the big advantage of sophisticated approaches is the overall view on environmental samples. Screening for thousands of compounds within one experiment is stunning and even retrospective analysis is furthermore possible. Indeed, pre-knowledge has to be given, or gained in upstream experiments, but the raise and future advantage of databases is improving the approach, excluding the pre-experimental part. Additionally, the more sophisticated method gives a better overall view combined with PCA approaches and as the interest in ECs rises in the community, related bioactive compounds like TPs, impurities, or glucuronides, are investigated and their occurrence in the environment can be confirmed. Nevertheless, TPs profiling provided and still provides adequate results of specific compounds, which are of greater interest for the scientific community. Therefore, it is of high significance to realize the importance of including these newly found TPs, however detected in environmental monitoring studies and, if they are associated with a high environmental risk, to consider them for inclusion in future water quality guidelines (39, 40). It seems to be still a long way to declare new bioactive substances to compounds of higher concern, especially if humankind does not discharge/ produce them, while these are transformed from parent compounds into the environment, as the EU-watch list approach shows. Some well known drug metabolites often exceed the parent compound concentration (17). Consequently, it should be noted that if metabolic routes of the parent compound are known, suspect screening is straightforward. If the metabolism, however, has not been revealed, new approaches like mass balance calculations, analysis of MS/MS fragmentations, and isotopic pattern analysis are useful methods that, supported by software solutions, open new windows of investigation. Most of these new approaches are associated with huge efforts and therefore not used by default. Furthermore, the collaboration between research institutions should be fostered to achieve common standards at the level of identification confidence and the generation of extensive MS/MS spectra libraries. HRMS helps provide an overview of anthropogenic pollutants and their TPs in the aquatic environment; a recent tendency is the combination of suspect screening with target analysis or non-target analysis. Computer-assisted data analysis has proved highly valuable and lab-scale batch experiments open the possibility to identify metabolites first under controlled settings and later their 39 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.

quantification in environmental samples. A breadth of new methods and software is creating sophisticated approaches for detection of ECs in water samples. Investigations based on non-target approaches combined with software, and the search in comprehensive HRMS spectra databases is a promising way in future environmental studies. Non-target analysis is complex and still in its early stages. However, computer aided suspect screening is a sophisticated approach with lots of potential, even if in years the non-target approaches should be the future leader, suspect screening is leading the non-target approach to its future achievements without any doubt.


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