Application of Orbitrap Mass Spectrometry for the Identification of Transformation Products of Trace Organic Contaminants Formed in the Environment

Chapter 9

Application of Orbitrap Mass Spectrometry for the Identification of Transformation Products of Trace Organic Contaminants Formed in the Environment C. Prasse1 and T.A. Ternes2, * 1 UC Berkeley, Berkeley CA, United States; 2Bundesanstalt fu¨r Gewa¨sserkunde (BfG), Koblenz, Germany *Corresponding author: E-mail: [email protected]

Chapter Outline 1. Short History of Orbitrap Mass Spectrometry 2. Application of Orbitrap MS for Nontarget, Suspect and Target Analysis 2.1 Nontarget and Suspect Analysis 2.2 Target Analysis 3. Orbitrap MS for the Identification of Transformation Products of Trace Organic Contaminants

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3.1 Example 1: Biodegradation of Penciclovir in Activated Sludge 3.2 Example 2: Ozonation of Carboxy-Acyclovir 4. Challenges Associated With Identification of TPs Using Orbitrap MS and Potential Future Solutions References

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1. SHORT HISTORY OF ORBITRAP MASS SPECTROMETRY High-resolution mass spectrometry (HRMS) has become a very powerful technology for a variety of environmental applications. Instrumental platforms currently include time-of-flight (TOF), Fourier-transform ion cyclotron Comprehensive Analytical Chemistry, Vol. 71. http://dx.doi.org/10.1016/bs.coac.2016.02.010 Copyright © 2016 Elsevier B.V. All rights reserved.

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resonance (FT-ICR) and Orbitrap mass spectrometry (MS) which all allow for the determination of accurate masses of detected ions, even though the accuracies vary significantly. This chapter specifically targets on the application of liquid chromatography (LC) Orbitrap MS for environmental applications, with the focus on the identification of transformation products (TPs) of trace organic contaminants formed in natural and engineered treatment systems as well as in the environment. For detailed information on TOF-MS and FT-ICRMS we refer the reader to various review articles [1,2]. Orbitrap MS was first described by Makarov in the year 2000 [3]. The name is derived from the mechanism of the analyser which uses orbital trapping of ions in an electrostatic field. The ions rotate around the axial central electrode by simultaneously oscillating along it. These oscillations are transformed by Fourier transformation into individual signals with mass resolution of up to 150,000, and high-accuracy mass measurements within 5 ppm [3]. Three years later, Hardman and Makarov described the coupling of the Orbitrap MS to an electrospray ionisation (ESI) source [4]. This was crucial to allow for its hyphenation with LC systems. Further improvements were achieved by coupling of a linear ion trap to a radio frequency (RF)-only ‘C-trap’ for intermediate storage of ions and of the Orbitrap mass analyser (LTQ Orbitrap) [5]. This also enabled determination of accurate masses of fragments formed in MSn experiments. Since the first instrument became commercially available about 10 years ago, Orbitrap MS has been used for a variety of applications, especially for ‘omics’ approaches including proteomics and metabolomics [6e8]. The first environmental application was described by Hogenboom et al. in 2009, who used Orbitrap MS for the identification of emerging contaminants in treated wastewater, surface water and drinking water [9]. In general, Orbitrap MS analysis of environmental samples can be applied for targeted, suspect and nontargeted analysis [10]. The ability of simultaneously acquiring full-scan MS data and fragmentation patterns of the most intense ions (MSn experiments), so-called data-dependent acquisition is particularly attractive for application of Orbitrap MS for suspect analysis and nontarget analysis.

2. APPLICATION OF ORBITRAP MS FOR NONTARGET, SUSPECT AND TARGET ANALYSIS 2.1 Nontarget and Suspect Analysis The acquisition speed and high mass accuracy together with the data-dependent acquisition possibilities make Orbitrap MS to a very powerful tool for the search of unknown or suspected compounds present in environmental samples [10]. Examples include the search for trace organic contaminants in wastewater and surface water [1,11], mussels and passive samplers [12], sediments [13], natural gas residual fluids [14] and oil sands process waters [15]. Nontarget analysis using

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Orbitrap MS has been combined with effect-directed analysis to screen for androgen-disrupting chemicals in river sediments [16], plastic bottles [17] and oil sands process waters [18]. Furthermore, it has been used to assess biodegradation in complex hydrocarbon fractions [19]. In order to facilitate data analysis and data processing, various computational tools such as MZMine [20], XCMS2 [21], MetFusion [22] or RMassbank [23] are available today. These allow for the identification of peaks using online databases, isotope pattern recognition, automatic recalibration and processing of mass spectra as well as automated MS and MS/MS data interpretation [24]. The development of spectra libraries as commonly used in GC/MS analysis is crucial, however, in contrast to electron ionisation, ESI MS/MS spectra have the disadvantage that the comparability between instruments from different vendors or even different models can differ substantially making it necessary to record MSn spectra at different fragmentation energies. Spectral databases, which are increasingly used including MassBank, Norman MassBank and NIST MS/MS [25e27]. In addition, in silico fragmentation software such as MS Fragmenter, Mass Frontier and MetFrag can be employed for the prediction of MS fragments [22,28,29]. Furthermore, comparison of retention time indices can be used as a complementary approach to accurate mass determinations and MS fragmentation [30,31]. In contrast to nontarget screening, suspect screening approaches use the HRMS data for searching compounds suspected to be present in the samples, but without a reference standard at hand [32]. In general, the fragmentation patterns of the detected analytes are compared with the spectral information of compounds contained in MS spectral databases. However, as indicated above, MS spectral libraries are only scarcely available, as fragmentation behaviour of compounds strongly depends on the type of instrument that is used [33]. To facilitate the search in complex environmental samples and limit the number of features, several studies have focussed on the elucidation of specific groups of trace organic contaminants, including sulphur-containing and perfluorinated compounds [34,35]. Overall, for both nontarget and suspect analyses the value of the obtained information highly depends on the quality of the HRMS data [36]. Ideally, the structural information obtained from the MS allows the assignment of a single chemical structure which can be confirmed by comparison with a reference standard. Otherwise only tentative structures of formed TPs can be proposed or complementary analytical techniques have to be used (this is discussed in greater detail in Section 3). Even though the analytical approaches have been found to be comparable between different laboratories, there is a need for further harmonisation of data processing workflows [27].

2.2 Target Analysis Even though target analysis is typically conducted using LC-MS/MS (using multiple reaction monitoring, MRM), the high accuracy of Orbitrap MS

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measurements and the increasing sensitivity of new-generation Orbitrap instruments have also led to rising efforts to apply this technology for the quantification of trace organic contaminants. The high resolution is particularly interesting for compounds that exhibit poor fragmentation in MSn experiments, which limits the applicability of MRM and substantially influences the sensitivity. In addition, HRMS also facilitates the differentiation between co-eluting compounds with similar masses (<1 m/z) as well as matrix components. This was applied in 2015 for the analysis of the artificial sweetener sucralose in different aqueous matrices, including seawater, in negative ESI (ESI) mode [37]. Even though the analysis in ESI provides the advantage of generally lower matrix suppression, especially in complex matrices such as seawater or wastewater, the applicability for sucralose is hindered by poor fragmentation of the parent anion. However, the determination of the exact mass full-scan mode together with the characteristic isotope patterns due to the presence of three chloride atoms in the molecule allowed for the quantification of sucralose down to 5.7 ng/L in seawater [37]. Other compounds that have been quantified in environmental waters using Orbitrap MS include nitrosamines [38], cytostatic drugs [39], poly- and perfluorinated compounds [40], brominated flame retardants [41], isoflavones [42], 1H-benzotriazoles and benzothiazoles [43], glucocorticoid compounds [44] and drugs of abuse [45e47]. In addition, several multimethods for the simultaneous analysis of trace organic contaminants in water were published in 2011, 2012 and 2015 [48e54].

3. ORBITRAP MS FOR THE IDENTIFICATION OF TRANSFORMATION PRODUCTS OF TRACE ORGANIC CONTAMINANTS The occurrence of a variety of trace organic contaminants has also resulted in efforts to elucidate the fate of these compounds in both engineered and natural systems. The identification of TPs of individual trace organic contaminants is of great relevance for the comprehensive assessment of their environmental fate and potential associated risks as well as for a detailed evaluation of (waste)water treatment technologies. This in particular includes the transformation of trace organic contaminants by biodegradation, oxidative treatment (ozonation, AOPs, chlorination) and photodegradation (direct and indirect photolysis) [55e58]. Trace organic compounds which have been investigated so far using Orbitrap MS include anticancer drugs [59,60], antimicrobials [61], antivirals [62e64], UV filters [65], opium alkaloids [66], biocides [67], perfluorinated and polyfluorinated compounds [68], phenazonetype pharmaceuticals [69], psychoactive compounds [70], erectile dysfunction drugs [71], artificial sweeteners [72], and carbamazepine, oxcarbazepine and their main human metabolites [73,74]. In general, the same setup is used as applied for the analysis of environmental samples described above using both full-scan and data-dependent

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acquisition to obtain exact mass and MS fragment information from formed products. To facilitate identification of TPs, elevated concentrations of parent compounds are typically used. Samples are taken at several time points to follow the degradation of parent compound and evolution of transformation products and to identify intermediate products that are transformed further. In addition, it is beneficial if a blank reactor (nonspiked control) is run in parallel to distinguish between relevant peaks and those formed by degradation of the matrix such as sewage sludge flocs or dissolved organic matter. To facilitate the search for TPs, different strategies have been employed including the prediction of bioTPs based on computational prediction tools [75e77]. However, as pointed out by Prasse et al. [62] these biodegradation prediction systems such as Eawag-PPS (formerly known as UM-PPS), as of 2015, still lack specificity and either largely over-predict the number of potentially formed TPs or, which is even more important, do not predict formed TPs at all. One advantage of using laboratory batch studies for the elucidation of TPs is that elevated concentrations of target compounds can be used, which drastically facilitates the identification of TPs. The following two examples illustrate the capabilities and the limitations of the Orbitrap MS with regard to the elucidation of chemical structures: (a) the identification of pencilovir TPs formed in contact with activated sludge and (b) the identification of COFA, the ozonation product of carboxyacyclovir.

3.1 Example 1: Biodegradation of Penciclovir in Activated Sludge Penciclovir is an antiviral drug used for the treatment of herpes infections. It has been detected in raw wastewater in concentrations in the low ng/L range and is widely eliminated during activated sludge treatment as indicated by significantly lower concentrations in treated wastewater [78]. To investigate its fate during activated sludge treatment, biodegradation experiments with activated sludge were performed in the laboratory [62]. Briefly, 200 mL of sludge taken from a nitrification unit of a municipal wastewater treatment plant (WWTP) was poured into 1 L amber glass bottles and filled up with 800 mL of WWTP effluent. In order to determine biodegradation kinetics, batch experiments were performed at environmental relevant concentration (10 mg/L). At each sampling time point, 50 mL were withdrawn, filtered (glass fibre filter with cut-off <1 mm) and subjected to solid-phase extraction prior to LC-MS/MS analysis. In order to elucidate the formation of TPs the same experimental setup was used but using substantially higher concentrations of penciclovir (20 mg/L). Samples (1 mL) were filtered through 0.45 mm syringe filters before injection into the LC-Orbitrap MS system. Experiments at elevated concentrations revealed the rapid elimination of penciclovir leading to the formation of eight different TPs (Fig. 1). Several of

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FIGURE 1 Biodegradation of penciclovir (left) and formation of TPs (right) in sewage sludge over 25 days (initial penciclovir concentration: 20 mg/L). Adapted from C. Prasse, M. Wagner, R. Schulz, T.A. Ternes, Biotransformation of the antiviral drugs acyclovir and penciclovir in activated sludge treatment, Environ. Sci. Technol. 45 (2011) 2761e2769.

the TPs which were initially formed (eg, penciclovir TP 267, TP233 and TP237) were decreasing during the experiments indicating that they are transformed further. Mass spectral information revealed the presence of fragment m/z 152.0564 for all TPs, indicating that biochemical reactions are limited to the side chain of the penciclovir molecules, leaving the guanine moiety unmodified. However, due to the fact that the NeC bond connecting the side chain to the guanine moiety was the main position of MS fragmentation, elucidation of the chemical structure of the modified side chains was challenging. Several Orbitrap MS instruments including the LTQ-Orbitrap Velos allow for the fragmentation of parent ions via two mechanisms, higher-energy collision dissociation (HCD) and ion-trapebased collision-induced dissociation (CID). HCD fragmentation thereby tends to produce electron ionisation (EI)elike fragmentation and records ions from multiple steps of collision, whereas CID enables a less stringent fragmentation, thus generally yielding fragments with higher masses [79]. In the case of the penciclovir transformation products, HCD led to the complete cleavage of the side chain attached to the guanine moiety, thus not providing any further insights into structural modifications. On the contrary, fragmentation experiments using CID allowed to further elucidate the chemical structure of the side chain. As an example, CID fragmentation of TP281 revealed the cleavage of two CO2 groups, clearly indicating the presence of two carboxylic acid moieties via oxidation of both terminal hydroxyl groups of the side chain (Fig. 2). This could be confirmed by comparison with a reference standard isolated from biodegradation experiments at elevated concentrations (see following paragraphs for further details). Similarly, for TP249A and TP249B which have the same exact mass (m/z 250.0921, sum formula C10H12O3N5; Fig. 3) only two fragments were observed using HCD, m/z 152.0564 and 99.0438, respectively. Hence, HCD did not allow a further identification of structural modifications at the side

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-C5H6O4 152.0564 C5H6ON5

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FIGURE 2 Fragmentation pattern of PCV TP281 observed in Orbitrap MSn experiments using CID and the postulated fragmentation pathway. Modified from C. Prasse, M. Wagner, R. Schulz, T.A. Ternes, Biotransformation of the antiviral drugs acyclovir and penciclovir in activated sludge treatment, Environ. Sci. Technol. 45 (2011) 2761e2769.

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Relative Abundance

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FIGURE 3 Fragmentation pattern of penciclovir TP249A (left) and TP249B (right). HCD fragments are given in green (grey in print versions), additional fragments observed using CID are provided in blue (dark grey in print versions). Compounds were analysed individually after separation using semipreparative HPLC-UV.

chain. In contrast, using CID additional fragments at m/z 222.0979 and 232.0826 were observed for TP249A and TP249B, respectively (Fig. 3). For TP249A this indicates the presence of an aldehyde moiety (eCO), whereas for TP249B formation of m/z 232.0826 (eH2O) the presence of a hydroxyl group is suggested. Even though this did not allow for an unambiguous identification of both TPs (which was, however, accomplished using NMR analysis), the information obtained from CID fragmentation strongly suggested that both compounds are isomers but not enantiomers. As a consequence, nuclear magnetic resonance (NMR) spectroscopy was applied as complementary analytical technique. To this end, additional experiments were performed at 150 mg/L initial penciclovir concentrations. After the complete degradation of the parent compound (about 25 days; monitored by analysis of samples using LC-MS/MS), the content of the flask (500 mL) was filtered through glass fibre filters (<1 mm) and the water phase was subjected to freeze-drying to reduce the sample volume to about 5e10 mL (Fig. 4). To enable the isolation of intermediate TPs, an additional bottle was used, and incubation was stopped after intermediate TPs peaks reached a maximum (monitored by full-scan LC-Orbitrap MS). After freeze-drying the remaining aqueous phase was injected into a semipreparative HPLC-UV system coupled to a fraction collector. Individual fractions were freeze-dried until complete dryness. Obtained solids were weighed using a microgram laboratory balance and purity of individual fractions was determined using HPLC-DAD. This procedure allows for the subsequent identification of TPs via NMR as well as to use the individual TPs as standards to quantify their concentrations in laboratory experiments as well as environmental samples via LC-MS/MS.

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FIGURE 4 Scheme of the approach used for the isolation of individual TPs.

The quantification of all eight TPs in laboratory experiments allowed for the calculation of the overall mass balance (Fig. 5). A close mass balance until 5 days indicates that all relevant initial transformation products were considered. However, the decrease of the mass balance to about 40% after 10 days either indicates a formation of additional TPs, an uptake of specific TPs into the microbial biomass or a complete mineralisation. The latter two possibilities are most likely due to a significant decrease of the dissolved organic carbon (DOC) observed during the experiments [62]. Based on the chemical structures of formed TPs as well as metabolic logic, this allowed for the proposal of a biotransformation pathway (Fig. 6).

FIGURE 5 Mass balance for biodegradation of penciclovir in activated sludge over 10 days (initial penciclovir concentration: 20 mg/L). Adapted from C. Prasse, M. Wagner, R. Schulz, T.A. Ternes, Biotransformation of the antiviral drugs acyclovir and penciclovir in activated sludge treatment, Environ. Sci. Technol. 45 (2011) 2761e2769.

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FIGURE 6 Biotransformation pathway of penciclovir in activated sludge. Modified from C. Prasse, M. Wagner, R. Schulz, T.A. Ternes, Biotransformation of the antiviral drugs acyclovir and penciclovir in activated sludge treatment, Environ. Sci. Technol. 45 (2011) 2761e2769.

3.2 Example 2: Ozonation of Carboxy-Acyclovir Acyclovir, another antiviral drug used for the treatment of herpes infections, has been shown to be present in WWTP influents in concentrations up to several mg/L [78]. Similar to results from penciclovir described in Example 1, acyclovir is extensively transformed during activated sludge treatment with elimination efficiencies generally >90%. However, this is leading to the formation of one main TP, carboxy-acyclovir which has been shown to be highly recalcitrant to further microbial degradation, leading to the contamination of surface waters, groundwater and finished drinking water [62]. In order to remove carboxy-acyclovir from wastewater and thus prevent its emission into receiving waters, its fate during advanced treatment via ozonation was investigated in bench-scale laboratory experiments [63]. As carboxy-acyclovir is not commercially available, the compound was isolated from biodegradation experiments with acyclovir in sewage sludge at high concentrations (150 mg/L). For the ozonation experiments, carboxy-acyclovir (2 mg/L) was spiked into buffered MilliQ water (50 mM phosphate buffer, pH 8) before aliquots of an ozone stock solution (1 mM O3) were added. Identification of formed TPs was accomplished using the same experimental setup as described in Example 1 for biodegradation of penciclovir. Results revealed the formation of one main oxidation product at m/z 274.0774 (OP273; C8H12O6N5), indicating the addition of two oxygens and the

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abstraction of two hydrogen atoms in comparison to carboxy-acyclovir (m/z 240.0728; C8H10O4N5). MSn experiments with TP273 using both HCD and CID revealed the formation of eight fragments (Fig. 7). The molecular ion peak at m/z 274.0774 was fragmented (MS2) into m/z 198.0620 and 134.0446. Cleavage of C2H4O3 from m/z 274.0774 leading to fragment m/z 198.0620 indicated that the side chain of the molecule remained intact; a similar behaviour has also been observed for carboxy-ACV [62]. On the contrary, the absence of fragment m/z 152.0564 revealed structural modification of the guanine moiety. Fragment m/z 198.0620 could be further fragmented (MS3) by cleavage of CO yielding m/z 170.0670. This is a strong indication for the presence of an aldehyde moiety in the TP. Furthermore, the presence of a primary amine moiety was indicated by the cleavage of NH3 from m/z 170.0670 yielding m/z 153.0406 (MS4). Despite the information from Orbitrap MS analysis it was impossible to identify the chemical structure of the oxidation product. Experiments conducted at elevated concentrations (100 mg/L) allowed us, however, to isolate sufficient quantities for NMR analysis which ultimately enabled the unambiguous identification of the chemical structure of the oxidation product (Fig. 7).

4. CHALLENGES ASSOCIATED WITH IDENTIFICATION OF TPS USING ORBITRAP MS AND POTENTIAL FUTURE SOLUTIONS Despite the great advantages Orbitrap MS offers for the identification of TPs, there are several challenges that can limit the capabilities for the identification of TPs. Even though it rather seems obvious, it is important to realise that not every compound can be detected by Orbitrap MS. In general, polar, nonvolatile compounds such as pharmaceuticals and polar pesticides are typically amenable to LC-HRMS, most frequently using reversed-phase (RP) chromatography for chromatographic separation. Lipophilic and/or volatile compounds such as polycyclic aromatic hydrocarbons (PAHs) and polybrominated diphenylethers (PBDEs) are typically analysed using gas chromatography (GC) analysis. However, ionisation in the Orbitrap source requires the presence of ionisable moieties within the molecules of interest. Three main ionisation techniques are available today, ESI, atmospheric pressure chemical ionisation (APCI) and atmospheric pressure photoionisation (APPI) (Fig. 8) with ESI being the most widely used ionisation source followed by APCI and APPI [80]. The ionisation efficiencies for individual compounds thereby can vary significantly, even between compounds with similar chemical structures. One main challenge, in particular when using ESI is the formation of adducts, especially in positive ionisation mode (including Naþ, Kþ, NH4 þ ), as well as the limited ionisation of target analytes due to competition with other compounds present in the sample (so-called matrix effects) [81]. As a result, the low intensity of a TP can either indicate that it is only formed to a minor

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FIGURE 7 MSn spectrum of COFA, the main oxidation product formed during ozonation of carboxy-acyclovir (top) as well as 1H-NMR (middle, left), 13C-NMR (middle, right), 1H,13CHSQC (bottom, left) and 1H,13N-HSQC (bottom, right) spectra of COFA. Adapted from C. Prasse, M. Wagner, R. Schulz, T.A. Ternes, Oxidation of the antiviral drug acyclovir and its biodegradation product carboxy-acyclovir with ozone: kinetics and identification of oxidation products, Environ. Sci. Technol. 46 (2012) 2169e2178.

extent or that it exhibits a low ionisation efficiency in the MS. For this reason, it is impossible to calculate mass balances based on peak areas as only slight changes of the molecular structure can significantly influence ionisation efficiencies. APCI and APPI have been shown to exhibit significantly lower matrix effect compared to ESI, but they are generally less suitable for the

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FIGURE 8 Polarityevolatility space of selected trace organic contaminants (left) and molecular weightepolarity space of different Orbitrap atmospheric pressure ionisation sources (right). WFD, Water Framework Directive.

detection of highly polar compounds. The latter are also particularly challenging in terms of the chromatographic separation as they typically show a low retardation on conventional RP columns. To tackle this problem, alternative chromatography techniques such as hydrophilic interaction LC (HILIC) and capillary electrophoresis (CE) are suggested [82e85]. Another aspect for the identification of TPs using Orbitrap MS is related to the structural information obtained from MSn experiments. Depending on the chemical structure and the presence of ionisable moieties, fragmentation can result in the formation of a variety of fragments or none at all. As a result the quality of the information that is obtained varies substantially. Even though the high mass accuracy allows for the assignment of a chemical formula, formula generation is frequently leading to several possibilities [86]. The number of possibilities is increasing with mass making the identification even more difficult. Another important aspect is spectral accuracy which is the accuracy with which an instrument measures the isotopic distribution of an ion [87,88]. Increasing spectral errors were observed at higher resolving power for most of the investigated compounds. This can most likely be attributed to the phenomena of isotopic beat patterns [87]. On the other hand, Kellmann et al. found that for consistent and reliable mass assignment (<2 ppm) of analytes at low concentration levels in complex matrices, a high resolving power (>50,000) is required [89]. This shows the challenges associated with HRMS for the determination of accurate masses on the one and spectral accuracy on the other hand. In order to facilitate the identification of TPs and to obtain additional information on their chemical structures, a variety of complementary approaches should be considered. First of all, information on retention times on C18 columns often allows narrowing down the different possibilities of chemical structures based on their physicochemical properties [90e92]. The investigation of structural analogues has further been shown to be valuable for both the identification of potential reactive moieties in organic trace contaminants and the elucidation of reaction mechanisms [66,73,93]. Furthermore, isotope patterns have been used to elucidate TPs being formed

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FIGURE 9 Chromatogram of lamivudine (retention time (RT) 16.93 min) and its photodegradation products cytosine (RT 2.86 min) and lamivudine S-oxide (RT 6.42 and 6.92 min) after extraction of mass range 112e112.1 from full-scan MS data.

from biodegradation of halogenated and sulphur-containing compounds [34,94]. In order to search for TPs in which certain moieties exhibiting characteristic MS fragments are conserved, the data obtained from data-dependent acquisition can be searched for the presence of specific fragments. The same can be obtained via full-scan MS for compounds for which fragmentation is already observed in the ionisation source. This is shown in Fig. 9 for the analysis of photodegradation products of the antiviral drug lamivudine [93]. Another example is the cleavage of iodine from iodinated compounds such as iodinated X-rayecontrast media [95]. Latest development in Orbitrap instruments also allows for all-ion fragmentation, that is, data-independent acquisition [96]. In contrast to the data-dependent acquisition approach mentioned in the preceding sections, data-independent acquisition enables fragmentation of the whole mass range of ions that are entering the MS. Even though data-dependent acquisition generally is sufficient to obtain MSn information on most intense TP peaks that are formed in cases where high concentrations of the parent compound are used, data-independent acquisition might be particularly valuable if a retrospective data analysis is needed (eg, to search for additional minor TPs) as well as for toxic compound for which only low concentrations can be used in biodegradation experiments.

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