The Challenge of the Identification and Quantification of Transformation Products in the Aquatic Environment Using High Resolution Mass Spectrometry
The environment is contaminated by a number of micropollutants and their degradation products, many of which still remain undetected. Nowadays, several European regulations require the inclusion of transformation products in environmental risk assessment and monitoring. In the last decade, intense efforts have been taken to recognize the identity, quantity, and toxicity of unknown transformation products. Liquid chromatography combined with mass spectrometry has become a key technique for environmental analysis, now allowing the development of screening, identification, confirmatory and quantitative methods for the trace analysis of polar compounds in complex environmental matrices. The combination of modern technologies comprising high resolution, high mass accuracy and mass fragmentation enables the identification of compounds without having the authentic standards or even the detection of unknown analytes. However, a reliable confirmation of proposed structures using NMR spectroscopy or available standards is still desirable. This chapter presents new analytical strategies to identify and quantify transformation products generated by human metabolism, microbial degradation, or other environmental breakdown processes. Various hyphenated mass spectrometric techniques used for structure elucidation, such as liquid chromatography coupled to time-of-flight mass spectrometry, quadrupole-time-of-flight and linear ion trap-Orbitrap hybrid mass spectrometry are presented on three case studies of pharmaceutical and pesticide transformation products in environmental matrices, such as wastewater and groundwater.
The environment is contaminated by a number of organic micropollutants released from urban, industrial, and agricultural activities, many of which still remain undetected. Although environmental monitoring includes more and more organic compounds, such as biocides, pesticides and pharmaceuticals, the analyses still mainly focus on parent compounds. However, the environmental exposure to their transformation products can be relevant as shown for pesticides in groundwater in the USA (Kolpin et al. 1997, 2004; Boxall et al. 2004) as well as in Switzerland (Hanke et al. 2007). In both studies, several pesticide transformation products (such as metolachlor-ESA or -OXA from the parent pesticide metolachlor) were found in higher concentrations in groundwater than the parent compounds. In the case of pharmaceuticals, human metabolites are excreted from the human body instead of or along with the parent compounds, often in considerable amounts. There is very limited knowledge on the environmental behaviour of those human metabolites. Some metabolites, such as conjugates of sulfamethoxazole and ethinylestradiol are cleaved back to the parent compound already in the sewer or in wastewater treatment plants (WWTP) (D’Ascenzo et al. 2003; Göbel et al. 2005). Few recent studies include the fate of persistent human metabolites of pharmaceuticals in the aquatic environment. Bendz et al. (2005) detected human ibuprofen metabolites not only in the WWTP as Buser et al. (1999), but also in the receiving river, while carbamazepine metabolites were found in WWTP effluent and even in drinking water (Miao et al. 2005; Hummel et al. 2006). In contrast to human metabolism of pharmaceuticals, which is studied in detail before pharmaceuticals are approved, their fate in the environment, including transformation pathways and formation of stable transformation products, has gained attention only recently. Only sparse information is currently available on transformation products of pharmaceuticals and their human metabolites formed in the environment or wastewater treatment plants (Kosjek et al. 2007).
As a consequence to findings of transformation products in the environment, the current European directive on drinking water as well as the guideline for groundwater quality with respect to pesticide contamination includes transformation products (Drinking Water Directive 1998; European Guidance Document 2003). Regarding chemical risk assessment, the need to identify and characterize relevant metabolites or transformation products is mentioned in several European directives and guidelines, for instance, in the EMEA guideline on the environmental risk assessment of medical products for human use (European Medicines Agency 2006) and the Council directive concerning the placing of plant protection products on the market (European Directive 1991). However, little concrete guidance on how to identify relevant transformation products is given.
Apart from the difficult selection of relevant transformation products for monitoring purposes, there are several challenges in analyzing transformation products in environmental samples such as surface and ground water. The first is, that the generally low but nevertheless potentially toxicologically relevant concentrations in the ng L−1 range require enrichment, separation from the matrix, and sensitive detection. The second challenge is the clear identification of transformation products without reference standards, which are often not available. An additional challenge is the identification of previously unidentified transformation products, which have never been described in the literature.
If the elemental composition is known to unequivocally identify the molecular structure of a transformation product without a reference standard, nuclear magnetic resonance (NMR) analysis coupled with liquid chromatography (LC) would be the method of choice. Although LC-NMR was successfully applied to environmental samples in a few cases (Levsen et al. 2000; Reineke et al. 2008), it requires costly equipment and is not yet sensitive enough for the low concentrations typically found in environmental samples. In contrast, GC-MS-(MS) and LC-MS-(MS) allow quantification in the concentration level down to a few ng L−1. Without reference standards, a complicated interpretation of the fragmentation pattern in MS/MS or MSn spectra is indispensable, which may give decisive hints for the identification of unknown transformation products. In modern GC-MS instruments, an electron impact (EI) ionization source is normally employed to provide a wealth of structural information in the mass spectra. EI is performed at 70 eV, thus yielding mass spectra which are identical over time and between instruments for a given compound. The resulting spectra can then be matched against spectra of authentic compounds which may be found in extensive GC-MS libraries. This ability to match analytical data to known spectra can significantly facilitate the structural elucidation of unknowns (Chiron et al. 1997). On the other hand, many transformation products are polar compounds containing hydroxy-, carboxy-, or amino-functional groups which enable GC-MS analysis only after derivatization. Derivatization can be avoided by employing LC separation, followed by electrospray or atmospheric pressure chemical ionization and tandem mass spectrometry, which is therefore the preferred identification technique for polar transformation products (Eichhorn et al. 2005). Ionization under different conditions results in a number of possible fragmentation patterns for a given compound, and consequently no large LC-MS libraries are commercially available which complicates the identification procedure.
Comparison of current mass spectrometers concerning resolving power, mass accuracy, and sensitivity
Resolving powera (FWHM)
Mass accuracya (ppm)
Quadrupole ion trap (QIT; linear, 3D)
Sector field (magnetic/electric)
Fourier transform ion cyclotron resonance (FT-ICR)
Combination of two or more m/z separation devices of different types, the so-called hybrid mass spectrometer, can combine the advantages of two techniques. A triple quadrupole mass spectrometer with the final quadrupole replaced by a time-of-flight tandem mass spectrometry (QTOF) or linear ion trap combined with an orbitrap mass spectrometry (LTQ-Orbitrap) have especially been shown to enable fast, sensitive and reliable detection and identification of low molecular weight substances thanks to their high mass accuracy and mass resolution (Van Bocxlaer et al. 2005; Lacorte and Fernandez-Alba 2006; Bueno et al 2007; Krauss and Hollender 2008). Full-scan mass spectra acquired with high mass accuracy and resolution allow selective searching for the molecular ions of transformation products based on their exact mass, while MS/MS technology provides structural information based on compound fragmentation.
Several studies report the use of high-resolution mass spectrometry to screen for transformation products in biodegradation experiments or photolysis studies carried out in the laboratory (Ibanez et al. 2004; Durand et al. 2006; Gomez et al. 2008; Ruan et al. 2008). In these studies, transformation of the parent compounds is studied at high initial concentrations in controlled matrix. In that case, classical techniques such as UV-VIS spectrometry can help characterizing the products as shown in Längin et al. (2009). Screening and identification of pesticides and their transformation products in environmental samples by the combination of LC-ion trap with LC-TOF instruments have also been described (Hernández et al. 2004, 2005; Thurman et al. 2005). A systematic procedure to screen for large numbers of transformation products in environmental samples containing a variety of organic compounds at low concentrations in the ng L−1 range using an LTQ-Orbitrap has only recently been reported (Kern et al. 2009).
The scope of this chapter is to present the potential of new hybrid tandem mass spectrometers to identify and quantify transformation products generated by human metabolism, microbial degradation, or other environmental breakdown processes. The strategies to identify transformation products by different hyphenated mass spectrometric techniques are presented in case studies where the advantages and the limitations of the structure elucidation procedure are also discussed. The first case study deals with the identification of a transformation product which is produced from a pharmaceutical during microbial degradation in the wastewater treatment process. The QTOF technology may enable identification of degradation products that are not yet described in the literature. In contrast, human metabolites are studied in detail in drug development procedure and are stated in pharmaceutical dossiers. Reference standards are sometimes not available and therefore clear identification must be carried out using, for instance, LC-TOF as presented in the second case study for a human metabolite in wastewater. The compound structure was confirmed by a QTRAP. Finally, we present the identification of a pesticide transformation product from groundwater samples. In this case study, Orbitrap technology enabled the identification of the transformation product in low environmental concentrations concurrently with a targeted screening. The case studies presented herein not only include the application of three different hybrid LC-MS techniques, but at the same time we show the identification of transformation products in very different matrices from relatively pure groundwater to highly contaminated wastewater.
11.2 Case Studies for Identification of Transformation Products by Different High Resolution Mass Spectrometric Techniques
11.2.1 Case Study 1: Identification of a Biotransformation Product of the Pharmaceutical Diclofenac in Wastewater by Ultra Performance Liquid Chromatography Hyphenated with Quadrupole-Time-of-Flight Mass Spectrometry
Among the most powerful instruments for the identification of unknown analytes is the quadrupole - time-of-flight mass spectrometer (QTOF), a hybrid mass spectrometric system that combines the advantages of ion separation and the detection principle of time-of-flight (TOF) systems and the fragmentation obtained with MS2 experiments. TOF instruments provide full-scan sensitivity, high mass resolution (10,000-20,000, full width at half maximum), good mass - accuracy (<3 ppm), and theoretically limitless scan range (Campbell et al. 1998). However, structural elucidation with the stand-alone TOF is primarily feasible for compounds with easy in-source fragmentation or those having a characteristic isotopic pattern (Petrovic´ and Barceló 2007). As an alternative, the hybrid QTOF, in which the final resolving mass filter of a triple quadrupole is replaced by a TOF analyzer, also enables the acquisition of high resolution mass spectra with accurate masses for the product ions. This gives the analyst a much higher degree of certainty when identifying compounds in non-target analyses, by positively and unequivocally confirming target compounds (Van Bocxlaer et al. 2005; Petrovic´ and Barceló 2006). While this instrument is already a well established tool for the confirmation of target micropollutants in environmental matrices, its use for the identification of complete unknowns or transformation products is still growing. So far only a few studies have reported the application of QTOF in this field (Eichhorn et al. 2005; Pérez et al. 2007; Kosjek et al. 2008).
With this study the key attributes of the QTOF instrument were confirmed: MS/MS fragmentation, high resolution, good mass accuracy, high sensitivity, and the ability to record a complete mass spectrum for each pulse of ions injected into the device. Further, this study implies that environmental and wastewater treatment processes yield different transformation products than human metabolism does.
11.2.2 Case Study 2: Identification of the Human Pharmaceutical Metabolite N-Acetyl-4-Aminoantipyrine by Liquid Chromatography Combined with Time-of-Flight Mass Spectrometry and Quantification by Quadrupole/Linear Iontrap Mass Spectrometry
Structural elucidation with a self-standing TOF is only feasible for compounds with easy in-source fragmentation or a characteristic isotopic pattern. If a hybrid system such as a QTOF is not available, additional measurements on a low resolution tandem mass spectrometer can be acquired to confirm the structure suggested based on the molecular ion obtained by TOF. As an additional benefit, target analysis based on MS/MS fragmentation by an triple quadrupole or ion trap provides excellent performance for quantitative analysis because of its inherent selectivity and sensitivity (Barceló and Petrovic´ 2007; Hernando et al. 2007a, b). Ion traps (IT) are particularly powerful for unequivocal confirmation or elucidation of molecular structures, since very fast and sensitive full scan modes (including MS2 and MSn) can be applied. The latest generation of linear ion trap (LIT) mass spectrometers enables the use of selected reaction monitoring (SRM) dwell times as low as 2 ms without loss of sensitivity enabling multi-target methods. QqLIT systems offer hybrid triple quadrupole/linear ion trap capabilities. Working in LIT mode, the QTRAP systems provide improved performance and enhanced sensitivity in full scan MS (EMS) and product ion scan (enhanced product ion (EPI)) modes. An extra operational mode of this hybrid system is the possibility of combining in the same run, SRM and EPI scans, by the built-in information-dependent acquisition (IDA) software, thus obtaining at the same time quantification and additional structural information.
This case study describes an analytical protocol that combines the use of QTRAP and TOF instruments to achieve both accurate and reliable target compound monitoring and identification of one of the major known metabolites of the antipyretic drug dipyrone (Bueno et al. 2007). The analytical strategy proposed in this work provides a comprehensive approach to increase the scope of a monitoring program for the identification of emerging contaminants (including transformation products and metabolites) in wastewater.
The chromatographic separations in both QTRAP and TOF systems were performed using an HPLC (series 1100, Agilent Technologies, Palo Alto, CA) equipped with a reversed-phase C-18 analytical column (Zorbax SB, Agilent Technologies) of 5-μm particle size, 250-mm length, and 3.0-mm i.d. Gradient LC elution was performed with 0.1% formic acid and 5% MilliQ water in acetonitrile as mobile phase A, and 0.1% formic acid in water (pH 3.5) as mobile phase B (for details see Bueno et al. 2007)
Identification of transformation products by QTRAP systems in wastewater samples was reinforced by the acquisition of three transitions. Additionally, confirmation by ratio of SRM transitions was also used as an identification criterion and as a way to detect possible contributions of matrix interferences to the transition intensities, thus avoiding overestimations or false positive findings in quantitative analysis. For instance, the metabolite of carbamazepine, carbamazepine 10,11-epoxide was confirmed by the acquisition of three SRM transitions (253.2 → 180.2; 253.2 → 236.2 and 253.2 → 210.2). By IDA software, QTRAP systems enable the application of survey scans in SRM mode and EPI mode in a single run. This alternative is useful for compounds for which the second transition is not detected or is present at low intensity and additional structural information is required for a suitable confirmation.
In summary, target analysis of contaminants by QTRAP provided quantitative results for a large group of selected compounds. The analyses by TOF-MS enabled the identification of non-target compounds in wastewater samples.
11.2.3 Case Study 3: Identification of A Transformation Product of the Pesticide Chloridazon in Groundwater by Liquid Chromatography Combined with Linear Iontrap-Orbitrap Mass Spectrometry
Orbitrap technology was introduced to the market in 2005. The hybrid system of linear ion trap combined with the new orbitrap technology (LTQ-Orbitrap) combines high sensitivity with high mass resolution (R > 100,000) and high mass accuracy (<2 ppm) (Hu et al. 2005; Makarov et al. 2006). In recent years several studies reported the use of this new technology to identify unknown micropollutants, metabolites, and transformation products in laboratory studies or environmental samples including surface and groundwater (Peterman et al. 2006; Ruan et al. 2008; Reineke et al. 2008; Kern et al. 2009).
As an example of the use of LTQ-Orbitrap, we describe the separation, detection, and successful identification of a transformation product of chloridazon in groundwater parallel to a multi-targeted screening. As part of a Swiss national survey in 2008, we screened approximately 20 groundwater samples from various catchments within both agricultural and urban areas for the occurrence of more than 200 pharmaceuticals, pesticides, biocides, and their transformation products. Additionally, the samples were analysed for non-target compounds using accurate mass screening. For this purpose, all samples were enriched using solid phase extraction (SPE). Subsequently, 20 μL of the SPE extract were injected into the LC system. Chromatographic separation of the extracts was achieved on a C-18 column (XBridge, Waters, 50 × 2.1 mm, particle size of 3.5 µm) using gradient elution with methanol and water (0.1% formic acid) at a flow rate of 200 µL min−1. After electrospray ionisation in the positive and negative mode, ions were detected by a LTQ- Orbitrap XL mass spectrometer (Thermo Fisher Scientific Corporation). High-resolution mass spectra (HR-MS) with a resolution of 60,000 were recorded to extract the chromatograms of target and non-target analytes. To confirm peak findings, data-dependent high-resolution product ion spectra (HR-MSMS) at a resolution of 7,500 were also produced. In order to receive more than ten HR-MS scans for each peak and simultaneously enough HR-MSMS within one chromatographic measurement, the resolution had to be set to this relatively low value. Mass calibration was carried out with external standard calibration compounds and a typical mass accuracy of <3 ppm was achieved.
As an example, the protonated molecule [M+H]+ with an accurate mass of 160.0272 occurred in nearly all groundwater samples with an intense peak at 2.2 min (see Fig. 11.4b). By taking the accurate mass and the isotope pattern into account, the elemental composition C5H6Cl1N3O1 could be unequivocally assigned to this peak by constraining the atoms to C, H, N, O, S, Cl, and Br for the elemental formula fit. The excellent match of the measured and theoretical isotope pattern is depicted in Fig. 11.4d and e. Searches in the Scifinder and Pubchem data base for C5H6Cl1N3O1 resulted in approximately 100 possible chemical structures. By comparing the measured HR-MSMS with predicted mass spectra proposed by the software Massfrontier (Thermo Fisher, USA) along with estimated retention times for all possible structures from the data base search, the best match for the identified elemental composition was determined to be chloridazon-methyl-desphenyl. Because a reference standard was available for this compound, the retention time and the HR-MSMS were matched between the sample (Fig. 11.4f) and the reference standard (Fig. 11.4g). Due to this comparison it could be unequivocally confirmed that the unknown compound is indeed chloridazon-methyl-desphenyl.
In summary, the LTQ-Orbitrap instrument concurrently enables a multi-targeted screening (with sensitivity comparable to a tandem mass spectrometer) and an identification of unknowns based on high mass resolution and mass accuracy for molecular ions and fragments.
The three case studies demonstrate that hybrid tandem mass spectrometry, which combines two mass spectrometric technologies including high resolution technique, opens possibilities for identification of polar transformation products without reference standards and even gives decisive hints for the identification of previously unknown transformation products. The hybrid mass spectrometry technology can be applied to different environmental matrices from relatively pure groundwater to highly contaminated wastewater. The new generation of instruments allows the detection of concentrations down to the low ng per liter range. Since the software tools for an automatic non-targeted screening mostly do not provide sufficient support and are demanding to work with, the detection of unknown transformation products is still time consuming and requires analysis by those with a high level of chemical expertise. The examples presented herein and described in the literature on the elucidation of transformation products are still scarce and more studies are needed to improve the knowledge about the occurrence of transformation products in the environment. Along with the identification and quantification of these compounds, the toxicity assessment is another important task, which may help to clarify the burden that the transformation products pose to human health and the environment.
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