The Challenge of the Identification and Quantification of Transformation Products in the Aquatic Environment Using High Resolution Mass Spectrometry

  • Juliane Hollender
  • Heinz Singer
  • Dolores Hernando
  • Tina Kosjek
  • Ester Heath
Chapter
Part of the Environmental Pollution book series (EPOL, volume 16)

Abstract

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.

11.1 Introduction

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.

A new approach to overcome the limitations discussed for GC-MS and LC-MS is to employ high-resolution mass spectrometry detection technology. Table 11.1 provides an overview of existing commercially available mass spectrometric techniques with respect to resolution, mass accuracy and sensitivity. The most common mass spectrometer in organic trace analytics is the triple quadrupole mass spectrometer, which selectively filters ions based on their mass-to-charge ratio (m/z) in two consecutive quadrupoles combined by a collision cell. It uses oscillating electrical fields to selectively stabilize or destabilize the paths of ions passing through a radio frequency (RF) quadrupole field. The quadrupole ion trap and linear quadrupole ion trap work on the same physical principles as the quadrupole mass analyzer, but the ions are trapped and sequentially ejected. In contrast, in time-of-flight mass spectrometry, ions are accelerated by an electrical field to the same kinetic energy with the velocity of the ion depending on its m/z. Thus, the time ions need to reach the detector can be used to determine the m/z. Sector field mass analyzers, which are nowadays rarely utilized in organic trace analytics, use an electric and/or magnetic field to affect the path and/or velocity of the ions. According to their m/z the ions are differently deflected. In the relatively new Orbitrap mass spectrometer ions are electrostatically trapped in an orbit and the mass is measured by detecting the image current produced by the ions oscillating in the presence of an electric field. The frequencies of these image currents depend on the m/z of the ions. Mass spectra are obtained by Fourier transformation of the recorded image currents. In very costly Fourier transform ion cyclotron resonance mass spectrometer the image current is produced in a magnetic field which enables superior resolution and mass accuracy.
Table 11.1

Comparison of current mass spectrometers concerning resolving power, mass accuracy, and sensitivity

Mass spectrometer

Resolving powera (FWHM)

Mass accuracya (ppm)

Sensitivitya (absolute)

Quadrupole (Q)

Unit resolutionb

100

fg-pg

Quadrupole ion trap (QIT; linear, 3D)

20,000

50

fg-pg

Time-of-flight (TOF)

20,000

3

pg

Sector field (magnetic/electric)

80,000

2

fg-pg

Orbitrap

100,000

2

fg-pg

Fourier transform ion cyclotron resonance (FT-ICR)

1,000,000

1

fg-pg

a  Common values for low mass range (about m/z of 400); mass resolution is dependent on different parameters like scan speed, mass, instrument design, etc.; special instruments can reach better values.

b  Unit mass resolution is the resolution for standard quadrupole instruments; with special hyperbolic quadrupole instruments a resolving power of 5,000 and a mass accuracy of 5 ppm can be achieved.

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).

As an example of use of QTOF-MS, in this paper we describe the separation, detection, and successful identification of a nitro-analogue of diclofenac, a biotransformation product produced in a pilot wastewater treatment plant. At the outset of the study, emphasis was placed on quality chromatographic separation, which is of great importance in a complex environmental matrix, such as wastewater. Thus, ultra performance liquid chromatography (UPLC) was employed and enabled elution of the analytes in narrow, concentrated bands resulting in improved resolution, increased peak capacity, and increased speed of chromatographic separation (Petrovic´ et al. 2006). This was performed with a Waters Acquity UPLC system (Waters Corp. Milford, MA, USA), equipped with a C-18 column with a 1.7 µm particle size (Waters Acquity 50 × 2.1 mm) using water / methanol gradient elution at a flow rate of 0.3 mL min−1. The UPLC system was hyphenated to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (QTOF Premier, Waters, Milford, Massachusetts, USA). To aid in the detection of biotransformation products, post-acquisition data processing was employed using the MetaboLynxTM software package. The algorithm, a part of MassLynx v4.1 software (Waters), searches extracted mass chromatograms for expected transformation products based on predicted or unpredicted molecular changes relative to the parent compound and thus aids in the detection and identification of unknowns, particularly those buried in spectral noise. The software compares mass spectral chromatograms between a control and a sample (Freed et al. 2004). Thus, the use of MetaboLynxTM software for comparison of treated and untreated wastewater samples spiked with diclofenac resulted in detection of diclofenac biotransformation product, eluted at 5.0 min. Accordingly, the isotopic cluster analysis was performed in order to determine the isotope ratio between cluster ion fragments and yielded the same results. Figure 11.1 illustrates a segment from the MetaboLynx report file with the extracted mass chromatogram of the biotransformation product (top right), its mass spectrum (top left), and the absence in the control sample chromatogram (bottom).
Fig. 11.1

Extracted from the MetaboLynx report file: analyte (top) and control (bottom) sample mass chromatograms and TOF-ESI(−) spectrum (left) of a peak eluting at tR 4.95 min

The high resolution and accurate mass measurements provided by the TOF mass analyser identified the deprotonated molecular mass [M − H] 338.9945. Using the “Elemental composition calculator” tool (±10 ppm mass error, C: 0-15, H: 0-20, N: 0-3, O: 0-10, Cl: 0-2) we were able to assign a highly probable elemental formula of the diclofenac biotransformation product (C14H10N2O4Cl2). Comparing to the elemental formula of diclofenac (C14H11NO2Cl2), the biotransformation product shows a substitution of a hydrogen atom for a NO2 group. The structure of the biotransformation product was studied based on its TOF-MS-(ESI(-)) and TOF-MSMS-(ESI(-)) fragmentation (Fig. 11.2). Besides the deprotonated molecule [M − H] (m/z 339), the elimination of CO2 to form m/z 295 also occurred under MS conditions due to in source fragmentation. The TOF-ESI(−) mass spectrum in Fig. 11.2a illustrates a dichloro isotopic pattern in both, m/z 339 and 295. The collision induced dissociation of m/z 339 (Fig. 11.2b) resulted in further losses of HCl with m/z 259 and homolytic cleavages of NO· with m/z 229 or NO2· with m/z 213. The product ion with m/z 223 is indicative of the loss of HCl from m/z 259, while m/z 193 corresponds to the loss of HCl from the product ion with m/z 229. The ion fragments proposed in Fig. 11.2 were compared with fragmentation pattern of parent compound and confirmed by accurate mass measurements in which the mass error did not exceed 0.7 mmu. The identification procedure is described in detail in Kosjek et al. (2008). In conclusion, the results indicate that the biotransformation yields a nitro-analogue of diclofenac. However, the exact position of the nitro group within the molecule could not be derived from the MS/MS data, and further investigations applying nuclear magnetic resonance (NMR) are necessary for complete structure elucidation. The incorporation of the NO2 group into the aromatic ring is a rather unusual transformation process, which does not occur during human metabolism. However, this transformation has previously been reported to occur on pesticides in the environment (Hernández et al. 2008) and is reasoned by the presence of nitrate in aquatic media (Hogenboom et al. 1999; Kosjek et al. 2008).
Fig. 11.2

Top: TOF-ESI(−) mass spectrum of biotransformation product (a); bottom: MS/MS spectrum of m/z 339 (b); proposed chemical structures of deprotonated molecule (m/z 339, c) and principal product ions 295 (d), 259 (e), 229 (f) and 213 (g) (Reproduced from Kosjek et al. (2008). With permission of Elsevier)

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)

As an example, the identification of N-acetyl-4-aminoantipyrine (4-AAA) by a TOF system is shown in Fig. 11.3. This major human metabolite of the antipyretic drug dypirone was identified for the first time in wastewater and surface water by Zuehlke et al. (2004). The strategy for identifying non-target analytes in the samples was based on three steps: (a) selection of the extracted ion chromatogram (XIC) for the target m/z, (20 mmu); (b) background-subtracted mass spectrum; (c) verification of accurate mass and elemental composition of the molecule and fragment ions. The agreement between the measured and calculated masses within a <5 ppm error level, along with matching retention times and mass spectra if reference standards are available, provided an unequivocal confirmation of the compounds in the samples. Analysis of spiked wastewater extracts resulted in errors lower than 2 ppm for the target compounds. Applying this strategy, other metabolites were also identified (4-dimethylaminoantipyrine; N-formyl-4-aminoantipyrine; 4-amino-antipyrine; antipyrine), confirmed with the acquisition of the appropriate standards, and finally included in the monitoring program.
Fig. 11.3

Identification of N-acetyl-4-aminoantipyrine (4-AAA), a major metabolite of the antipyretic drug dipyrone, by TOF system

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.

For the identification of more than 200 compounds, the accurate masses were extracted from the HR-MS-TIC with a mass filter of 5 ppm and confirmed by matching the HR-MSMS and the retention time with the related reference standards. To find unknowns, non-target compound detection was performed by filtering the total acquired mass range (115-1,000 m/z) of the HR-MS-TIC (Fig. 11.4a) with a 5 ppm mass extraction window using the Formulator software (Thermo Fisher, USA). As a result, up to 5,000 extracted ion chromatograms containing peaks with a signal-to-noise ratio greater than 5 were collected per sample. After sorting the data set by retention time and peak intensity, compound peaks with high signal intensity and distinct isotopic patterns were processed further.
Fig. 11.4

Chromatograms and spectra of an enriched ground water sample for the identification of chloridazon-methyl-desphenyl as a mobile and persistent transformation product of the herbicide chloridazon (a): Total ion chromatogram of HR-MS scans (R 60’000, 115-2,000 m/z) (b): Extracted ion chromatogram (5 ppm) of [M + H]+ 160.0272 m/z (c): Data dependent HR-MSMS scan of 160.0272 (R 7’500, 50-175 m/z) (d): Measured HR-MS at 2.2 min (R 60’000, 115-2,000 m/z) (e): Theoretical HR-MS of the elemental composition C5H6ClN3O ([M + H]+=160.0272) (f): Measured HR-MSMS of 160.0272 at 2.2 min, marked fragments (*) were predicted by Massfrontier G: Measured HR-MSMS of a standard solution of chloridazon-methyl-desphenyl

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.

Chloridazon is a systemic herbicide which is widely used for sugarbeet and beet crops. The biological formation of chloridazon-methyl-desphenyl from chloridazon takes place in soil (Roberts and Hutson 2002; EU DG 2006). Chloridazon (5-amino-4-chloro-2-phenylpyridazin-3(2H)-one) is first degraded to chloridazon-desphenyl which is further transformed to chloridazon-methyl-desphenyl (Fig. 11.5). Both transformation products were detected in many of the investigated groundwater samples in concentrations up to several 100 ng L−1. This is in agreement with findings of Weber et al. (2007), who described the occurrence of these compounds in surface, ground, and drinking water in Germany. The transformation products were most often found in higher concentrations than the parent compound which reinforces the need to include transformation products in environmental quality monitoring.
Fig. 11.5

Proposed formation of the transformation product methyl-desphenyl-chloridazon from chloridazon in soil (Weber et al. 2007)

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.

11.3 Conclusions

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.

References

  1. Barceló, D., & Petrovic´, M. (2007). Challenges and achievements of LC-MS in environmental analysis: 25 years on. Trends in Analytical Chemistry, 26, 2–11.CrossRefGoogle Scholar
  2. Bendz, D., Paxéus, N. A., Ginn, T. R., & Loge, F. J. (2005). Occurrence and fate of pharmaceutically active compounds in the environment, a case study: Höje River in Sweden. Journal of Hazardous Materials, 122, 195–204.CrossRefGoogle Scholar
  3. Boxall, A. B. A., Sinclair, C. J., Fenner, K., Kolpin, D., & Maud, S. J. (2004). When synthetic chemicals degrade in the environment. Environmental Science and Technology, 38, 368A-375A.CrossRefGoogle Scholar
  4. Bueno, M. J. M., Aguera, A., Gomez, M. J., Hernando, M. D., Garcia-Reyes, J. F., & Fernandez-Alba, A. R. (2007). Application of liquid chromatography/quadrupole-linear ion trap mass spectrometry and time-of-flight mass spectrometry to the determination of pharmaceuticals and related contaminants in wastewater. Analytical Chemistry, 79, 9372–9384.CrossRefGoogle Scholar
  5. Buser, H.-R., Poiger, T., & Muller, M. D. (1999). Occurrence and environmental behavior of the chiral pharmaceutical drug ibuprofen in surface waters and in wastewater. Environmental Science and Technology, 33, 2529–2535.CrossRefGoogle Scholar
  6. Campbell, J. M., Collings, B. A., & Douglas, D. J. (1998). A new linear ion trap time-of-flight system. Rapid Communications in Mass Spectrometry, 12, 1463–1474.CrossRefGoogle Scholar
  7. Chiron, S., Fernandez-Alba, A. R., & Rodriguez, A. (1997). Pesticide chemical oxidation an analytical approach. Trends in Analytical Chemistry, 15, 518–527.CrossRefGoogle Scholar
  8. D’Ascenzo, G., Di Corcia, A., Gentili, A., Mancini, R., Mastropasqua, R., Nazzari, M., et al. (2003). Fate of natural estrogen conjugates in municipal sewage transport and treatment facilities. Science of the Total Environment, 302, 199–209.CrossRefGoogle Scholar
  9. Drinking Water Directive. (1998). Council Directive 98/83/EC on the quality of water intended for human consumption. Google Scholar
  10. Durand, S., Legeret, B., Martin, A. S., Sancelme, M., Delort, A. M., Besse-Hoggan, P., et al. (2006). Biotransformation of the triketone herbicide mesotrione by a Bacillus strain. Metabolite profiling using liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry, 20, 2603–2613.Google Scholar
  11. Eichhorn, P., Ferguson, L., Pérez, S., & Aga, D. S. (2005). Application of ion trap-MS with H/D exchange and QqTOF-MS in the identification of microbial degradates of trimethoprim in nitrifying activated sludge. Analytical Chemistry, 77, 4176–4184.CrossRefGoogle Scholar
  12. EU DG of health and consumer protection, Plant Protection Products. (2006). Existing active substances decisions and review reports. Health and Protection Consumer DG. From http://ec.europa.eu/food/plant/protection/evaluation/exist_subs_rep_en.htm.
  13. European Directive. Concerning the placing of plant protection products on the market. 91/414/EEC (1991). OJ L 230, ISSN 0378 6978.Google Scholar
  14. European Guidance Document on the assessment of the relevance of metabolites in groundwater of substances regulated under council directive 91/414/EEC. (2003). Health and Consumer Protection Directorate-General, Sanco/221/2000.Google Scholar
  15. European Medicines Agency (EMEA). (2006). Guideline to the environmental risk assessment of medicinal products for human use. Committee for Medicinal Products for Human Use, EMEA/SWP/4447/00.Google Scholar
  16. Freed, A. L., Kale, U., Ando, H., Rossi, D. T., & Kingsmill, C. A. (2004). Improving the detection of degradants and impurities in pharmaceutical drug products by applying mass spectral and chromatographic searching. Journal of Pharmaceutical and Biomedical Analysis, 35, 727–738.CrossRefGoogle Scholar
  17. Göbel, A., Thomsen, A., McArdell, C. S., Joss, A., & Giger, W. (2005). Occurrence and sorption behavior of sulfonamides, macrolides, and trimethoprim in activated sludge treatment. Environmental Science and Technology, 39, 3981–3989.CrossRefGoogle Scholar
  18. Gomez, M. J., Sirtori, C., Mezcua, M., Fernandez-Alba, A. R., & Aguera, A. (2008). Photodegradation study of three dipyrone metabolites in various water systems: Identification and toxicity of their photodegradation products. Water Research, 42, 2698–2706.CrossRefGoogle Scholar
  19. Hanke, I., Singer, H., McArdell, C. S., Brennwald, M., Traber, D., Muralt, R., et al. (2007). Arzneimittel und Pestizide im Grundwasser. GWA - Gas und Wasserwirtschaft, 3, 187–196.Google Scholar
  20. Hernández, F., Ibá˜nez, M., Pozo, Ó. J., & Sancho, J. V. (2008). Investigating the presence of pesticide transformation products in water by using liquid chromatography-mass spectrometry with different mass analyzers. Journal of Mass Spectrometry, 43, 173–184.CrossRefGoogle Scholar
  21. Hernández, F., Ibánez, M., Sancho, J. V., & Pozo, O. J. (2004). Comparison of different mass spectrometric techniques combined with liquid chromatography for confirmation of 21 pesticides in environmental water based on the use of identification points. Analytical Chemistry, 76, 4349–4357.CrossRefGoogle Scholar
  22. Hernández, F., Pozo, Ó. J., Sancho, J. V., López, F. J., Marín, J. M., & Ibánez, M. (2005). Strategies for quantification and confirmation of multi-class polar pesticides and transformation products in water by LC-MS using triple quadrupole and hybrid quadrupole time-of-flight analyzers. Trends in Analytical Chemistry, 24, 596–612.CrossRefGoogle Scholar
  23. Hernando, M. D., Agüera, A., & Fernández-Alba, A. R. (2007a). LC-MS analysis and environmental risk of lipid regulators. Analytical and Bioanalytical Chemistry, 387, 1269–1285CrossRefGoogle Scholar
  24. Hernando, M. D., Gómez, M. J., Agüera, A., & Fernández-Alba, A. (2007b). LC-MS analysis of basic pharmaceuticals (beta-blockers and anti-ulcer agents) in wastewater and surface water. Trends in Analytical Chemistry, 26, 581–594.CrossRefGoogle Scholar
  25. Hogenboom, A. C., Niessen, W. M. A., & Brinkman, U. A. T. (1999). On-line solid-phase extraction-short-column liquid chromatography combined with various tandem mass spectrometric scanning strategies for the rapid study of transformation of pesticides in surface water. Journal of Chromatography A, 841, 33–44.CrossRefGoogle Scholar
  26. Hu, Q., Noll, R. J., Li, H., Makarov, A., Hardman, M., & Cooks, R. G. T. (2005). The Orbitrap: A new mass spectrometer. Journal of Mass Spectrometry, 40, 430–443.CrossRefGoogle Scholar
  27. Hummel, D., Löffler, D., Fink, G., & Ternes, T. A. (2006). Simultaneous determination of psychoactive drugs and their metabolites in aqueous matrices by liquid chromatography mass spectrometry. Environmental Science and Technology, 40, 7321–7328.CrossRefGoogle Scholar
  28. Ibanez, M., Sancho, J. V., Pozo, O. J., & Hernandez, F. (2004). Use of quadrupole time-of-flight mass spectrometry in environmental analysis: elucidation of transformation products of triazine herbicides in water after UV exposure. Analytical Chemistry, 76, 1328–1335.CrossRefGoogle Scholar
  29. Ibanez, M., Sancho, J. V., Pozo, O. J., Niessen, W., & Hernandez, F. (2005). Use of quadrupole time-of-flight mass spectrometry in the elucidation of unknown compounds present in environmental water. Rapid Communications in Mass Spectrometry, 19, 169–178.CrossRefGoogle Scholar
  30. Kern, S., Fenner, F., Singer, H. P., Schwarzenbach, R.P., & Hollender, J. (2009). Identification of transformation products of organic contaminants in natural waters by computer-aided prediction and high-resolution mass spectrometry. Environmental Science and Technology, 43, 7039–7046.CrossRefGoogle Scholar
  31. Kolpin, D. W., Kalkhoff, S. J., Goolsby, D. A., Sneck-Fahrer, D. A., & Thurman, E. M. (1997). Occurrence of selected herbicides and herbicide degradation products in Iowa’s Ground Water, 1995. Ground Water, 35, 679–688.CrossRefGoogle Scholar
  32. Kolpin, D. W., Schnoebelen, D. J., & Thurman, E. M. (2004). Degradates provide insight to spatial and temporal trends of herbicides in ground water. Ground Water, 42, 601–608.CrossRefGoogle Scholar
  33. Kosjek, T., Heath, E., Petrovic´, M., & Barceló, D. (2007). Mass spectrometry for identifying pharmaceutical biotransformation products in the environment. Trends in Analytical Chemistry, 26, 1076–1085.CrossRefGoogle Scholar
  34. Kosjek, T., Žigon, D., Kralj, B., & Heath, E. (2008). The use of quadrupole-time-of-flight mass spectrometer for the elucidation of diclofenac biotransformation products in wastewater. Journal of Chromatography A, 1215, 57–63.CrossRefGoogle Scholar
  35. Krauss, M., & Hollender, J. (2008). Analysis of nitrosamines in wastewater: Exploring the trace level quantification capabilities of a hybrid linear ion trap/orbitrap mass spectrometer. Analytical Chemistry, 80, 834–842.CrossRefGoogle Scholar
  36. Lacorte, S., & Fernandez-Alba, A. (2006). Time of flight mass spectrometry applied to the liquid chromatographic analysis of pesticides in water and food. Mass Spectrometry Reviews, 25, 866–880.CrossRefGoogle Scholar
  37. Längin, A., Alexy, R., König, A., & Kümmerer, K. (2009). Deactivation and transformation products in biodegradability testing of ss-lactams amoxicillin and piperacillin. Chemosphere, 75, 347–354.CrossRefGoogle Scholar
  38. Levsen, K., Preiss, A., & Godejohann, M. (2000). Application of high-performance liquid chromatography coupled to nuclear magnetic resonance and high-performance liquid chromatography coupled to mass spectrometry to complex environmental samples. Trends in Analytical Chemistry, 19, 27–48.CrossRefGoogle Scholar
  39. Makarov, A., Denisov, E., Kholomeev, A., Balschun, W., Lange, O., Strupat, K., et al. (2006). Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer. Analytical Chemistry, 78, 2113–2120.CrossRefGoogle Scholar
  40. Miao, X.-S., Yang, J.-J., & Metcalfe, C. D. (2005). Carbamazepine and its metabolites in wastewater and in biosolids in a municipal wastewater treatment plant. Environmental Science and Technology, 39, 469–7475.CrossRefGoogle Scholar
  41. Pérez, S., Eichhorn, P., & Barceló, D. (2007). Structural characterization of photodegradation products of enalapril and its metabolite enalaprilat obtained under simulated environmental conditions by hybrid quadrupole-linear ion trap-MS and quadrupole-time-of-flight-MS. Analytical Chemistry, 79, 8293–8300.CrossRefGoogle Scholar
  42. Peterman, S. M., Duczak, N., Jr., Kalgutkar, A. S., Lame, M. E., & Soglia, J. R. (2006). Application of a linear ion trap/Orbitrap mass spectrometer in metabolite characterization studies: examination of the human liver microsomal Metabolism of the non-tricyclic anti-depressant nefazodone using data-dependent accurate mass measurements. Journal of the American Society for Mass Spectrometry, 17, 363–375.CrossRefGoogle Scholar
  43. Petrovic´, M., & Barceló, D. (2006). Application of liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QqTOF-MS) in the environmental analysis. Journal of Mass Spectrometry, 41, 1259–1267.CrossRefGoogle Scholar
  44. Petrovic´, M., & Barceló, D. (2007). LC-MS for identifying photodegradation products of pharmaceuticals in the environment. Trends in Analytical Chemistry, 26, 486–493.CrossRefGoogle Scholar
  45. Petrovic´, M., Gros, M., & Barceló, D. (2006). Multi-residue analysis of pharmaceuticals in wastewater by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Journal of Chromatography A, 1124, 68–81.CrossRefGoogle Scholar
  46. Reineke, A., Preiss, M., Elend, M., & Hollender, J. (2008). Detection of methylquinoline transformation products in microcosm experiments and in tar oil contaminated groundwater using LC-NMR. Chemosphere, 70, 2118–2126.CrossRefGoogle Scholar
  47. Roberts, T., & Hutson, D. (2002). Metabolic pathways of agrochemicals on CD-ROM. Cambridge: The Royal Society of Chemistry.Google Scholar
  48. Ruan, Q., Peterman, S., Szewc, M. A., Ma, L., Cui, D., Humphreys, G. W., et al. (2008). An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection. Journal of Mass Spectrometry, 43, 251–261.CrossRefGoogle Scholar
  49. Thurman, E. M., Ferrer, I., Zweigenbaum, J. A., García-Reyes, J. F., Woodman, M., & Fernández-Alba, A. R. (2005). Discovering metabolites of post-harvest fungicides in citrus with liquid chromatography/time-of-flight mass spectrometry and ion trap tandem mass spectrometry. Journal of Chromatography A, 1082, 71–80.CrossRefGoogle Scholar
  50. Van Bocxlaer, J. F., Casteele, S. R. V., Van Poucke, C. J., & Van Peteghem, C. H. (2005). Confirmation of the identity of residues using quadrupole time-of-flight mass spectrometry. Analytica Chimica Acta, 529, 65–73.CrossRefGoogle Scholar
  51. Weber, W. H., Seitz, W., Schulz, W., & Wagener, H.-A. (2007). Detection of the metabolites desphenyl-chloridazon and methyldesphenyl-chloridazon in surface water, groundwater and drinking water. Vom Wasser, 105, 7–14.Google Scholar
  52. Zuehlke, S., Duennbier, U., & Heberer, T. (2004). Determination of polar drug residues in sewage and surface water applying liquid chromatography-tandem mass spectrometry. Analytical Chemistry, 76, 6548–6554.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Juliane Hollender
    • 1
  • Heinz Singer
    • 1
  • Dolores Hernando
    • 2
    • 3
  • Tina Kosjek
    • 4
  • Ester Heath
    • 4
  1. 1.EawagSwiss Federal Institute of Aquatic Science and TechnologyDübendorfSwitzerland
  2. 2.National Reference Centre for Persistent Organic Pollutants and Spanish REACH Reference Centre – University of AlcaláMadridSpain
  3. 3.University of AlmeriaAlmeriaSpain
  4. 4.Jožef Stefan InstituteLjubljanaSlovenia

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