Science of the Total Environment 493 (2014) 588–595
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Studies on the microbial biotransformation of the novel psychoactive substance methylenedioxypyrovalerone (MDPV) in wastewater by means of liquid chromatography-high resolution mass spectrometry/ mass spectrometry Marie Mardal, Markus R. Meyer ⁎ Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Saar, Germany
H I G H L I G H T S • • • •
strategy for studying wastewater biotransformation of drugs of abuse presented. MDPV was used to apply the novel approach on microbial biotransformation. MDPV phase I and II metabolites were studies concerning microbial biotransformation. biotransformation data as prerequisite for sewage profiling studies.
a r t i c l e
i n f o
Article history: Received 24 April 2014 Received in revised form 4 June 2014 Accepted 4 June 2014 Available online xxxx Editor: D. Barcelo Keywords: Sewage profiling Wastewater analysis Drugs of abuse MDPV High-resolution mass spectrometry Microbial biotransformation
a b s t r a c t Sewage profiling as a mean to estimate consumption of drugs of abuse is gaining increasing attention. However, only scarce data are available so far on the impact of microbial biotransformation on the presence and hence detectability of drugs of abuse and their metabolites in wastewater (WW) samples. The aim of this work was therefore to study the biotransformation pathways of the novel psychoactive substance 3,4-methylenedioxypyrovalerone (MPDV) in WW by incubating it, based on the OECD guideline 314 A. MDPV was incubated (100 μg/L) for 10 d at 22 °C in WW from a local WW treatment plant. Furthermore, urine and feces collected from rats administered 20 mg MDPV/kg BW were incubated correspondingly. Samples were worked-up either by centrifugation/filtration and solid-phase (HCX) extraction or QuEChERS. High resolution (HR) mass spectra (MS) were recorded using an Orbitrap mass spectrometer. All products were identified via their HR-MS2 spectra and chromatographic properties. The observed biotransformations in WW were: demethylenation and subsequent O-methylation, hydroxylation at the phenyl part, hydroxylation at the pyrrolidine part with subsequent methylation or oxidation, Ndemethylation, and hydroxylation at the alkyl part as well as combination of them. In total, 12 biotransformation products were identified after 10 days of incubation. Three of these biotransformation products were previously reported to be also rat and human metabolites. No additional MDPV biotransformation products could be found after incubating the rat urine and feces samples. Instead, the urinary phase II glucuronides were nearly completely cleaved after one day of WW incubation. The presented study indicates that demethylenyl-methyl MDPV, the most abundant metabolite in human urine, should be the best indicator in WW to estimate its use. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Studies on the biotransformation of compounds are critical for their thorough analysis in final matrices such as urine or wastewater (WW). After intake, a drug can be extensively metabolized in human and often only metabolites are excreted in urine and/or feces (Grigoryev et al.,
⁎ Corresponding author. Tel.: +49 6841 16 26430; fax: +49 6841 16 26051. E-mail address: [email protected] (M.R. Meyer).
http://dx.doi.org/10.1016/j.scitotenv.2014.06.016 0048-9697/© 2014 Elsevier B.V. All rights reserved.
2012; Moran et al., 2011). Therefore, analytical targets in urinalysis are often mainly or exclusively metabolites. These metabolites an the parent compound can be further biotransformed in the WW environment, leading to other targets than those for urinalysis. For pharmaceuticals and personal care products, biotransformation and biodegradation studies are regularly conducted using different models such as activated sludge (EMEA, 2006; Prasse et al., 2011; Quintana et al., 2005; Ternes et al., 1999; Wick et al., 2011). For emerging drugs of abuse or nowadays referred as novel psychoactive substances (NPS), such studies are still sparse. It was recently concluded that studies should be encouraged
M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595
on the metabolism of NPS such as 3,4-methylenedioxypyrovalerone (MDPV) and that the unchanged forms may be present only in a low amount in human urine and hence WW due to extensive metabolism (van Nuijs et al., 2013). Future research should therefore focus on metabolism and biodegradation of NPS to help select the most representative analytes for WW analysis. These data would also allow to identify possible overlapping microbial and mammalian biotransformation pathways. Several authors presented data on the stability of selected drugs in the WW microcosm and concluded that biotransformation pathways of the individual compounds should be taken into account when performing back-calculations in sewage epidemiology (Baker and Kasprzyk-Hordern, 2011a; Bisceglia and Lippa, 2013; Chen et al., 2013; Reid et al., 2013; van Nuijs et al., 2012). Therefore, the first aim of this study was to provide target analytes for WW screening procedures via identification of microbial biotransformation products (MBPs) exemplified for the NPS MDPV and its mammalian phase I and II metabolites. They should be incubated in WW under aerobic conditions based on the modified OECD 314 A guideline, extracted via solid-phase extraction (SPE) and analyzed by liquid-chromatography and high-resolution tandem mass spectrometry (LC-HR-MS/MS). The second aim was to provide semi-quantitative time-profiling data on the formation of the most abundant MBPs using QuEChERS and LC-HR-MS/MS. 2. Experimental 2.1. Reagents and materials R,S-Methylenedioxypyrovalerone HCl (MDPV) was obtained from LGC Standards (Wesel, Germany), RoQ QuEChERS kit from Phenomenex (California, USA), tramadol from Grunenthal (Stolberg, Germany), Syringe filters (1–2 μm) from Carl Roth (Karlsruhe, Germany), and syringes from Braun (Bad Arolsen, Germany). All other reagents and chemicals were purchased from VWR (Darmstadt, Germany). 2.2. Wastewater samples Samples were collected as grab samples of influent WW at the head of a local WW treatment plant (WWTP) in accordance to the OECD guideline 314A. WW was spiked with MDPV, urine, or feces within 3 h from sampling. At the day of WW sampling for the time profile experiments, the COD was 448 mg/L and the temperature was 12.9 °C (data from WWTP). 2.3. Incubation of MDPV Incubations were based on the OECD guideline 314 A for biodegradation in a sewer test system (OECD, 2008) with modifications. Briefly, the OECD guideline refers to low dissolved oxygen, continuous slow mixing, and mercuric chloride for poisoning prior to autoclaving. Modifications were as follows: reactors were closed by a syringe filter with a pore size of 0.2 μm allowing free flow of sterile air. Dissolved oxygen was measured to be 8.5 ± 0.6 mg/L over the course of the time profile study. Temperature was set to 22 °C and reactors were continuously and vigorously shaken. The biotic, abiotic, and control samples were prepared as follows: the biotic and the abiotic samples consisted of 150 mL WW and the clean sample of 150 mL demineralized water in amber glass bottles. All samples were then spiked with MDPV to achieve a final concentration of 100 μg/L. The abiotic and clean samples were additionally treated with NaN3 (final concentration, 0.2% w/v) according to Gonzalez-Marino et al. (2010). Individual samples were drawn through a plastic tube connecting a sterile plastic syringe and the reactor minimizing disturbance of microbial community and cross-contamination. Samples were collected once per day for the first seven days and at day 10.
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2.4. Incubation of rat urine and feces samples Urine and feces samples were collected from two male rats (Wistar, Ch. River, Sulzfeld, Germany) after administration (20 mg/kg body weight) of MDPV for toxicological diagnostic reasons (Meyer et al., 2010) according to the corresponding German law (http://www. gesetze-im-internet.de/tierschg/). Samples were collected once after 24 h and pooled afterwards. More details can be found in Meyer et al. (2010). Aliquots of 10 mL urine and 4 g feces were mixed with 40 mL WW and incubated as mentioned before, only using control and biotic reactors. Experiments were only conducted once due to limited amount of urine and feces. 2.5. Sample preparation 2.5.1. SPE for biotransformation studies All samples spiked with MDPV were filtered prior to SPE and all samples spiked with rat urine or feces were centrifuged for 1 min (18,407 g) prior to SPE. Volume filtered/centrifuged was 6 mL per sampling, ensuring 5 mL filtrate/supernatant available for further sample preparation. After incubation, a 5 mL aliquot was loaded on a Biotage Isolute HCX 200 mg column previously conditioned with 2 mL methanol and 2 mL distilled water. The column was washed with 2 mL distilled water, 2 mL 0.01 M HCl, and 2 mL methanol. Basic fractions were eluted using 1.5 mL methanol/NH3 33% (98/2 v/v). The eluate was then evaporated to dryness and reconstituted in 100 μL mobile phase A:B (50:50 v/v). Finally, 10 μL was injected onto the LC-HRMS/MS system. 2.5.2. QuEChERS for time profiling studies QuEChERS was done using an extracted 5 mL aliquot of the sample, which was mixed over ice with 2 g MgSO4, 0.5 g NaCl, and 5 mL acetonitrile containing 10 μg/L tramadol as internal standard (IS). The mixture was then shaken by hand and centrifuged for 5 min at 5 °C and 3076 g. A volume of 1.5 mL supernatant was then transferred to a tube containing 150 mg MgSO4 and 50 mg PSA (primary secondary amine). Samples were vigorously shaken and centrifuged for 5 min at 18,407 g. Finally, 10 μL of the supernatant was injected onto the LCHR-MS/MS. GraphPad Prism 5.0 (La Jolla, USA) was used for statistical analysis. 2.6. Identification of biotransformation products by LC-HR-MS/MS Biotransformation products were analyzed using a ThermoFisher Scientific Instruments (TF, Dreieich, Germany) Accela LC system consisting of a degasser, a quaternary pump, and an HTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) coupled to a TF Q-Exactive system equipped with a heated electrospray ionization II (HESI-II) source. 2.6.1. LC conditions Analytes were separated on a TF Accucore RP-MS column (150 × 2.1 mm, 2.6 μm) using gradient elution mode, with 0.1% (v/v) formic acid in Millipore water as mobile phase A and 0.1% (v/v) formic acid in acetonitrile as mobile phase B. The flow rate was 500 μL/min and the gradient programmed as follows: starting at 98% A, 5 min ramp to 45% A, hold 3 min at 10% A and ending with 2 min at 98% A. 2.6.2. MS settings Positive ion scan mode (MS1) from m/z 66.7–1000; resolution, 35,000; microscans, 1; sheath gas, 60 arbitrary units (AU); auxiliary gas, 10 AU; vaporizer temperature, 320 °C; spray voltage, 3 kV; ion transfer capillary temperature, 320 °C; maximum injection time, 120 ms; data-dependent mode (MS2); resolution, 17,500; ACG target, 2e5; maximum injection time, 250 ms; loop count, 5; normalized collision energy (NCE), 35%; stepped NCE, 50%; dynamic exclusion,
Δ from MDPV
−C +2H +O, −2H +O +O +O +O +O, +2H +O, +2H +O, +C, +2H +O, +C, +2H +2O
Most important fragment ions
84.0813, 126.1279, 135.0441, 149.0233, 175.0753, 205.0860 84.0814, 123.0443, 126.1279, 137.0235, 175.0755, 193.0861 84.0813, 126.1279, 137.0598, 151.0391, 175.0754, 207.1016 135.0442, 140.1071, 149.0236, 175.0756, 205.0856 84.0814, 135.0442, 142.1227, 149.0235, 221.0805 126.1279, 151.0390, 165.0183, 221.0808 100.0761, 135.0441, 142.1292, 149.0233, 175.0753, 205.0859, 84.0813, 98.0968, 135.0441, 149.0233, 203.0703, 232.0967, 274.1436 137.0596, 142.1226, 151.0388, 175.0750, 207.1010 135.0443, 149.0236, 174.0916, 204.1022, 276.1599 114.0915, 135.0441, 149.0233, 156.1384, 175.0755, 205.0863 135.0441, 149.0234, 174.0914, 204.1021, 274.1437 98.0605, 135.0443, 149.0236, 174.0915, 247.0839, 290.1388
Mass error ppm
8 s; microscans, 1. Targeted mode using a predefined m/z list was additionally used for recording the product ion spectra of less abundant products. TF Xcalibur 2.2 MS software was used for data analysis and RAW file data was exported to TF SIEVE® version 2.0, used for differential analysis-aided identification of biotransformation products. 3. Results and discussion 3.1. Sample preparation SPE was previously shown to be an appropriate sample preparation procedure not only for metabolism studies but also for analyzing low concentrated drugs in WW samples (Baker and Kasprzyk-Hordern, 2011a,b; Meyer et al., 2010; van Nuijs et al., 2013). Therefore, SPE was used for preparation of the WW incubations to ensure also extraction and up concentration of low abundant MBPs and to minimize possible matrix bias. However, as QuEChERS was a less labor-intensive and quicker preparation procedure, this was used to monitor the four most abundant MBPs and MDPV over a specific time course. Additionally, the presence of minor biotransformation products was also checked in the abiotic and control samples by QuEChERS. Its application in the field of biotransformation studies is particularly new but it was previously successfully used for analysis of drug and drugs of abuse in human body fluids and sewage sludge (Peysson and Vulliet, 2013; Usui et al., 2012). Using the QuEChERS procedure, seven out of 12 MBPs could be detected in comparison to the SPE preparation (Table 1). Nevertheless, as sample enrichment (either via SPE or liquid–liquid extraction) has been used, it cannot be excluded that some formed MBPs are missing (especially those with critical extraction behavior such as zwitterionic compounds).
Also detected in the biotic samples after QuEChERS.
C16H21NO3 C15H21NO3 C16H23NO3 C16H19NO4 C16H21NO4 C16H21NO4 C16H21NO4 C16H21NO4 C16H23NO4 C16H23NO4 C17H23NO4 C17H23NO4 C16H21NO5 a
Molecular formula MBP of MDPV
MDPV Demethylenyla Demethylenyl-methyla Oxo a Hydroxy-alkyl Hydroxy-aryla Hydroxy-pyrrolidinea Oxo-alkyl-N-dealkyl Demethylenyl-methyl-hydroxy-pyrrolidine Hydroxy-alkyl-N-dealkyl-a Methoxy-pyrrolidine Methoxy-dedihydro-N-dealkyl Hydroxy-alkyl-oxo-N-dealkyl a
No
276.1599 264.1594 278.1750 290.1386 292.1543 292.1543 292.1543 292.1543 294.1699 294.1699 306.1699 306.1699 308.1497
276.1594 264.1595 278.1751 290.1388 292.1544 292.1542 292.1541 292.1541 294.1702 294.1702 306.1701 306.1703 308.1493
3.2. Identification of microbial biotransformation products
I II III IV V VI VII VIII IX X XI XII XIII
Exact mass [M + H]+
Accurate mass [M + H]+
−1.81 0.38 0.36 0.69 0.34 −0.34 −0.68 −0.68 1.02 1.02 0.65 1.31 −1.62
M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595 Table 1 Molecular formulas, exact masses, accurate masses, mass error in ppm, most important fragment ions, and microbial biotransformation reactions of the individual MBPs observed after wastewater incubation of MDPV. MBPs are arranged to increasing molecular masses.
590
MDPV was incubated at a high concentration of 100 μg/L to allow also detection of minor MBPs. MBPs were tentatively identified via interpretation of their full product ion HR mass spectra (HRMS2) acquired in the data-dependent and targeted mode and their retention times in relation to the unchanged parent compound. A total of 12 MBPs could be tentatively identified by interpreting their HRMS2 spectra depicted in Fig. S1 (supplementary data). The exact masses of the protonated molecules, main fragment ions, and their corresponding elemental compositions are summarized in Table 1. All MBPs were exclusively detected in the biotic samples indicating that they should have been formed by microbial biotransformations and not due to unspecific chemical reactions. Since a higher scan rate was favored over a higher resolution for the MS method, all assigned fragments should have had a mass deviation less than 10 ppm and the protonated molecule less than 5 ppm. The parent compound formed mainly the six fragment ions A–F depicted in detail in Fig. 1. The chemical structure of the detected MBPs could then be postulated via interpretation of the mass shifts in their HRMS2 spectra in relation to the HRMS2 spectrum of the parent compound and in accordance to previously published data (Meyer et al., 2010). Important fragmentation patterns together with selected mass spectra of MBPs are shown in Fig. 2. Again, the HRMS2 data of all MBPs can be found in Table 1 and in Fig. S1 (supplementary data). 3.2.1. Pattern I Demethylenation without or with subsequent O-methylation was observed in MBP nos. II and III and IX, respectively. The demethylenation (MBP II) is indicated by a shift (−12.0000 u) in fragments A and D to fragment ions at 137.0223 and 193.0859, respectively. Stepwise further fragmentation of the fragment ion at m/z 193.0859 leads then to the fragment ions at 175.0753 and 123.0440. Odemethylation and O-methylation have been described to be mediated
M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595
by microorganisms in activated sludge (Chen et al., 2011; Quintana et al., 2005).
3.2.2. Pattern II Additional methylation (III and IX) is indicated by shift of +14.0156 (+ carbon, + 2*hydrogen) to fragment ions at m/z 151.0389 and 207.1015, respectively. Further stepwise fragmentation of the fragment ion at m/z 207.1015 leads to the fragment ions at m/z 175.0753 and 137.0597. These fragmentation pathways are in correspondence to previously reported results (Meyer et al., 2010). Oxidation of the pyrrolidine ring of MDPV to the corresponding lactam (MBP IV) is clearly indicated by a shift of 13.9792 u (+oxygen, − 2*hydrogen) in the fragment F (m/z 126.1277 to 140.1069) and in the mass of the protonated molecule (m/z 276.1594 to 290.1388). Such oxidations were also reported by Prasse et al. (2011) and Wick et al. (2011). Hydroxylation of the aryl (VI) or alkyl (V) part could be deduced via the shift corresponding to the introduction of one oxygen observed in fragment ions A (to m/z 221.0808), C (to m/z 151.0390), D (to m/z 165.0183), or F (to m/z 142.1227), respectively. Also the conserved fragment ion at m/z 126.1279 (VI) clearly indicated the hydroxylation to have occurred at the aromatic system. Hydroxylation of the pyrrolidine ring (VII) is indicated by a corresponding mass shift in fragment ions E and F (m/z 84.0813 to m/z 100.0761 and m/z 126.1277 to 142.1292) and the conserved fragment ions A, B, C, and D. Microbial hydroxylations of micropollutants were frequently reported under aerobic conditions (Helbling et al., 2010; Kern et al., 2009; Li et al., 2013; Perez and Barcelo, 2007; Quintana et al., 2005; Wick et al., 2011). The subsequent methylation of the hydroxy pyrrolidine part to methoxy-pyrrolidine MDPV (XI) is indicated by a further shift of fragment ions E and F by 14.0156 u (to m/z 114.0915 and 156.1384). N-Dealkylation of MDPV led to ring open structures present in MBPs VIII, X, XII, and XIII. Solely N-dealkylated MDPV was not detected but might have been formed as intermediate.
591
3.2.3. Pattern III In MBP no. X loss of water leads to the fragment ion at m/z 276.1594 and further fragmentation via amine-induced beta-cleavage and elimination of the methylenedioxy part to the fragment ion at 174.0913. Corresponding reactions were observed in the mass spectra of MBP nos. XII and XIII, which can be found in Fig. S1. Elimination of formaldehyde leads to the fragment ion at m/z 247.1437 in MBP no. XII. N-Dealkylation was reported to be a common reaction in activated sludge (Helbling et al., 2010; Wick et al., 2011). 3.2.4. Pattern IV The unique fragment ion at m/z 232.0967 in MBP no. VIII in contrast to MBPs X, XII, and XIII resulted from a combination of the amineinduced beta-cleavage and the carbonyl-induced alpha-cleavage. The combination of alkyl hydroxylation, oxidation of the pyrrolidine ring, and N-dealkylation observed in MBP no. XIII could be deduced by a fragmentation of the compound, after loss of water, comparable to MBP no. IV (oxo MDPV). In the spectrum of MBP no. IX, the aforementioned fragment ions indicated a demethylenation and subsequent methylation as well as a pyrrolidine ring hydroxylation. Finally, MBP no. X should be a combination of alkyl hydroxylation and N-dealkylation, documented by the initial loss of water (m/z 294.1702 to 276.1599) and further fragmentation in analogy to the other MBPs, considering the preferred alpha cleavage (nitrogen) and the respective mass shifts. In summary, the following biotransformation steps could be observed. Hydroxylations at the alkyl side chain, the pyrrolidine ring, and the aryl ring, demethylenation, N-dealkylation, reduction of the alcohol to the ketone and O-methylations as well as combinations of them. 3.3. Proposed biotransformation pathway The biotransformation pathways for microbial biotransformation of MDPV in WW are presented in Fig. 3 whereas the specific organisms and enzymes responsible for these reactions are not known under the
+
CH2 N
E
O C
O
HC
+ m/z 84.0813
CH3
+
F
N
D O
m/z 126.1277
m/z 149.0233
O CH3
O H + N
O
100
ms2 m/z 276
126.1279
m/z 276.1594
135.0441
+
C
O
O m/z 135.0440
O
+
CH
A O
O C
+
CH3
m/z 205.0859
relative abundance, %
CH2
O
149.0233
50
175.075 3 276.1594 84.0813 121.0285
CH3
205.0860
B HO
m/z 175.0754
233.1051
0 100
200
300
m/z
Fig. 1. ESI+ fragmentation scheme of methylenedioxypyrovalerone (MDPV) leading to the six characteristic fragment ions A–F (left side) and its full product ion HR ESI+ mass spectrum. Signals with relative abundances less than 3.5% were filtered out.
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M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595
pattern I
100
O CH
HO
+
C
HO HO
HO
HO
m/z 175.0753
m/z 193.0859
+ CH 2
relative abundance,%
HO
+
m/z 123.0440
O C
HO
+
ms2 m/z 264 123.0443
126.1279
II
72.0815 264.1595 137.0235
50 70.0658
175.0755 193.0861
84.0814
165.0913
HO
m/z 137.0223
0
221.1049
97.0891
100
pattern II
100
ms2 m/z 278
200 126.1279
III
137.0598
175.075 4
O CH
O
+
C
HO
m/z 175.0753
m/z 207.1015
CH 2
O
HO
HO
m/z 137.0597
O C
O
m/z
+
+
relative abundance, %
O
300
+
278.1751
151.0391
50
160.0516 207.1016
72.0815 84.0813
HO
m/z 151.0389
179.1069
119.0855
235.1208
0 100 100
200
X OH
O
O
CH3
O
CH3
+ NH2
O
H2O
m/z 294.1699
+
C
N
H2C
CH3
+
NH2
m/z 174.0913
m/z 204.1019
CH3
relative abundance, %
pattern III O
300
m/z
ms2 m/z 294 294.1702
174.0916
135.0443
50
276.1599
204.1022
149.0236 216.1023 246.1491 258.1500
84.0812
0 100 100
O
O
O CH3
O + NH2
O
m/z 292.1543
relative abundance, %
pattern IV O O
C
+
N
m/z 232.0968
CH3
200
ms2 m/z 292
VIII
300
m/z
232.096 7
292.1541
50 135.0441 98.0968
84.0813 70.0658
CH3
230.1 174
149.0233 172.1 120 203.0703
214.0862
110.0603
274.1436
0 100
200
300
m/z
Fig. 2. Important ESI+ fragmentation patterns and selected HR ESI+ mass spectra of microbial biotransformation products of MDPV.
incubation circumstances. In addition, the potential reversibility of particular steps such as the hydroxylation and oxidation to the lactam as well as the O-methylation and O-demethylation cannot be excluded. Again, the reactions could be attributed to microbial activity, since the MBPs were not observed in the abiotic and clean samples. As demethylenation and subsequent methylation are also a common metabolic step observed for other methylenedioxy drugs of abuse (Meyer and Maurer, 2010; Meyer et al., 2014; Staack and Maurer, 2005), corresponding MBPs should be expected. 3.4. Time profile of biotransformation products MDPV and the formation of its four most abundant MBPs were monitored over 10 days of incubation. The relative amounts of MDPV and the MBPs were estimated by integrating the peaks of their monoisotopic masses versus IS, using a mass tolerance of 5 ppm. Biotic incubations
were prepared in biological triplicates (biotic 1–3) to address biological variances. None of the four MBPs were present in abiotic and control samples and all were verified by their MS2 spectra. The time profiles for MDPV and its four most abundant metabolites are presented in Fig. 4. Demethylenyl-methyl MDPV (III) showed an increase over the time course of the study, except for biotic 1 reactor after 10 days. This reactor showed high biotransformation rate to hydroxy-pyrrolidine MDPV (VII) already after 2 days, whereas the signals from biotic 1 and biotic 2 increased after day 3. These findings are in accordance to the study by Quintana et al., who observed a lag phase of five days before the formation of metabolites of ketoprofen (Quintana et al., 2005). After four days, there was no clear increase or decrease of the hydroxy-pyrrolidine MDPV (VII) indicating a steady-state of bioformation and/or further biotransformation via reactions such as the N-dealkylation. Resulting hydroxy-alkyl-N-dealkyl MDPV (X) was decreasing towards the end of
M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595
the incubations and was possibly further transformed via oxidation of the pyrrolidine ring to hydroxy-alkyl-oxo-N-dealkyl MDPV (XIII), which set off after day 3. However, formation of MBPs did significantly (t-test) decrease the concentration of MDPV over 10 days only in biotic 1 and not in biotic 2 and 3. These findings are in line with previously presented data, showing the stability of MDPV in untreated WW and reported 15% degradation over 3 days (Chen et al., 2013). Furthermore, time profiling indicated an altered microbial composition and/or an altered metabolizing performance as expected. The present study was run over 10 days without added nutrients other than MDPV. Vital parameters over time are therefore different compared to sewer conditions e.g. oxygen levels and temperatures were higher than those mentioned in the OECD guideline 314 A. However, dissolved oxygen and biomass are even higher in activated sludge, which should therefore show a higher biocatalytic activity. Furthermore, the residence time for compounds in the sewer system would be much lower than 10 days. Despite aeration, pH did not change considerably throughout the test duration. The aim of this study was to investigate the possible biocatalytic fate of discharged drugs and their metabolites to present most representative analytes for WW analysis. Nevertheless, the study setup could only address the biotransformation in WW by suspended WW microbes and not the impact of biofilms also present in sewer systems. As no significant decrease of MDPV was observed in two of the three replicates (Fig. 4), the detected MBPs might be of minor relevance concerning analytical strategies. During the incubation, hydrolysis of biomass (toilet paper etc.) can release readily biodegradable substrates, absence of new nutrients can promote growth of microbes capable of utilizing specific carbon sources for energy, and dead microbes can lyse and serve as nutrients for other bacteria etc. (Jacobsen et al., 2013). The importance of biological variance was addressed by making the incubations in three different reactors. The reactors were treated alike and different time curves should be the result of different biocatalytic performance, analytical variations, and variations in sample preparation. The tested conditions should be seen as maximum degradation capacity rather than a simulation of real systems as experiments were aerated constantly throughout the duration of the test, which is in contrast to the OECD guideline.
3.5. Stability of rat urine/feces metabolites monitored in wastewater In WW incubated with rat urine/feces, caffeine was used as intrinsic IS as caffeine showed less than 5% dissipation in WW from primary effluent incubated at 20 °C for 2 days (Buerge et al., 2003). Caffeine was thus found suitable for IS for 2 days to correct for inter-day instrument performance. Previously identified rat phase I metabolites (Meyer et al., 2010) appeared to be stable over two days of incubation although it is not clear at this point in what extent phase I and phase II metabolites can be transformed into each other mimicking their stability. The stability of the previously reported four most abundant phase II metabolites, the glucuronides of demethylenyl MDPV, demethylenylmethyl MDPV, demethylenyl-methyl-oxo-pyrrolidine MDPV, and demethylenyl-methyl-hydroxy-aryl MDPV, was monitored in detail over two days of incubation. In the present study, all monitored signals from these four glucuronides were decreased by more than 99% after one day. Glucuronidase activity in activated sludge has been reported by monitoring the stability of 4-methylumbelliferyl-β-D-glucuronide, where 19% of the spiked glucuronides were cleaved after 6.7 h (Ternes et al., 1999). In another study, estrone 3-glucuronide and estriol 16α-glucuronide were no more detectable after 8 h of incubation in WW (Tucker et al., 1998). However, the detection of metabolites in samples drawn from the sewer systems with short residence time could be improved by enzymatic cleavage of the glucuronides and other conjugates during samples workup. Therefore, further studies will show this impact. In comparison to previously reported human and rat metabolism, microbial biotransformation of MDPV partly followed similar pathways (Meyer et al., 2010). Of the 12 tentatively identified MBPs, three have also been detected in human and/or rat urine samples. As demethylenyl-methyl MDPV was the most abundant MDPV metabolite in human urine and only the fourth most abundant in the presented study, it should thus be the most suitable target for sewage profiling. However, neither hydroxy-pyrrolidine MDPV (VII), hydroxyl-alkyl-Ndealkyl MDPV (X) nor hydroxyl-alkyl-oxo-N-dealkyl MDPV (XIII), the three most abundant MBPs in the presented study, were previously O HO
O
O NH
O
These conditions were chosen to allow thorough detection of biotransformation products and to assess the degradation potential of MDPV.
O
O
XII
593
demethylenation
I
N
O
N HO
II, h
O OH O
hydroxylation
n
tio
la
xy
ro
O
O
O O
N
IV, h
O
O
O
oxidation
O
VII
N
O
N
hydroxylation
HO
methylation
O
III, h
O hydroxylation
OH
O
N
O
V
dealkylation
methylation
d hy
O
OH
O NH
O
X
O O
O
O
VI
O
O N
XI
N
HO
OH O
oxidation
N
IX OH
O
O
OH
O
O
O NH
O
XIII
O
NH
VIII
O
Fig. 3. Pathways for microbial biotransformation of MDPV in wastewater. The numbering is in accordance to Table 1 and Fig. 1. h: also detected as human metabolite.
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Fig. 4. Time profile of relative MDPV concentration and formation of the four most abundant MBPs over 10 days.
detected in human or rat urine (Meyer et al., 2010). This offers the opportunity to distinguish between human metabolism and microbial biotransformation and shows again the importance to additionally gain knowledge about WW biotransformations as this can be different from mammalian metabolism. 4. Conclusions QuEChERS was successfully implemented in a biotransformation study in WW. In total, 12 microbial biotransformation products were tentatively identified after incubation of MDPV in WW for 10 days. However, biotransformation only leads to a significant decrease of the MDPV concentrations in one of the 3 biotic reactors and there was a lag phase of two days before biotransformation products could be detected. Urinary glucuronides of MDPV metabolites were almost completely cleaved after one day of WW incubation, which will help in phase I metabolite detection for sewage profiling. Finally, the presented study indicates that demethylenyl-methyl MDPV, the most abundant metabolite in human urine, should be the best biomarker in WW to estimate human consumption. It is expected that the presented data will help to develop analytical methods for MDPV analysis in WW for sewage epidemiology, which can be used in addition to classical studies such as surveys to estimate illicit drug use in populations Further studies are encouraged to investigate the rate of microbial cleaving of not only glucuronides, but also sulfates and other conjugates, of excreted metabolites. Acknowledgments The authors would like to thank Hans H. Maurer, Veit Flockerzi, Markus Bischoff, Julia Dinger, Andreas Helfer, Golo Magnus Meyer, Julian Michely, Martin-Simon Thomas, Armin A. Weber, Jessica Welter, Ann-Kathrin Ostermeyer, and Carina Wink for their support and/or discussion. This project has received funding from the European Union's Seventh Framework Programme for research, technological
development and demonstration under grant agreement no Marie Curie-FP7-PEOPLE Grant #317205. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2014.06.016. References Baker DR, Kasprzyk-Hordern B. Critical evaluation of methodology commonly used in sample collection, storage and preparation for the analysis of pharmaceuticals and illicit drugs in surface water and wastewater by solid phase extraction and liquid chromatography-mass spectrometry. J Chromatogr A 2011a;1218:8036–59. Baker DR, Kasprzyk-Hordern B. Multi-residue analysis of drugs of abuse in wastewater and surface water by solid-phase extraction and liquid chromatography-positive electrospray ionisation tandem mass spectrometry. J Chromatogr A 2011b;1218: 1620–31. Bisceglia KJ, Lippa KA. Stability of cocaine and its metabolites in municipal wastewater — the case for using metabolite consolidation to monitor cocaine utilization. Environ Sci Pollut Res Int 2013;21(6):4453–60. Buerge II, Poiger T, Muller MD, Buser HR. Caffeine, an anthropogenic marker for wastewater contamination of surface waters. Environ Sci Technol 2003;37:691–700. Chen X, Nielsen JL, Furgal K, Liu Y, Lolas IB, Bester K. Biodegradation of triclosan and formation of methyl-triclosan in activated sludge under aerobic conditions. Chemosphere 2011;84:452–6. Chen C, Kostakis C, Irvine RJ, White JM. Increases in use of novel synthetic stimulant are not directly linked to decreased use of 3,4-methylenedioxy-N-methylamphetamine (MDMA). Forensic Sci Int 2013;231:278–83. EMEA. Committee for medicinal products for human use: guideline on the environmental risk assessment of medicinal products for human use. emea/chmp/swp/4447/00 corr 1*; 2006. Gonzalez-Marino I, Quintana JB, Rodriguez I, Cela R. Determination of drugs of abuse in water by solid-phase extraction, derivatisation and gas chromatography-ion traptandem mass spectrometry. J Chromatogr A 2010;1217:1748–60. Grigoryev A, Kavanagh P, Melnik A. The detection of the urinary metabolites of 3[(adamantan-1-yl)carbonyl]-1-pentylindole (AB-001), a novel cannabimimetic, by gas chromatography-mass spectrometry. Drug Test Anal 2012;4:519–24. Helbling DE, Hollender J, Kohler HP, Singer H, Fenner K. High-throughput identification of microbial transformation products of organic micropollutants. Environ Sci Technol 2010;44:6621–7. Jacobsen TH, Vollertsen J, Nielson ARH. Sewer processes: microbial and chemical process engineering of sewer networks. CRC Press; 2013.
M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595 Kern S, Fenner K, Singer HP, Schwarzenbach RP, Hollender J. Identification of transformation products of organic contaminants in natural waters by computer-aided prediction and high-resolution mass spectrometry. Environ Sci Technol 2009;43:7039–46. Li ZZ, Li XF, Yang W, Dong X, Yu J, Zhu SL, et al. Identification and functional analysis of cytochrome P450 complement in Streptomyces virginiae IBL14. BMC Genomics 2013;14:130. Meyer MR, Maurer HH. Metabolism of designer drugs of abuse: an updated review. Curr Drug Metab 2010;11:468–82. Meyer MR, Du P, Schuster F, Maurer HH. Studies on the metabolism of the alphapyrrolidinophenone designer drug methylenedioxy-pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC-MS and LC-highresolution MS and its detectability in urine by GC-MS. J Mass Spectrom 2010;45: 1426–42. Meyer MR, Richter LHJ, Maurer HH. Methylenedioxy designer drugs: mass spectrometric characterization of their glutathione conjugates by means of liquid chromatographyhigh-resolution mass spectrometry/mass spectrometry and studies on their glutathionyl transferase inhibition potency. Anal Chim Acta 2014;822:37–50. Moran CL, Le VH, Chimalakonda KC, Smedley AL, Lackey FD, Owen SN, et al. Quantitative measurement of JWH-018 and JWH-073 metabolites excreted in human urine. Anal Chem 2011;83:4228–36. OECD. Test no. 314: simulation tests to assess the biodegradability of chemicals discharged in wastewater; 2008. Perez S, Barcelo D. Application of advanced MS techniques to analysis and identification of human and microbial metabolites of pharmaceuticals in the aquatic environment. TrAC Trends Anal Chem 2007;26:494–514. Peysson W, Vulliet E. Determination of 136 pharmaceuticals and hormones in sewage sludge using quick, easy, cheap, effective, rugged and safe extraction followed by analysis with liquid chromatography-time-of-flight-mass spectrometry. J Chromatogr A 2013;1290:46–61.
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Prasse C, Wagner M, Schulz R, Ternes TA. Biotransformation of the antiviral drugs acyclovir and penciclovir in activated sludge treatment. Environ Sci Technol 2011;45: 2761–9. Quintana JB, Weiss S, Reemtsma T. Pathways and metabolites of microbial degradation of selected acidic pharmaceutical and their occurrence in municipal wastewater treated by a membrane bioreactor. Water Res 2005;39:2654–64. Reid MJ, Derry L, Thomas KV. Analysis of new classes of recreational drugs in sewage: synthetic cannabinoids and amphetamine-like substances. Drug Test Anal 2013;6(1-2): 72–9. Staack R, Maurer H. Metabolism of designer drugs of abuse. Curr Drug Metab 2005;6: 259–74. Ternes TA, Kreckel P, Mueller J. Behaviour and occurrence of estrogens in municipal sewage treatment plants—II. Aerobic batch experiments with activated sludge. Sci Total Environ 1999;225:91–9. Tucker K, Adams M, Shaw L, Smith AJ. Human enamel as a substrate for in vitro acid dissolution studies: influence of tooth surface and morphology. Caries Res 1998;32:135–40. Usui K, Hayashizaki Y, Hashiyada M, Funayama M. Rapid drug extraction from human whole blood using a modified QuEChERS extraction method. Leg Med (Tokyo) 2012;14:286–96. van Nuijs AL, Abdellati K, Bervoets L, Blust R, Jorens PG, Neels H, et al. The stability of illicit drugs and metabolites in wastewater, an important issue for sewage epidemiology? J Hazard Mater 2012;239–240:19–23. van Nuijs AL, Gheorghe A, Jorens PG, Maudens K, Neels H, Covaci A. Optimization, validation, and the application of liquid chromatography-tandem mass spectrometry for the analysis of new drugs of abuse in wastewater. Drug Test Anal 2013. (Epub ahead of print). Wick A, Wagner M, Ternes TA. Elucidation of the transformation pathway of the opium alkaloid codeine in biological wastewater treatment. Environ Sci Technol 2011;45: 3374–85.
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Studies on the microbial biotransformation of the novel psychoactive substance methylenedioxypyrovalerone (MDPV) in wastewater by means of liquid chromatography-high resolution mass spectrometry/ mass spectrometry Marie Mardal, Markus R. Meyer ⁎ Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Saar, Germany
H I G H L I G H T S • • • •
strategy for studying wastewater biotransformation of drugs of abuse presented. MDPV was used to apply the novel approach on microbial biotransformation. MDPV phase I and II metabolites were studies concerning microbial biotransformation. biotransformation data as prerequisite for sewage profiling studies.
a r t i c l e
i n f o
Article history: Received 24 April 2014 Received in revised form 4 June 2014 Accepted 4 June 2014 Available online xxxx Editor: D. Barcelo Keywords: Sewage profiling Wastewater analysis Drugs of abuse MDPV High-resolution mass spectrometry Microbial biotransformation
a b s t r a c t Sewage profiling as a mean to estimate consumption of drugs of abuse is gaining increasing attention. However, only scarce data are available so far on the impact of microbial biotransformation on the presence and hence detectability of drugs of abuse and their metabolites in wastewater (WW) samples. The aim of this work was therefore to study the biotransformation pathways of the novel psychoactive substance 3,4-methylenedioxypyrovalerone (MPDV) in WW by incubating it, based on the OECD guideline 314 A. MDPV was incubated (100 μg/L) for 10 d at 22 °C in WW from a local WW treatment plant. Furthermore, urine and feces collected from rats administered 20 mg MDPV/kg BW were incubated correspondingly. Samples were worked-up either by centrifugation/filtration and solid-phase (HCX) extraction or QuEChERS. High resolution (HR) mass spectra (MS) were recorded using an Orbitrap mass spectrometer. All products were identified via their HR-MS2 spectra and chromatographic properties. The observed biotransformations in WW were: demethylenation and subsequent O-methylation, hydroxylation at the phenyl part, hydroxylation at the pyrrolidine part with subsequent methylation or oxidation, Ndemethylation, and hydroxylation at the alkyl part as well as combination of them. In total, 12 biotransformation products were identified after 10 days of incubation. Three of these biotransformation products were previously reported to be also rat and human metabolites. No additional MDPV biotransformation products could be found after incubating the rat urine and feces samples. Instead, the urinary phase II glucuronides were nearly completely cleaved after one day of WW incubation. The presented study indicates that demethylenyl-methyl MDPV, the most abundant metabolite in human urine, should be the best indicator in WW to estimate its use. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Studies on the biotransformation of compounds are critical for their thorough analysis in final matrices such as urine or wastewater (WW). After intake, a drug can be extensively metabolized in human and often only metabolites are excreted in urine and/or feces (Grigoryev et al.,
⁎ Corresponding author. Tel.: +49 6841 16 26430; fax: +49 6841 16 26051. E-mail address: [email protected] (M.R. Meyer).
http://dx.doi.org/10.1016/j.scitotenv.2014.06.016 0048-9697/© 2014 Elsevier B.V. All rights reserved.
2012; Moran et al., 2011). Therefore, analytical targets in urinalysis are often mainly or exclusively metabolites. These metabolites an the parent compound can be further biotransformed in the WW environment, leading to other targets than those for urinalysis. For pharmaceuticals and personal care products, biotransformation and biodegradation studies are regularly conducted using different models such as activated sludge (EMEA, 2006; Prasse et al., 2011; Quintana et al., 2005; Ternes et al., 1999; Wick et al., 2011). For emerging drugs of abuse or nowadays referred as novel psychoactive substances (NPS), such studies are still sparse. It was recently concluded that studies should be encouraged
M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595
on the metabolism of NPS such as 3,4-methylenedioxypyrovalerone (MDPV) and that the unchanged forms may be present only in a low amount in human urine and hence WW due to extensive metabolism (van Nuijs et al., 2013). Future research should therefore focus on metabolism and biodegradation of NPS to help select the most representative analytes for WW analysis. These data would also allow to identify possible overlapping microbial and mammalian biotransformation pathways. Several authors presented data on the stability of selected drugs in the WW microcosm and concluded that biotransformation pathways of the individual compounds should be taken into account when performing back-calculations in sewage epidemiology (Baker and Kasprzyk-Hordern, 2011a; Bisceglia and Lippa, 2013; Chen et al., 2013; Reid et al., 2013; van Nuijs et al., 2012). Therefore, the first aim of this study was to provide target analytes for WW screening procedures via identification of microbial biotransformation products (MBPs) exemplified for the NPS MDPV and its mammalian phase I and II metabolites. They should be incubated in WW under aerobic conditions based on the modified OECD 314 A guideline, extracted via solid-phase extraction (SPE) and analyzed by liquid-chromatography and high-resolution tandem mass spectrometry (LC-HR-MS/MS). The second aim was to provide semi-quantitative time-profiling data on the formation of the most abundant MBPs using QuEChERS and LC-HR-MS/MS. 2. Experimental 2.1. Reagents and materials R,S-Methylenedioxypyrovalerone HCl (MDPV) was obtained from LGC Standards (Wesel, Germany), RoQ QuEChERS kit from Phenomenex (California, USA), tramadol from Grunenthal (Stolberg, Germany), Syringe filters (1–2 μm) from Carl Roth (Karlsruhe, Germany), and syringes from Braun (Bad Arolsen, Germany). All other reagents and chemicals were purchased from VWR (Darmstadt, Germany). 2.2. Wastewater samples Samples were collected as grab samples of influent WW at the head of a local WW treatment plant (WWTP) in accordance to the OECD guideline 314A. WW was spiked with MDPV, urine, or feces within 3 h from sampling. At the day of WW sampling for the time profile experiments, the COD was 448 mg/L and the temperature was 12.9 °C (data from WWTP). 2.3. Incubation of MDPV Incubations were based on the OECD guideline 314 A for biodegradation in a sewer test system (OECD, 2008) with modifications. Briefly, the OECD guideline refers to low dissolved oxygen, continuous slow mixing, and mercuric chloride for poisoning prior to autoclaving. Modifications were as follows: reactors were closed by a syringe filter with a pore size of 0.2 μm allowing free flow of sterile air. Dissolved oxygen was measured to be 8.5 ± 0.6 mg/L over the course of the time profile study. Temperature was set to 22 °C and reactors were continuously and vigorously shaken. The biotic, abiotic, and control samples were prepared as follows: the biotic and the abiotic samples consisted of 150 mL WW and the clean sample of 150 mL demineralized water in amber glass bottles. All samples were then spiked with MDPV to achieve a final concentration of 100 μg/L. The abiotic and clean samples were additionally treated with NaN3 (final concentration, 0.2% w/v) according to Gonzalez-Marino et al. (2010). Individual samples were drawn through a plastic tube connecting a sterile plastic syringe and the reactor minimizing disturbance of microbial community and cross-contamination. Samples were collected once per day for the first seven days and at day 10.
589
2.4. Incubation of rat urine and feces samples Urine and feces samples were collected from two male rats (Wistar, Ch. River, Sulzfeld, Germany) after administration (20 mg/kg body weight) of MDPV for toxicological diagnostic reasons (Meyer et al., 2010) according to the corresponding German law (http://www. gesetze-im-internet.de/tierschg/). Samples were collected once after 24 h and pooled afterwards. More details can be found in Meyer et al. (2010). Aliquots of 10 mL urine and 4 g feces were mixed with 40 mL WW and incubated as mentioned before, only using control and biotic reactors. Experiments were only conducted once due to limited amount of urine and feces. 2.5. Sample preparation 2.5.1. SPE for biotransformation studies All samples spiked with MDPV were filtered prior to SPE and all samples spiked with rat urine or feces were centrifuged for 1 min (18,407 g) prior to SPE. Volume filtered/centrifuged was 6 mL per sampling, ensuring 5 mL filtrate/supernatant available for further sample preparation. After incubation, a 5 mL aliquot was loaded on a Biotage Isolute HCX 200 mg column previously conditioned with 2 mL methanol and 2 mL distilled water. The column was washed with 2 mL distilled water, 2 mL 0.01 M HCl, and 2 mL methanol. Basic fractions were eluted using 1.5 mL methanol/NH3 33% (98/2 v/v). The eluate was then evaporated to dryness and reconstituted in 100 μL mobile phase A:B (50:50 v/v). Finally, 10 μL was injected onto the LC-HRMS/MS system. 2.5.2. QuEChERS for time profiling studies QuEChERS was done using an extracted 5 mL aliquot of the sample, which was mixed over ice with 2 g MgSO4, 0.5 g NaCl, and 5 mL acetonitrile containing 10 μg/L tramadol as internal standard (IS). The mixture was then shaken by hand and centrifuged for 5 min at 5 °C and 3076 g. A volume of 1.5 mL supernatant was then transferred to a tube containing 150 mg MgSO4 and 50 mg PSA (primary secondary amine). Samples were vigorously shaken and centrifuged for 5 min at 18,407 g. Finally, 10 μL of the supernatant was injected onto the LCHR-MS/MS. GraphPad Prism 5.0 (La Jolla, USA) was used for statistical analysis. 2.6. Identification of biotransformation products by LC-HR-MS/MS Biotransformation products were analyzed using a ThermoFisher Scientific Instruments (TF, Dreieich, Germany) Accela LC system consisting of a degasser, a quaternary pump, and an HTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) coupled to a TF Q-Exactive system equipped with a heated electrospray ionization II (HESI-II) source. 2.6.1. LC conditions Analytes were separated on a TF Accucore RP-MS column (150 × 2.1 mm, 2.6 μm) using gradient elution mode, with 0.1% (v/v) formic acid in Millipore water as mobile phase A and 0.1% (v/v) formic acid in acetonitrile as mobile phase B. The flow rate was 500 μL/min and the gradient programmed as follows: starting at 98% A, 5 min ramp to 45% A, hold 3 min at 10% A and ending with 2 min at 98% A. 2.6.2. MS settings Positive ion scan mode (MS1) from m/z 66.7–1000; resolution, 35,000; microscans, 1; sheath gas, 60 arbitrary units (AU); auxiliary gas, 10 AU; vaporizer temperature, 320 °C; spray voltage, 3 kV; ion transfer capillary temperature, 320 °C; maximum injection time, 120 ms; data-dependent mode (MS2); resolution, 17,500; ACG target, 2e5; maximum injection time, 250 ms; loop count, 5; normalized collision energy (NCE), 35%; stepped NCE, 50%; dynamic exclusion,
Δ from MDPV
−C +2H +O, −2H +O +O +O +O +O, +2H +O, +2H +O, +C, +2H +O, +C, +2H +2O
Most important fragment ions
84.0813, 126.1279, 135.0441, 149.0233, 175.0753, 205.0860 84.0814, 123.0443, 126.1279, 137.0235, 175.0755, 193.0861 84.0813, 126.1279, 137.0598, 151.0391, 175.0754, 207.1016 135.0442, 140.1071, 149.0236, 175.0756, 205.0856 84.0814, 135.0442, 142.1227, 149.0235, 221.0805 126.1279, 151.0390, 165.0183, 221.0808 100.0761, 135.0441, 142.1292, 149.0233, 175.0753, 205.0859, 84.0813, 98.0968, 135.0441, 149.0233, 203.0703, 232.0967, 274.1436 137.0596, 142.1226, 151.0388, 175.0750, 207.1010 135.0443, 149.0236, 174.0916, 204.1022, 276.1599 114.0915, 135.0441, 149.0233, 156.1384, 175.0755, 205.0863 135.0441, 149.0234, 174.0914, 204.1021, 274.1437 98.0605, 135.0443, 149.0236, 174.0915, 247.0839, 290.1388
Mass error ppm
8 s; microscans, 1. Targeted mode using a predefined m/z list was additionally used for recording the product ion spectra of less abundant products. TF Xcalibur 2.2 MS software was used for data analysis and RAW file data was exported to TF SIEVE® version 2.0, used for differential analysis-aided identification of biotransformation products. 3. Results and discussion 3.1. Sample preparation SPE was previously shown to be an appropriate sample preparation procedure not only for metabolism studies but also for analyzing low concentrated drugs in WW samples (Baker and Kasprzyk-Hordern, 2011a,b; Meyer et al., 2010; van Nuijs et al., 2013). Therefore, SPE was used for preparation of the WW incubations to ensure also extraction and up concentration of low abundant MBPs and to minimize possible matrix bias. However, as QuEChERS was a less labor-intensive and quicker preparation procedure, this was used to monitor the four most abundant MBPs and MDPV over a specific time course. Additionally, the presence of minor biotransformation products was also checked in the abiotic and control samples by QuEChERS. Its application in the field of biotransformation studies is particularly new but it was previously successfully used for analysis of drug and drugs of abuse in human body fluids and sewage sludge (Peysson and Vulliet, 2013; Usui et al., 2012). Using the QuEChERS procedure, seven out of 12 MBPs could be detected in comparison to the SPE preparation (Table 1). Nevertheless, as sample enrichment (either via SPE or liquid–liquid extraction) has been used, it cannot be excluded that some formed MBPs are missing (especially those with critical extraction behavior such as zwitterionic compounds).
Also detected in the biotic samples after QuEChERS.
C16H21NO3 C15H21NO3 C16H23NO3 C16H19NO4 C16H21NO4 C16H21NO4 C16H21NO4 C16H21NO4 C16H23NO4 C16H23NO4 C17H23NO4 C17H23NO4 C16H21NO5 a
Molecular formula MBP of MDPV
MDPV Demethylenyla Demethylenyl-methyla Oxo a Hydroxy-alkyl Hydroxy-aryla Hydroxy-pyrrolidinea Oxo-alkyl-N-dealkyl Demethylenyl-methyl-hydroxy-pyrrolidine Hydroxy-alkyl-N-dealkyl-a Methoxy-pyrrolidine Methoxy-dedihydro-N-dealkyl Hydroxy-alkyl-oxo-N-dealkyl a
No
276.1599 264.1594 278.1750 290.1386 292.1543 292.1543 292.1543 292.1543 294.1699 294.1699 306.1699 306.1699 308.1497
276.1594 264.1595 278.1751 290.1388 292.1544 292.1542 292.1541 292.1541 294.1702 294.1702 306.1701 306.1703 308.1493
3.2. Identification of microbial biotransformation products
I II III IV V VI VII VIII IX X XI XII XIII
Exact mass [M + H]+
Accurate mass [M + H]+
−1.81 0.38 0.36 0.69 0.34 −0.34 −0.68 −0.68 1.02 1.02 0.65 1.31 −1.62
M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595 Table 1 Molecular formulas, exact masses, accurate masses, mass error in ppm, most important fragment ions, and microbial biotransformation reactions of the individual MBPs observed after wastewater incubation of MDPV. MBPs are arranged to increasing molecular masses.
590
MDPV was incubated at a high concentration of 100 μg/L to allow also detection of minor MBPs. MBPs were tentatively identified via interpretation of their full product ion HR mass spectra (HRMS2) acquired in the data-dependent and targeted mode and their retention times in relation to the unchanged parent compound. A total of 12 MBPs could be tentatively identified by interpreting their HRMS2 spectra depicted in Fig. S1 (supplementary data). The exact masses of the protonated molecules, main fragment ions, and their corresponding elemental compositions are summarized in Table 1. All MBPs were exclusively detected in the biotic samples indicating that they should have been formed by microbial biotransformations and not due to unspecific chemical reactions. Since a higher scan rate was favored over a higher resolution for the MS method, all assigned fragments should have had a mass deviation less than 10 ppm and the protonated molecule less than 5 ppm. The parent compound formed mainly the six fragment ions A–F depicted in detail in Fig. 1. The chemical structure of the detected MBPs could then be postulated via interpretation of the mass shifts in their HRMS2 spectra in relation to the HRMS2 spectrum of the parent compound and in accordance to previously published data (Meyer et al., 2010). Important fragmentation patterns together with selected mass spectra of MBPs are shown in Fig. 2. Again, the HRMS2 data of all MBPs can be found in Table 1 and in Fig. S1 (supplementary data). 3.2.1. Pattern I Demethylenation without or with subsequent O-methylation was observed in MBP nos. II and III and IX, respectively. The demethylenation (MBP II) is indicated by a shift (−12.0000 u) in fragments A and D to fragment ions at 137.0223 and 193.0859, respectively. Stepwise further fragmentation of the fragment ion at m/z 193.0859 leads then to the fragment ions at 175.0753 and 123.0440. Odemethylation and O-methylation have been described to be mediated
M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595
by microorganisms in activated sludge (Chen et al., 2011; Quintana et al., 2005).
3.2.2. Pattern II Additional methylation (III and IX) is indicated by shift of +14.0156 (+ carbon, + 2*hydrogen) to fragment ions at m/z 151.0389 and 207.1015, respectively. Further stepwise fragmentation of the fragment ion at m/z 207.1015 leads to the fragment ions at m/z 175.0753 and 137.0597. These fragmentation pathways are in correspondence to previously reported results (Meyer et al., 2010). Oxidation of the pyrrolidine ring of MDPV to the corresponding lactam (MBP IV) is clearly indicated by a shift of 13.9792 u (+oxygen, − 2*hydrogen) in the fragment F (m/z 126.1277 to 140.1069) and in the mass of the protonated molecule (m/z 276.1594 to 290.1388). Such oxidations were also reported by Prasse et al. (2011) and Wick et al. (2011). Hydroxylation of the aryl (VI) or alkyl (V) part could be deduced via the shift corresponding to the introduction of one oxygen observed in fragment ions A (to m/z 221.0808), C (to m/z 151.0390), D (to m/z 165.0183), or F (to m/z 142.1227), respectively. Also the conserved fragment ion at m/z 126.1279 (VI) clearly indicated the hydroxylation to have occurred at the aromatic system. Hydroxylation of the pyrrolidine ring (VII) is indicated by a corresponding mass shift in fragment ions E and F (m/z 84.0813 to m/z 100.0761 and m/z 126.1277 to 142.1292) and the conserved fragment ions A, B, C, and D. Microbial hydroxylations of micropollutants were frequently reported under aerobic conditions (Helbling et al., 2010; Kern et al., 2009; Li et al., 2013; Perez and Barcelo, 2007; Quintana et al., 2005; Wick et al., 2011). The subsequent methylation of the hydroxy pyrrolidine part to methoxy-pyrrolidine MDPV (XI) is indicated by a further shift of fragment ions E and F by 14.0156 u (to m/z 114.0915 and 156.1384). N-Dealkylation of MDPV led to ring open structures present in MBPs VIII, X, XII, and XIII. Solely N-dealkylated MDPV was not detected but might have been formed as intermediate.
591
3.2.3. Pattern III In MBP no. X loss of water leads to the fragment ion at m/z 276.1594 and further fragmentation via amine-induced beta-cleavage and elimination of the methylenedioxy part to the fragment ion at 174.0913. Corresponding reactions were observed in the mass spectra of MBP nos. XII and XIII, which can be found in Fig. S1. Elimination of formaldehyde leads to the fragment ion at m/z 247.1437 in MBP no. XII. N-Dealkylation was reported to be a common reaction in activated sludge (Helbling et al., 2010; Wick et al., 2011). 3.2.4. Pattern IV The unique fragment ion at m/z 232.0967 in MBP no. VIII in contrast to MBPs X, XII, and XIII resulted from a combination of the amineinduced beta-cleavage and the carbonyl-induced alpha-cleavage. The combination of alkyl hydroxylation, oxidation of the pyrrolidine ring, and N-dealkylation observed in MBP no. XIII could be deduced by a fragmentation of the compound, after loss of water, comparable to MBP no. IV (oxo MDPV). In the spectrum of MBP no. IX, the aforementioned fragment ions indicated a demethylenation and subsequent methylation as well as a pyrrolidine ring hydroxylation. Finally, MBP no. X should be a combination of alkyl hydroxylation and N-dealkylation, documented by the initial loss of water (m/z 294.1702 to 276.1599) and further fragmentation in analogy to the other MBPs, considering the preferred alpha cleavage (nitrogen) and the respective mass shifts. In summary, the following biotransformation steps could be observed. Hydroxylations at the alkyl side chain, the pyrrolidine ring, and the aryl ring, demethylenation, N-dealkylation, reduction of the alcohol to the ketone and O-methylations as well as combinations of them. 3.3. Proposed biotransformation pathway The biotransformation pathways for microbial biotransformation of MDPV in WW are presented in Fig. 3 whereas the specific organisms and enzymes responsible for these reactions are not known under the
+
CH2 N
E
O C
O
HC
+ m/z 84.0813
CH3
+
F
N
D O
m/z 126.1277
m/z 149.0233
O CH3
O H + N
O
100
ms2 m/z 276
126.1279
m/z 276.1594
135.0441
+
C
O
O m/z 135.0440
O
+
CH
A O
O C
+
CH3
m/z 205.0859
relative abundance, %
CH2
O
149.0233
50
175.075 3 276.1594 84.0813 121.0285
CH3
205.0860
B HO
m/z 175.0754
233.1051
0 100
200
300
m/z
Fig. 1. ESI+ fragmentation scheme of methylenedioxypyrovalerone (MDPV) leading to the six characteristic fragment ions A–F (left side) and its full product ion HR ESI+ mass spectrum. Signals with relative abundances less than 3.5% were filtered out.
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M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595
pattern I
100
O CH
HO
+
C
HO HO
HO
HO
m/z 175.0753
m/z 193.0859
+ CH 2
relative abundance,%
HO
+
m/z 123.0440
O C
HO
+
ms2 m/z 264 123.0443
126.1279
II
72.0815 264.1595 137.0235
50 70.0658
175.0755 193.0861
84.0814
165.0913
HO
m/z 137.0223
0
221.1049
97.0891
100
pattern II
100
ms2 m/z 278
200 126.1279
III
137.0598
175.075 4
O CH
O
+
C
HO
m/z 175.0753
m/z 207.1015
CH 2
O
HO
HO
m/z 137.0597
O C
O
m/z
+
+
relative abundance, %
O
300
+
278.1751
151.0391
50
160.0516 207.1016
72.0815 84.0813
HO
m/z 151.0389
179.1069
119.0855
235.1208
0 100 100
200
X OH
O
O
CH3
O
CH3
+ NH2
O
H2O
m/z 294.1699
+
C
N
H2C
CH3
+
NH2
m/z 174.0913
m/z 204.1019
CH3
relative abundance, %
pattern III O
300
m/z
ms2 m/z 294 294.1702
174.0916
135.0443
50
276.1599
204.1022
149.0236 216.1023 246.1491 258.1500
84.0812
0 100 100
O
O
O CH3
O + NH2
O
m/z 292.1543
relative abundance, %
pattern IV O O
C
+
N
m/z 232.0968
CH3
200
ms2 m/z 292
VIII
300
m/z
232.096 7
292.1541
50 135.0441 98.0968
84.0813 70.0658
CH3
230.1 174
149.0233 172.1 120 203.0703
214.0862
110.0603
274.1436
0 100
200
300
m/z
Fig. 2. Important ESI+ fragmentation patterns and selected HR ESI+ mass spectra of microbial biotransformation products of MDPV.
incubation circumstances. In addition, the potential reversibility of particular steps such as the hydroxylation and oxidation to the lactam as well as the O-methylation and O-demethylation cannot be excluded. Again, the reactions could be attributed to microbial activity, since the MBPs were not observed in the abiotic and clean samples. As demethylenation and subsequent methylation are also a common metabolic step observed for other methylenedioxy drugs of abuse (Meyer and Maurer, 2010; Meyer et al., 2014; Staack and Maurer, 2005), corresponding MBPs should be expected. 3.4. Time profile of biotransformation products MDPV and the formation of its four most abundant MBPs were monitored over 10 days of incubation. The relative amounts of MDPV and the MBPs were estimated by integrating the peaks of their monoisotopic masses versus IS, using a mass tolerance of 5 ppm. Biotic incubations
were prepared in biological triplicates (biotic 1–3) to address biological variances. None of the four MBPs were present in abiotic and control samples and all were verified by their MS2 spectra. The time profiles for MDPV and its four most abundant metabolites are presented in Fig. 4. Demethylenyl-methyl MDPV (III) showed an increase over the time course of the study, except for biotic 1 reactor after 10 days. This reactor showed high biotransformation rate to hydroxy-pyrrolidine MDPV (VII) already after 2 days, whereas the signals from biotic 1 and biotic 2 increased after day 3. These findings are in accordance to the study by Quintana et al., who observed a lag phase of five days before the formation of metabolites of ketoprofen (Quintana et al., 2005). After four days, there was no clear increase or decrease of the hydroxy-pyrrolidine MDPV (VII) indicating a steady-state of bioformation and/or further biotransformation via reactions such as the N-dealkylation. Resulting hydroxy-alkyl-N-dealkyl MDPV (X) was decreasing towards the end of
M. Mardal, M.R. Meyer / Science of the Total Environment 493 (2014) 588–595
the incubations and was possibly further transformed via oxidation of the pyrrolidine ring to hydroxy-alkyl-oxo-N-dealkyl MDPV (XIII), which set off after day 3. However, formation of MBPs did significantly (t-test) decrease the concentration of MDPV over 10 days only in biotic 1 and not in biotic 2 and 3. These findings are in line with previously presented data, showing the stability of MDPV in untreated WW and reported 15% degradation over 3 days (Chen et al., 2013). Furthermore, time profiling indicated an altered microbial composition and/or an altered metabolizing performance as expected. The present study was run over 10 days without added nutrients other than MDPV. Vital parameters over time are therefore different compared to sewer conditions e.g. oxygen levels and temperatures were higher than those mentioned in the OECD guideline 314 A. However, dissolved oxygen and biomass are even higher in activated sludge, which should therefore show a higher biocatalytic activity. Furthermore, the residence time for compounds in the sewer system would be much lower than 10 days. Despite aeration, pH did not change considerably throughout the test duration. The aim of this study was to investigate the possible biocatalytic fate of discharged drugs and their metabolites to present most representative analytes for WW analysis. Nevertheless, the study setup could only address the biotransformation in WW by suspended WW microbes and not the impact of biofilms also present in sewer systems. As no significant decrease of MDPV was observed in two of the three replicates (Fig. 4), the detected MBPs might be of minor relevance concerning analytical strategies. During the incubation, hydrolysis of biomass (toilet paper etc.) can release readily biodegradable substrates, absence of new nutrients can promote growth of microbes capable of utilizing specific carbon sources for energy, and dead microbes can lyse and serve as nutrients for other bacteria etc. (Jacobsen et al., 2013). The importance of biological variance was addressed by making the incubations in three different reactors. The reactors were treated alike and different time curves should be the result of different biocatalytic performance, analytical variations, and variations in sample preparation. The tested conditions should be seen as maximum degradation capacity rather than a simulation of real systems as experiments were aerated constantly throughout the duration of the test, which is in contrast to the OECD guideline.
3.5. Stability of rat urine/feces metabolites monitored in wastewater In WW incubated with rat urine/feces, caffeine was used as intrinsic IS as caffeine showed less than 5% dissipation in WW from primary effluent incubated at 20 °C for 2 days (Buerge et al., 2003). Caffeine was thus found suitable for IS for 2 days to correct for inter-day instrument performance. Previously identified rat phase I metabolites (Meyer et al., 2010) appeared to be stable over two days of incubation although it is not clear at this point in what extent phase I and phase II metabolites can be transformed into each other mimicking their stability. The stability of the previously reported four most abundant phase II metabolites, the glucuronides of demethylenyl MDPV, demethylenylmethyl MDPV, demethylenyl-methyl-oxo-pyrrolidine MDPV, and demethylenyl-methyl-hydroxy-aryl MDPV, was monitored in detail over two days of incubation. In the present study, all monitored signals from these four glucuronides were decreased by more than 99% after one day. Glucuronidase activity in activated sludge has been reported by monitoring the stability of 4-methylumbelliferyl-β-D-glucuronide, where 19% of the spiked glucuronides were cleaved after 6.7 h (Ternes et al., 1999). In another study, estrone 3-glucuronide and estriol 16α-glucuronide were no more detectable after 8 h of incubation in WW (Tucker et al., 1998). However, the detection of metabolites in samples drawn from the sewer systems with short residence time could be improved by enzymatic cleavage of the glucuronides and other conjugates during samples workup. Therefore, further studies will show this impact. In comparison to previously reported human and rat metabolism, microbial biotransformation of MDPV partly followed similar pathways (Meyer et al., 2010). Of the 12 tentatively identified MBPs, three have also been detected in human and/or rat urine samples. As demethylenyl-methyl MDPV was the most abundant MDPV metabolite in human urine and only the fourth most abundant in the presented study, it should thus be the most suitable target for sewage profiling. However, neither hydroxy-pyrrolidine MDPV (VII), hydroxyl-alkyl-Ndealkyl MDPV (X) nor hydroxyl-alkyl-oxo-N-dealkyl MDPV (XIII), the three most abundant MBPs in the presented study, were previously O HO
O
O NH
O
These conditions were chosen to allow thorough detection of biotransformation products and to assess the degradation potential of MDPV.
O
O
XII
593
demethylenation
I
N
O
N HO
II, h
O OH O
hydroxylation
n
tio
la
xy
ro
O
O
O O
N
IV, h
O
O
O
oxidation
O
VII
N
O
N
hydroxylation
HO
methylation
O
III, h
O hydroxylation
OH
O
N
O
V
dealkylation
methylation
d hy
O
OH
O NH
O
X
O O
O
O
VI
O
O N
XI
N
HO
OH O
oxidation
N
IX OH
O
O
OH
O
O
O NH
O
XIII
O
NH
VIII
O
Fig. 3. Pathways for microbial biotransformation of MDPV in wastewater. The numbering is in accordance to Table 1 and Fig. 1. h: also detected as human metabolite.
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Fig. 4. Time profile of relative MDPV concentration and formation of the four most abundant MBPs over 10 days.
detected in human or rat urine (Meyer et al., 2010). This offers the opportunity to distinguish between human metabolism and microbial biotransformation and shows again the importance to additionally gain knowledge about WW biotransformations as this can be different from mammalian metabolism. 4. Conclusions QuEChERS was successfully implemented in a biotransformation study in WW. In total, 12 microbial biotransformation products were tentatively identified after incubation of MDPV in WW for 10 days. However, biotransformation only leads to a significant decrease of the MDPV concentrations in one of the 3 biotic reactors and there was a lag phase of two days before biotransformation products could be detected. Urinary glucuronides of MDPV metabolites were almost completely cleaved after one day of WW incubation, which will help in phase I metabolite detection for sewage profiling. Finally, the presented study indicates that demethylenyl-methyl MDPV, the most abundant metabolite in human urine, should be the best biomarker in WW to estimate human consumption. It is expected that the presented data will help to develop analytical methods for MDPV analysis in WW for sewage epidemiology, which can be used in addition to classical studies such as surveys to estimate illicit drug use in populations Further studies are encouraged to investigate the rate of microbial cleaving of not only glucuronides, but also sulfates and other conjugates, of excreted metabolites. Acknowledgments The authors would like to thank Hans H. Maurer, Veit Flockerzi, Markus Bischoff, Julia Dinger, Andreas Helfer, Golo Magnus Meyer, Julian Michely, Martin-Simon Thomas, Armin A. Weber, Jessica Welter, Ann-Kathrin Ostermeyer, and Carina Wink for their support and/or discussion. This project has received funding from the European Union's Seventh Framework Programme for research, technological
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