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Electrochemistry Combined with LC–HRMS: Elucidating Transformation Products of the Recalcitrant Pharmaceutical Compound Carbamazepine Generated by the White-Rot Fungus Pleurotus ostreatus Bettina Seiwert, Naama Golan-Rozen, Cindy Weidauer, Christina Riemenschneider, Benny Chefetz, Yitzhak Hadar, and Thorsten Reemtsma Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02229 • Publication Date (Web): 08 Sep 2015 Downloaded from http://pubs.acs.org on September 16, 2015
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Environmental Science & Technology
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Electrochemistry Combined with LC–HRMS: Elucidating Transformation
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Products of the Recalcitrant Pharmaceutical Compound Carbamazepine
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Generated by the White-Rot Fungus Pleurotus ostreatus
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Bettina Seiwert1, Naama Golan-Rozen2, Cindy Weidauer1, Christina Riemenschneider1,
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Benny Chefetz2, Yitzhak Hadar2, Thorsten Reemtsma1*
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Permoserstrasse 15, 04318 Leipzig, Germany
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Department of Analytical Chemistry, Helmholtz Centre for Environmental Research – UFZ,
Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
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*Corresponding author:
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Thorsten Reemtsma
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Department of Analytical Chemistry
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Helmholtz Centre for Environmental Research – UFZ
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Permoserstrasse 15
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04318 Leipzig, Germany
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Tel.: +49 (0341) 235 1261
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Fax: +49 (0341) 235 450822
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Keywords
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wastewater, drug, analysis, biodegradation, antiepileptic
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Abstract
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Transformation products (TPs) of environmental pollutants must be identified to
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understand biodegradation processes and reaction mechanisms, and to assess the efficiency of
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treatment processes. The combination of oxidation by an electrochemical cell (EC) with
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analysis by liquid chromatography–high-resolution mass spectrometry (LC–HRMS) is a rapid
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approach for the determination and identification of TPs generated by natural microbial
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processes. Electrochemically generated TPs of the recalcitrant pharmaceutical carbamazepine
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(CBZ) were used for a target screening for TPs formed by the white-rot fungus Pleurotus
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ostreatus. EC with LC–HRMS facilitates detection and identification of TPs since the product
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spectrum is not superimposed with biogenic metabolites and elevated substrate concentrations
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can be used. Ten TPs formed in the microbial process were detected by target screening for
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molecular ions, and another four by screening based on characteristic fragment ions. Three of
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these TPs have never been reported before. For CBZ, EC with LC–HRMS was found to be
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more effective in defining targets for the screening than software tools and faster in TP
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identification than non-target screening alone. EC with LC-HRMS may be used to feed MS
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databases with spectra of possible TPs of larger numbers of environmental contaminants for
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an efficient target screening.
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Environmental Science & Technology
Introduction
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Identification of transformation products (TPs) of environmental contaminants formed by
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living organisms is crucial to improving our understanding of biodegradation processes and
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better assessing contaminant fate in the environment and biota. Knowledge of TPs is also
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essential for assessing their toxicity. Because biologically generated TPs tend to be more
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polar than their parent compound, much of the work on TP identification relies on liquid
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chromatography coupled to mass spectrometry (LC–MS). Today, high-resolution (HR) MS is
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becoming popular due to its high selectivity and high mass accuracy, mostly in the form of
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Orbitrap or QqTOF mass spectrometers [1].
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There are two obstacles to analyzing unknown TPs from complex biological samples: (a)
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determination of the chromatographic signals that correspond to TPs of the contaminant under
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study and (b) the (tentative) identification of these TPs, i.e., the determination of their
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molecular formula from the exact mass of the molecular ions or their adducts and structure
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elucidation based on the product ion spectra. Multiple TPs can be formed from one
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contaminant in biota when a molecule exhibits different sites for enzymatic attack or when
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one initial transformation enables consecutive enzymatic processing, including formation of
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phase II metabolites [2, 3].
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Determination of all relevant TPs of a contaminant in biota is often hampered by the
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metabolome—a complex set of low-molecular-weight biogenic compounds which are co-
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extracted with the transformation products. While it is obvious that one should attempt to
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detect all TPs generated by the organism to establish the correct transformation pathway, this
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task becomes increasingly challenging when the number of TPs increases.
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TPs can be detected by two different approaches: (a) suspect or target screening, which
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starts with the structure of the parent compound and first defines possible TPs which are then
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sought, and (b) non-target screening, in which the study is starting from the sample and
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interesting MS signals that may be related to TPs are sought.
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Targets or suspects for the first approach, i.e., TPs that are sought by LC–HRMS, may be
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known from previous studies, or derived from the molecular structure of the parent compound
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based on transfer of knowledge or software prediction. When using the targeted approach, it
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seems essential to consider all possible transformation reactions and not to miss TPs of
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relevance; excessively long lists of target compounds to search for do, however, increase the
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risk of false positive findings and may, therefore, be counter-productive.
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The non-targeted approach always requires control samples and it selects signals of
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interest from the data assuming that they are only present in the sample containing the parent
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compound (and therefore also TPs) and not in the control. Considering that extracts of biota
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samples contain a large number of biogenic metabolites at much higher concentrations than
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the TPs, this selection of signals of interest in non-target screening can become difficult [4, 5].
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The analysis of samples taken repeatedly during a transformation process rather than solely at
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its end facilitates selection of interesting signals [6]. Therefore the combination of both, target
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and non-target screening seems advantageous.
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Identification of TPs by HRMS relies on molecular formula identification based on the
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exact mass of the molecular ion, its isotopic pattern and possible adduct ions, and on structure
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elucidation based on the product ion spectrum. Software tools that help compare a product ion
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spectrum with the suggested structure are available, but the fragmentation processes can be
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complex, and the rearrangement of the carbon skeleton upon fragmentation is difficult to
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predict. In any case, the identification of a TP according to its mass spectrometric data
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remains tentative as long as no reference standard is available for comparison. Such standard
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materials are rarely available for TPs of environmental contaminants.
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Electrochemistry (EC) coupled, off-line or on-line, to MS may support both, detection
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and identification of TPs. This method has been used mainly to simulate transformation of 4 ACS Paragon Plus Environment
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drugs by phase I and phase II metabolism in higher organisms [7-9]. Hydroxylation of
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activated aromatics, benzylic hydroxylation, N-dealkylation of amines, dealkylation of ethers
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and thioethers, S- and P-oxidation, oxidation of alcohols to aldehydes and dehydration may be
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simulated by this method [8]. Electrochemical transformation does not require the addition of
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any reagent so that the treated solutions can be analyzed directly without any need for clean-
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up, which may lead to loss of TPs. However, EC oxidation is less site-specific than enzymatic
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oxidation so that a broader range of TPs may be formed. Thus the set of TPs generated by EC
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may differ greatly from the set generated by higher organisms. EC as an analytical tool may
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be more suitable to simulate microbial transformations which involve other enzymes than
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those known found in higher organisms.
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Biodegradation of organic substrates by white-rot fungi is often mediated by a variety of
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peroxidases, such as lignin peroxidases and manganese peroxidases [10]. These enzymes,
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while acting in different ways depending on the specific enzyme, the substrate and available
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cofactors, often form intermediate radicals in their multistep electron transfer as outlined in
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detail by Dashtban et al. [11]. In terms of the variability and complexity of oxidation
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processes this appears comparable to what is known for electrochemical oxidation. Also there
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multistep electron transfer at the electrode surface [8], oxidation by (hydroxy-) radicals in
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solution or bound to the electrode surface at pH < 9 [9, 12] may occur.
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Therefore, in this study, we explored the potential of EC to support detection and
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identification of TPs in screening approaches using LC–HRMS. Transformation by the white-
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rot fungus Pleurotus ostreatus [13] was studied and the highly persistent pharmaceutical
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compound carbamazepine (CBZ) was chosen as the parent compound. CBZ is considered a
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marker of anthropogenic input into surface and groundwater [14]. CBZ is a well-studied
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compound [15-21], where numerous TPs are already known and some of these are
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commercially available. By that it is ideally suited to show how the use of electrochemistry
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supports the identification of TPs in complex mixtures. 5 ACS Paragon Plus Environment
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Experimental section
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Chemicals. Acetonitrile, ammonium acetate and acetic acid (all ULC/MS grade) were
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from Biosolve (Valkenswaard, Netherlands). Ozonated CBZ samples in different proportions
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(1:1 and 1:4.5) stabilized with t-BuOH were a gift from Uwe Hübner (TU Berlin). CBZ, CBZ
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10,11-epoxide and 9-carboxylic acid acridine were purchased from Sigma-Aldrich (Munich,
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Germany), cis-10,11-dihydroxy-10,11-dihydrocarbamazepine (cis-diOH-CBZ), rac trans-
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10,11-dihydro-10,11-dihydroxy carbamazepine (trans-diOH-CBZ) and acridone were
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obtained from Biozol (Eching, Germany).
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UPLC-Q-TOF-MS analysis. The analysis was performed on an ACQUITY UPLCTM
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system connected to a Synapt G2STM equipped with an electrospray ionization source (Waters
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Corp., Milford, USA). A 10-µL aliquot of each sample was injected into the column. UPLC
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separation was achieved using an ACQUITY UPLCTM BEH C18 column (100 x 2.1, 1.7 µm)
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at a flow rate of 0.45 mL min-1 and the column temperature was set to 45 °C. The mobile
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phase consisted of (A) ACN (0.01 M NH4OAc, pH 5) and (B) 0.01 M NH4OAc pH 5. The
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following gradient was applied: 0–12.25 min, 2–99% B; 12.25–13.00 min, 99% B; 13.00–
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13.01 min, 99–2% B; 13.01–14.00 min, 2% B. Ionization source conditions were as follows:
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capillary voltage of 0.7 kV, source temperature 140 °C, and desolvation temperature 550 °C.
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The sampling cone voltage was set to 35 V, source offset at 50 V. Nitrogen and argon were
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used as cone and collision gases. The desolvation gas flow was 950 L h-1.
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MS data were collected from m/z 50 to m/z 1200 in negative and positive continuum
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mode with a 0.15 s scan time. To ensure accuracy during MS analysis, leucine enkephalin was
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infused via the reference probe as the lockspray and a two-point calibration was applied. Two
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sets of data were collected in parallel using MSE acquisition. One dataset contained low-
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collision-energy data (4 eV, MS, effectively the accurate mass of precursors) and the second
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dataset elevated-collision-energy data (15–35 eV, MSE, all of the fragments). 6 ACS Paragon Plus Environment
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Culture conditions and extraction. Pleurotus ostreatus strain PC9 was used in this
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study. Culture conditions are detailed in Golan-Rozen et al. [13]. Briefly, solid-state
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fermentation was conducted in 100-mL Erlenmeyer flasks using 2 g of chopped (
