Workflow for the identification of biotransformation products of amine-containing psychotropic drugs in the aquatic environment

Pharmaceuticals are continuously discarded Pharmaceuticals are continuously discarded into the aquatic system through wastewater treatment plants (WWTPs). The microbial degradation of these organic micropollutants and formation of transformation products (TPs) under aerobic conditions is the fundamental process for their elimination. It is of paramount importance to understand the microbial metabolic pathways so as to obtain knowledge of how fast micropollutants degraded and to assess the exposure to their potential TPs as they can be more polar and consequently environmentally persistent. In this study, batch reactors seeded with activated sludge from the WWTP of Athens were set up to assess biotic, abiotic and sorption losses of selective psychotropic drugs, containing amine moieties. Biodegradation and transformation products were identified using liquid chromatography quadrupole-time-offlight mass spectrometry (LC-QToF-MS). A workflow for target, suspect and non-target screening was developed. Data treatment was performed by using metabolite tools accompanying Bruker’s maxis impact ESI-QToF-MS and the structure elucidation of the candidate transformation products was based on accurate mass and isotopic pattern measurements by HRMS and tentative interpretation of MS/MS spectra. Finally four biotransformation products were identified for both lidocaine and ephedrine. Despite the structure similarities, different degradation constants were calculated for each compound

Targeted determination of more than 1500 micropollutants & transformation products in wastewater samples by liquid chromatography quadrupole-time-of-flight mass spectrometry with an accurate-mass database

High resolution mass spectrometry has dramatically improved the possibilities of the environmental analysis. The present study describes the development of an analytical method, based on liquid chromatography quadrupole-time-of-flight mass spectrometry (LC–QToF-MS) for the target determination of more than 1500 contaminants of emerging concern (CECs) and transformation products (TPs) including, among others, pharmaceuticals, illicit drugs, personal care products, pesticides, industrial chemicals, and sweeteners in wastewater. Analytes were extracted from wastewater samples by mixed mode solid-phase extraction, and data were acquired through broad-band Collision Induced Dissociation (bbCID) mode, providing MS and MS/MS spectra, simultaneously, in both positive and negative ionization mode (two separate runs). The in-house mass spectral database was built by injection of standard solution of the analytes and it includes information of the retention time, parent ions and adducts, as well as fragment ions. The raw data were analyzed with Bruker Target Analysis 1.3 software. Retention time, accurate mass of the precursor ion and adducts, isotopic pattern, in combination with absence of the peak in the procedural blank were the parameters used for confirmation of the target compounds. Experimental fragment ions were also considered, along with the ion ratio, intensity and isotopic pattern. Furthermore, semi-quantitation of these contaminants was possible. The method herein presented, in addition of providing accurate information about the presence of a large number of relevant substances, has the advantage that the data generated can be further processed for suspect and non-target screening, expanding the information on the samples. An important advantage of this method is that retrospective investigation of the data is available to look for the presence of additional CECs and their TPs, which were not considered at the time of the analysi

Biotransformation of citalopram: Insights from identification of transformation products by LC-QToF-MS

Biodegradation is considered to be the key process for the elimination of the majority of pharmaceuticals in the environment. During wastewater treatment or once they are disposed in the aquatic environment, pharmaceuticals may transformed to new, structurally-related compounds which are called transformation products (TPs). Since most of these compounds are unknowns, their identification is essential not only to provide a comprehensive risk assessment on micropollutants in the environment, but also to design improved removal technologies for (pseudo)persistent trace contaminants. In this study, batch reactors seeded with activated sludge from the WWTP of Athens were set up to assess biotic, abiotic and sorption losses of a SSRI drug, citalopram. TPs were identified by reversed-phase liquid chromatography quadrupole-time-of-flight mass spectrometry (RPLC-QToF-MS). Hydrophilic interaction liquid chromatography (HILIC) was also used as a complementary, orthogonal, technique for the identified TPs, instead of NMR. A workflow for suspect and non-target screening was developed. A suspect list of possible TPs was compiled by literature and in silico prediction tools (EAWAG-BBD Pathway Prediction System and Bruker’s Metabolite Predict). Structure elucidation of TPs was based on accurate mass and isotopic pattern measurements and interpretation of MS/MS spectra by the observed fragmentation pattern and library-spectrum match. In total, thirteen TPs were identified. Four out of them were fully identified and confirmed by reference standards (desmethylcitalopram, citalopram amide, citalopram carboxylic acid and 3-oxo-citalopram). A probable structure based on diagnostic evidence and tentative candidates were proposed for the additional five and four TPs, respectively. Finally, a transformation pathway based on the identified compounds was presented

Identification of biotransformation products of citalopram formed in activated sludge

Citalopram (CTR) is a highly consumed antidepressant which is removed incompletely by conventional wastewater treatment. Although it is highly detected in effluent wastewaters, little is known about its behavior and transformation processes that undergo during wastewater treatment. The present study aims to expand the knowledge on fate and transformation of CTR during the biological breakdown process. For this purpose, biotransformation batch reactors were set up to assess biotic, abiotic and sorption losses of this compound. One of the main objectives of the study was the identification of the formed transformation products (TPs) by applying suspect and non-target strategies based on liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). In this regard, the complementary use of reversed phase chromatography (RP) and hydrophilic interaction liquid chromatography (HILIC) in the identification of polar TPs, the deep evaluation of the obtained MS/MS spectra, as well as the use of in-house developed quantitative structure-retention relationship (QSRR) retention time prediction models provided valuable support to identification. Fourteen TPs were detected. Thirteen of them were tentatively identified. Four compounds were confirmed (N-desmethylCTR, CTR amide, CTR carboxylic acid and 3-oxo-CTR) through the purchase of the corresponding reference standard. Probable structures based on diagnostic evidence were proposed for the additional nine TPs. A transformation pathway for the biotransformation of CTR was proposed. The presence of the identified TPs was assessed in real wastewater samples through retrospective analysis resulting in the detection of five compounds. Finally, the potential ecotoxicological risk posed by CTR and its TPs to different trophic levels of aquatic organisms, exposed to the studied effluents, was evaluated by means of risk quotients

Identification of biotransformation products of citalopram formed in activated sludge

Citalopram (CTR) is a worldwide highly consumed antidepressant which has demonstrated incomplete removal by conventional wastewater treatment. Despite its global ubiquitous presence in different environmental compartments, little is known about its behaviour and transformation processes during wastewater treatment. The present study aims to expand the knowledge on fate and transformation of CTR during the biological treatment process. For this purpose, batch reactors were set up to assess biotic, abiotic and sorption losses of this compound. One of the main objectives of the study was the identification of the formed transformation products (TPs) by applying suspect and non-target strategies based on liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). The complementary use of reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) for the identification of polar TPs, and the application of in-house developed quantitative structure-retention relationship (QSRR) prediction models, in addition to the comprehensive evaluation of the obtained MS/MS spectra, provided valuable information to support identification. In total, fourteen TPs were detected and thirteen of them were tentatively identified. Four compounds were confirmed (N-desmethylCTR, CTR amide, CTR carboxylic acid and 3-oxo-CTR) through the purchase of the corresponding reference standard. Probable structures based on diagnostic evidence were proposed for the additional nine TPs. Eleven TPs are reported for the first time. A transformation pathway for the biotransformation of CTR was proposed. The presence of the identified TPs was assessed in real wastewater samples through retrospective analysis, resulting in the detection of five compounds. Finally, the potential ecotoxicological risk posed by CTR and its TPs to different trophic levels of aquatic organisms was evaluated by means of risk quotients. © 2016 Elsevier Lt

Identification of biotransformation products of citalopram formed in activated sludge

Citalopram (CTR) is a worldwide highly consumed antidepressant which has demonstrated incomplete removal by conventional wastewater treatment. Despite its global ubiquitous presence in different environmental compartments, little is known about its behaviour and transformation processes during wastewater treatment. The present study aims to expand the knowledge on fate and transformation of CTR during the biological treatment process. For this purpose, batch reactors were set up to assess biotic, abiotic and sorption losses of this compound. One of the main objectives of the study was the identification of the formed transformation products (TPs) by applying suspect and non-target strategies based on liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). The complementary use of reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) for the identification of polar TPs, and the application of in-house developed quantitative structure-retention relationship (QSRR) prediction models, in addition to the comprehensive evaluation of the obtained MS/MS spectra, provided valuable information to support identification. In total, fourteen TPs were detected and thirteen of them were tentatively identified. Four compounds were confirmed (N-desmethylCTR, CTR amide, CTR carboxylic acid and 3-oxo-CTR) through the purchase of the corresponding reference standard. Probable structures based on diagnostic evidence were proposed for the additional nine TPs. Eleven TPs are reported for the first time. A transformation pathway for the biotransformation of CTR was proposed. The presence of the identified TPs was assessed in real wastewater samples through retrospective analysis, resulting in the detection of five compounds. Finally, the potential ecotoxicological risk posed by CTR and its TPs to different trophic levels of aquatic organisms was evaluated by means of risk quotients.This project was implemented under the Greek Operational Program “Education and Lifelong Learning” and funded by the European Union (European Social Fund) and Greek National Resources (ARISTEIA 624, TREMEPOL project, http://tremepol.chem.uoa.gr/). Authors gratefully acknowledge the contribution of Dr. Rebekka Gulde for reviewing the manuscript, as well as Dr. Anna Bletsou and Nikiforos Alygizakis for the analysis of the WWTP samples.Peer reviewe