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The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for health, food and environmental matters. Monensin (MON) is an ionophore antibiotic widely used to cure and prevent coccidiosis by chicken especially in broiler farming. Residues can be found in food products (chicken and eggs) and in the environment (manure, soil, water). Several transformation processes can alter the parent compound MON, ranging from biotransformation in living organism to biotic/abiotic and microbial processes in environmental matters.
The main objective of this work was to investigate the potential of electrochemistry (EC) to simulate oxidative transformation processes of MON and to predict TPs. An electrochemical reactor was used consisting of a flow-through cell with a glassy carbon working electrode. Derived TPs were analyzed by online coupling of EC and high-resolution mass spectrometry (HRMS) and LC/HRMS offline measurements. Among the generated TPs already known as well as unknown TPs of MON could be found.
Additionally, MON was subjected also to other transformation experiments like metabolism tests with rat microsomes or the pH-dependent hydrolysis. As a result, different targeted and suspected TPs could be identified by analysis with LC/HRMS.
An overview of detected/identified TPs from this study will be presented in comparison to literature known metabolites and TPs.
Online coupling of electrochemistry with mass spectrometry (EC/MS) is highly promising for prediction and simulation of metabolic processes of xenobiotics in living organisms. Less time and cost of analysis, matrix free detection, and automation make EC/MS-based metabolomics superior over traditional in-vivo and in-vitro methods. Furthermore, EC/MS has a special feature to identify reactive intermediates and reaction mechanisms.
The main objective of this work was to simulate biotransformation processes of pesticides by EC/MS and to elucidate the Transformation products (TPs). We have studied the oxidative phase I metabolism processes of selected pesticides by EC/MS or with liquid chromatography (EC/LC/MS) and compared the derived TPs with cytochrome based metabolites. The electrochemical TPs were produced by boron-doped diamond electrode, separated by LC, and detected by single quadrupole ESI-MS online. Structural identification of both electrochemical oxidation and liver microsome metabolites were based on accurate mass measurements by FT-ICR high-resolution mass spectrometry, isotopic pattern, MS/MS fragmentation, and Retention time alignments.
Main phase I oxidative metabolites by P-oxidation, N- & O- dealkylation, dechlorination, hydroxylation, and -OH- oxidation have been identified. Many targeted and untargeted metabolites have been identified by EC/(LC)/MS. Additionally, reactive species have been trapped online by biomolecules to study phase II conjugative reactions. Furthermore, we synthesized TP standards by EC/MS and applied them for pesticide's TPs occurrence investigation in foodstuf matrices.
The identification of transformation products (TPs) of pesticides in food is a crucial task difficult to tackle, due to the lack of standards. In this work, we present a novel methodology to synthesize five main TP standards of the insecticide chlorpyrifos (CPF) and to investigate their occurrence in selected fruits and spices. TPs were electrochemically (EC) synthesized using a boron-doped diamond electrode (BDD) and identified by EC coupled online to mass spectrometry, LC-MS/MS, and high-resolution mass spectrometry. CPF and its TPs were analyzed in the food samples by LC-MS/MS on multiple reaction monitoring (MRM) after dispersive solid-phase extraction. A good recovery of 83–103% for CPF and 65–85% for TPs was obtained. Matrix effects, which cause signal suppression, ranged between 81 to 95% for all targeted analytes. The limit of detection and quantification for CPF were 1.6–1.9 and 4.9–5.7 μg/kg, respectively. Among investigated samples, CPF was determined in fresh lemon (104 μg/kg), fenugreek seed (40 μg/kg), and black pepper (31 μg/kg). CPF content in all samples was lower than the EU maximum residue level (MRL). The most frequently detected TPs were diethylthiophosphate and diethylphosphate. Other TPs, CPF oxon and trichloropyridinol, were also detected. Hence, EC is a versatile tool to synthesize TP standards which enables the determination of contaminants and residues in foodstuffs even if no commercial standards are available.
The drug salinomycin (SAL) is a polyether antibiotic and used in veterinary Medicine as coccidiostat and growth promoter. Recently, SAL was suggested as a potential anticancer drug.
However, transformation products (TPs) resulting from metabolic and environmental degradation of SAL are incompletely known and structural information is missing. In this study, we therefore systematically investigated the formation and identification of SAL derived TPs using electrochemistry (EC) in an electrochemical reactor and rat and human liver microsome incubation (RLM and HLM) as TP generating methods. Liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) was applied to determine accurate masses in a suspected target analysis to identify TPs and to deduce occurring modification reactions of derived TPs. A total of 14 new, structurally different TPs were found (two EC-TPs, five RLM-TPs, and 11 HLM-TPs). The main modification reactions are decarbonylation for EC-TPs and oxidation (hydroxylation) for RLM/HLM-TPs. Of particular interest are potassium-based TPs identified after liver microsome incubation because these might have been overlooked or declared as oxidated sodium adducts in previous, non-HRMS-based studies due to the small mass difference between K and O + Na of 21 mDa. The MS fragmentation pattern of TPs was used to predict the position of identified modifications in the SAL molecule. The obtained knowledge regarding transformation reactions and novel TPs of SAL will contribute to elucidate SAL-metabolites with regards to structural prediction.
The formation of transformation products (TPs) from contaminants and residues is becoming an increasing focus of scientific community. All organic compounds can form different TPs, thus demonstrating the complexity and interdisciplinarity of this topic. The properties of TPs could stand in relation to the unchanged substance or be more harmful and persistent. To get important information about the generated TPs, methods are needed to simulate natural and manmade transformation processes. Current tools are based on metabolism studies, photochemical methods, electrochemical methods, and Fenton's reagent. Finally, most transformation processes are based on redox reactions. This review aims to compare these methods for structurally different compounds. The groups of pesticides, pharmaceuticals, brominated flame retardants, and mycotoxins were selected as important residues/contaminants relating to their worldwide occurrence and impact to health, food, and environmental safety issues. Thus, there is an increasing need for investigation of transformation processes and identification of TPs by fast and reliable methods.
Pesticides including fungicides, herbicides and insecticides are among primary residues detected in food and feed. They are transformed to a variety of products due to metabolic reactions in living organisms, microbial activities, industrial processes and photochemical reactions. To understand metabolic transformation products (TPs), in-vitro and in-vivo methods were used for a long period of time. However, these conventional methods are hampered by the long-time of analysis and by the matrix complexity. Nowadays, online coupling of electrochemistry with mass spectrometry is a technique of interest for fast prediction/ simulation of metabolic TPs and to understand the mechanism of metabolic processes.
The main objective of this work was to understand the mechanism of fluopyram (fungicide) and chlorpyrifos (insecticide) metabolism and to identify TPs by electrochemistry coupled to liquid chromatography-mass spectrometry (EC-LC-MS). Additionally, TPs of fluopyram by photochemical reaction have been investigated. Furthermore, the TPs and parent compounds in real food matrices were investigated by LC-MS/MS.
Phase-I metabolism via N- and O-dealkylation, P-oxidation and hydroxylation mechanisms were successfully simulated/predicted by EC-LC-MS. Additionally, metabolites produced by human and rat liver microsomes were identified by LC-MS/MS and high resolution mass spectrometry (HR-MS) and simulated with EC oxidation products. It is known that some phase-I metabolites are further conjugated with different biomolecules such as glucoside and glutathione. Phase-II metabolism was simulated by trapping the oxidized products (phase-I) online by biomolecules and allowing them to react in the loop before the electrospray ionization interface of the MS. Standard solution of fluopyram was irradiated with a medium pressure Hg-lamp (150 W) at 12.5 0C for 2 hrs and aliquots were characterized by LC-MS/MS.
In conclusion, the EC-LC-MS method enables fast, cost effective and matrix free detection and prediction of metabolic pathways compared to in-vitro assays. Its versatilities to synthesis reference substances and metabolites for off-line characterization (such as NMR and HR-MS) and possibilities of determining fast reactive intermediates make EC-LC-MS more advantageous than in-vitro assays.