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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.
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.