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Among major food contaminants agrochemicals (including insecticides, fungicides and herbicides) are a threat for food safety in many countries. Once they entered the food chain or the environment, the parent compound can be transformed into different products by manmade and natural processes. The transformation products (TPs) might be more toxic and stable than the parent compound. In addition to this the transformation products might undergo conjugation with different compounds and/or changed to phase II metabolites.
Conventional in-vivo or in-vitro methods to study phase I and II metabolism have drawbacks of long-time sample preparation and matrix complexity. Online coupling of an electrochemical cell with liquid chromatography-mass spectrometry (EC–LC–MS) is a promising technique to study metabolites, fate and transformation products of pesticides. The new approach enables to identify metabolic transformation products by oxidizing analyte of interest on the EC, separate the metabolites on HPLC and identifying them by MS.
The aim of the present study is to identify/mimic pesticides phase I metabolites using electrochemical cell coupled to liquid chromatography-mass spectrometry (EC-LC-MS). Metabolic transformed products of two model compounds, fluopyram (fungicide) and chlorpyrifos (insecticide) were studied by EC upfront with MS. The results of EC-LC-MS are compared and discussed with those derived from in-vitro assays and the metabolites identified by LC-MS/MS.
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.
Within a class of agrochemical residues, pesticides are significant concern due to their various adverse effects on people, animals and the environmental systems. Once they entered to food or to the environment, the parent compound is transformed to metabolites which have different toxicity profiles. Activities like food cooking and waste water treatment (WWT) may lead to produce more toxic metabolites than the parent substances.
Online coupling of electrochemistry with liquid chromatography mass spectrometry (EC-HPLC-MS) is one of the most promising and powerful techniques for metabolite studies which exploited widely for pharmaceuticals so far [1]. However, data on the application of EC-HPLC-MS to simulate environmental transformation products, the fate of contaminants and their metabolic pathways are rare. The oxidation of analyte of interest on the EC, separate the metabolites on HPLC and identifying them by MS gives enormous advantages to identify the contaminants transformation products [2]. Thus, applying this technique (EC-MS, EC-HPLC-MS, and HPLC-EC-MS) for transformation products of pesticide residues is urgently needed.
The main interest of this study will be investigating the electrochemical oxidative degradation products, simulating to in-vitro metabolites and transformation processes of selected pesticides in food and environmental samples. The parent pesticides’, like carbamates and abamectin, oxidation by EC, product metabolites identification and their fates will be discussed using electrochemistry coupled to HPLC-MS techniques.
Chlorpyrifos (CPF), an anticholinesterase organophosphate insecticide, is commonly used to control pests in agricultural sectors. In recent years, it is one of the most frequently detected residues in fruits and vegetables. On the other hand, pesticides including chlorpyrifos undergo extensive abiotic (industrial processes, waste treatments and photodegradations) and/or biotic (metabolism and microbial activities) processes which lead to transformation products (TPs) with different toxicity. Furthermore, lack of representative standards and complexity of transformation mechanisms make monitoring of TPs in real samples difficult.
The aim of this work was to investigate CPF and its TPs in selected food matrices. Representative standards of TPs were synthesized by electrochemistry coupled online to liquid chromatography-mass spectrometry (EC/LC/MS) that equipped with a follow-through and/or synthesis cell with boron doped diamond working electrode. The TPs were characterized by LC-MS/MS and high resolution mass spectrometry (HRMS) and used for real sample investigations. Different fruit and spice samples (fortified by TPs standards and blank) were extracted by dispersive solid phase extraction (dSPE) and analyzed by LC-MS/MS.
Recoveries were obtained ranging between 94 – 101% (with matrix effect 85 – 97%). The method limit of detection (LOD) and quantification (LOQ) for CPF were 1.9 and 5.7 µg/kg, respectively. Among investigated samples CPF was detected in fresh lemon, black pepper and fenugreek seed with a content of 104, 31 and 4 µg/kg, respectively. Coriander and cinnamon samples also contained trace levels of CPF (<LOD). Transformation products of CPF mainly diethylthiophosphate (DETP), chlorpyrifos oxon (CPF oxon) and trichloropyridinol (TCP) were detected alongside of the parent compound in different samples. Hence, by synthesizing reference standards using EC/LC/MS we were able to detect the main TPs of CPF in real food samples. The results show that not only parent residues but also monitoring of TPs is vital to ensure future food safety.