Analytische Chemie
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Mycotoxins are secondary metabolites produced by fungi contaminating food and feed worldwide. Intake of these foodborne toxins can cause several diseases in humans and animals highlighting the need to understand metabolic pathways of mycotoxins. Methods of choice have been in vitro and in vivo approaches, so far. Beside hundreds of documented mycotoxins numerous new ones need to be elucidated and enhance the demand for fast and reliable methods. Here, we present electrochemistry coupled to mass spectrometry (EC/MS set up Fig. 1) as novel and promising tool in mycotoxin research. Electrochemical oxidation of mycotoxins like zearalenone, citrinin or dihydroergocristine lead to several oxidation products known from phase I biotransformation as well as new interesting reaction products analyzed by EC/MS, LC MS/MS and ESI-HRMS. To ensure a comparative overview results obtained from electrochemical oxidation experiments were compared to Fenton reaction, UV irradiation and microsomal experiments. The presentation will point out the benefits and drawbacks of EC/MS in mycotoxin research on the basis of selected food relevant mycotoxins.
An automated method is presented for fast simulation of (bio)transformation products (TPs) of the organophosphate insecticide chlorpyrifos CPF)based on electrochemistry coupled online to liquid chromatography-mass spectrometry (EC-LC-MS). Oxidative TPs were produced by a boron doped diamond (BDD) electrode, separated by reversed phase HPLC and online detected by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, EC oxidative TPs were investigated by HPLC-tandem mass spectrometry (LC-MS/MS) and FT-ICR high resolution mass spectrometry (HRMS) and compared to in-vitro assay metabolites (rat and human liver microsomes). Main phase I metabolites of CPF: chlorpyrifos oxon (CPF oxon), trichloropyridinol (TCP), diethylthiophosphate (DETP), diethylphosphate (DEP), desethyl chlorpyrifos (De-CPF), and desethyl chlorpyrifos oxon (De-CPF oxon), were successfully identified by the developed EC-LC-MS method. The EC-LC-MS method showed similar metabolites compared to the in-vitro assay with possibilities of determining reactive species. Our results reveal that online EC-(LC)-MS brings an advantage on time of analysis by eliminating sample preparation steps and Matrix complexity compared to conventional in-vivo or in-vitro 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.
Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth.