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Mycotoxins can be found worldwide in food and feed and cause a variety of mold-related health risks which makes it necessary to further examine their metabolic fate in human and other mammals. Beside standard in vitro assays with liver cell preparations an increasing interest in new simulation methods are playing a growing role. The online coupling of electrochemistry with mass spectrometry (EC/MS) is one of these novel techniques, successfully applied in pharmacological and drug research for several years now. The primary objective of this study was to investigate the capability of EC/MS to elucidate metabolic pathways of the mycotoxin citrinin as relevant food contaminant. For this purpose, a coulometric flow through cell equipped with a glassy carbon working electrode was used by applying a ramped potential between 0 and 2 V vs Pd/H2. The electrochemically generated oxidation products analyzed by EC/MS were compared to those obtained from in vitro assays. To receive a comprehensive assessment of EC/MS other non-microsomal oxidation techniques such as Fenton-like reaction and UV irradiation were applied. Several hydroxylated derivatives of citrinin were generated by EC/MS and Fenton-like reaction which are similar to microsomal biotransformation products. These data show that EC/MS is a versatile tool that can be easily applied in mycotoxin research to support metabolic investigations of known and unknown mycotoxins.
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.
Mycotoxins occur widely in foodstuffs and cause a variety of mold-related health risks to humans and animals. Elucidation of the metabolic fate of mycotoxins and the growing number of newly discovered mycotoxins have enhanced the demand for fast and reliable simulation methods. The viability of electrochemistry coupled with mass spectrometry (EC/ESI-MS), Fenton-like oxidation, and UV irradiation for the simulation of oxidative phase I metabolism of the mycotoxins citrinin (CIT) and dihydroergocristine (DHEC) was investigated. The specific reaction products are compared with metabolites produced by human and rat liver microsomes in vitro. Depending on the applied potential between 0 and 2000 mV vs. Pd/H-2 by using a flow-through cell, CIT and DHEC are oxidized to various products. Besides dehydrogenation and dealkylation reactions, several hydroxylated DHEC and CIT species are produced by EC and Fenton-like reaction, separated and analyzed by LC-MS/MS and ESI-HRMS. Compared to reaction products from performed microsomal incubations, several mono- and dihydroxylated DHEC species were found to be similar to the reaction products of EC, Fenton-like reaction, and UV-induced oxidation. Consequentially, nonmicrosomal efficient and economic simulation techniques can be useful in early-stage metabolic studies, even if one-to-one simulation is not always feasible.
To understand the metabolic fate of food relevant mycotoxins in vitro systems were mainly used as the method of choice, so far. Yet, in recent years coupling of electrochemistry mass spectrometry (EC-MS) gained increasing importance as promising technique for fast simulation of metabolic processes and was successfully applied in particular for drug metabolism [1].
The aim of our work was to investigate the potential of EC-MS to predict phase I metabolites of priority mycotoxins and to compare the results with in vitro experiments. Hence, the EU-regulated Fusarium mycotoxins zearalenone (ZEN) and patulin as well as dihydroergocristine (DHEC) as model compound of ergot alkaloids were electrochemically oxidized and analyzed by EC MS for the first time.
Electrochemical conditions were set-up individually for each of the three mycotoxins. By using a coulometric flow through cell with a diamond working electrode oxidation of the chosen mycotoxins was observed after applying potentials between 1.7 and 2.0 V vs. Pd/H2. The electrochemically generated reaction products were analyzed online by mass-spectrometric detection.
All of the three chosen mycotoxins were electrochemically converted to mono- and/or dihydroxylated products confirming the results of ZEN related metabolism studies [2, 3] and in case of DHEC own results from in vitro assays. Due to a lack of metabolism studies concerning the oxidative fate of patulin, interpretation of EC-MS data and performing microsomal studies is of particular relevance.
Beside the identified products from electrochemical oxidation of ZEN, patulin and DHEC there is still a number of yet unknown compounds. Additional structural characterization of detected compounds by NMR and X-ray analysis will be facilitated by their large-scale production using preparative EC cells.
Mycotoxins are secondary metabolites of fungi which have diverse detrimental effects on humans, animals and crops. Traceable worldwide in foods and animal feeds, these contaminants cause manifold diseases and extensive economic losses. Therefore, European legislation set maximum levels of distinct mycotoxins to minimize the risks for the buying public. But standardized food analysis techniques fail to detect masked mycotoxins, whose research increasingly moves to the fore in recent years. They are formed from detoxification metabolism of plants as well as from fungi, which conjugate for example with glucosides or dihexosides. All masked mycotoxins have one thing in common: They are not detectable with standard methods, thereby contributing to the overall exposure and pose an additional health risk for the consumer.
The dissertation work will focus on the following potential new group of masked toxins. Food safety relevant mycotoxins like zearalenone and ochratoxin A possess one or more 1,3-dicarbonyl moieties. Latter are principally able to form thermodynamically stable chelate complexes with metal cations. First investigations at BAM showed interactions between zearalenone and copper ions and it is conceivable that they possibly build a complex. Our main focus is now to identify, characterize and quantify 1,3-dicarbonyl mycotoxin metal complexes as potential candidates within the group of conjugated mycotoxins.
We will simulate processes of biotransformation and identify distinct metabolites by electrochemistry coupled to liquid chromatography/mass spectrometry (EC-HPLC-MS). The obtained knowledge contributes to a better understanding of masked mycotoxins and an improved monitoring of foods and feeds, to ensure food safety.
Mycotoxins can be found worldwide in foods and feed and cause a variety of mold-related health risks which makes it necessary to further examine their toxic effects and metabolic fate in human and other mammals. Beside standard in vitro and in vivo assays with liver cell preparations or rodents an increasing interest in new simulation methods are playing a growing role. Electrochemistry (EC) is one of these novel techniques and has been used successfully and efficiently in pharmacological and drug research for several years now.
The primary objective of this study was to determine the capability of EC as a supportive and versatile instrument to elucidate metabolic pathways of mycotoxins.
On the example of the food relevant mycotoxin Citrinin a coulometric flow through cell equipped with a carbon working electrode was used to oxidize Citrinin by applying potential between 0.7 and 2.5 V vs. Pd/H2. The electrochemically generated oxidation products were then analyzed by mass-spectrometric detection coupled online to EC (EC-MS) and compared with data from a standard in vitro model with human and rat liver microsomes preparations. To receive a comprehensive assessment of oxidative techniques chemical oxidation by Fenton´s reaction was performed as well.
The obtained LC-MS/MS data confirmed the production of Dihydrocitrinone by all of the three tested oxidation systems and demonstrates the potential of EC-MS for the successful prediction of the main phase I metabolic reactions of mycotoxins, since Dihydrocitrinone is the mainly formed metabolite by humans after intake of Citrinin.
Beside the identified Dihydrocitrinone from electrochemical, enzymatic and chemical oxidation of Citrinin there is still a number of yet unknown compounds. As the next step structural characterization of the generated oxidation products by NMR and X-ray analysis will be enabled by their large-scale production using preparative EC cells.
Mycotoxins cause a variety of mold-related health risks which makes it necessary to further examine their metabolic pathways in human and other mammals. Beside standard in vitro assays with liver cell microsomes an increasing interest in new and rapid simulation techniques are playing a growing role in mycotoxin research.
Herein, the coupling of electrochemistry with liquid chromatography and mass spectrometry (EC/LC/MS) is presented as fast and simple method to investigate the oxidative fate of mycotoxins. For this case study, two food relevant mycotoxins (zearalenone and citrinin) were selected. Experiments were performed by using an electrochemical flow through cell integrated in the flow path of the autosampler of the chromatographic system. The reaction mixture was separated by a RP-C18 column and analyzed by a single quadrupole MS (Figure 1). Oxidation products were generated by applying potentials of 400, 800, 1200 and 1600 mV vs Pd/H2 using a glassy carbon working electrode. Different oxidation reactions like hydroxylation, dehydrogenation and dimerization lead to a diverse product pattern of the investigated mycotoxins.
In a comparative study, electrochemical generated reaction products were compared with metabolites produced by human and rat liver microsomes in vitro. The obtained data show that EC/LC/MS is a versatile and promising tool in mycotoxin research to support metabolic investigations of known and unknown mycotoxins.
A new dimer of the food-relevant mycotoxin zearalenone was isolated after electrochemical and chemical oxidation. The structure was determined as a 16-O-15'-biaryl ether-linked dimer based on spectroscopic analyses (1H- and 13C-NMR, COSY, HMBC, and HSQCAD) and high-Resolution mass spectrometry analysis (Q-TOF).
To keep pace with the rising number of detected mycotoxins, there is a growing need for fast and reliable toxicity tests to assess the potential threat to food safety. Toxicity tests with the bacterial-feeding nematode Caenorhabditis elegans as model organism are well established. In this study the C. elegans wildtype strain N2 (var. Bristol) was used to investigate the toxic effects of the food-relevant mycotoxins citrinin (CIT) and zearalenone-14-sulfate (ZEA-14-S) and zearalenone (ZEA) on different life cycle parameters including reproduction, thermal and oxidative stress resistance and lifespan. The metabolization of the mycotoxins by the nematodes in vivo was investigated using HPLC-MS/MS. ZEA was metabolized in vivo to the reduced isomers α-zearalenol (α-ZEL) and β-ZEL. ZEA 14-S was reduced to α-/β-ZEL 14-sulfate and CIT was metabolized to mono-hydroxylated CIT. All mycotoxins tested led to a significant decrease in the number of nematode offspring produced. ZEA and CIT displayed negative effects on stress tolerance levels and for CIT an additional shortening of the mean lifespan was observed. In the case of ZEA-14-S, however, the mean lifespan was prolonged. The presented study shows the applicability of C. elegans for toxicity testing of emerging food mycotoxins for the purpose of assigning potential health threats.