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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.
Development of analytical method and certified reference material for zearalenone in edible oils
(2017)
Quality and safety of food products require their reliable analysis. Contaminants, in particular mycotoxins, are key-components for food safety. About 25 % of the world's food crops are contaminated with mycotoxins posing a severe health risk to humans. In order to strengthen food safety and consumer protection the European Commission (EC) set maximum levels for priority mycotoxins in certain foods for human consumption. In 2013, the EC and CEN (European Committee for Standardization) started an initiative to standardize analytical methods for mycotoxins in food which gained increasing relevance, e.g. zearalenone (ZEN).[1] ZEN, an estrogenic mycotoxin produced by several Fusarium species, contaminates cereal crops worldwide. Due to its lipophilic nature ZEN is often found in edible oils (particularly in maize germ oils) derived from contaminated plants. Therefore, an European maximum level of 400 µg/kg is currently in force.[2] To perform reliable food analysis a sustainable metrological infrastructure is of major importance enabling the quantification of priority mycotoxins (here: ZEN). To achieve this goal an integrated approach is needed targeted at the development of validated analytical methods and certified reference materials (CRM).
A highly selective method for ZEN in edible oils will be presented, based on solid phase extraction (SPE) using hydrazine-functionalized particles. This method was developed for manual application using commercial SPE cartridges as well as for automated SPE-HPLC online coupling. While ZEN is covalently coupled to the solid phase by means of a hydrazone bond, undesired matrix components can be removed very efficiently. Finally, ZEN is decoupled from the solid phase, leading to highly purified extracts which are measured by HPLC-FLD.
The development of the first European Reference Material (ERM®) for ZEN in maize germ oil (ERM®-BC715) will be presented and discussed. This ERM®-project underpins the urgent need for mycotoxin-CRMs to support food safety and public health.
[1] European Commission (EC) Mandate M/520 (2013) for standardisation addressed to CEN for methods of analysis for mycotoxins in food. [2] Commission Regulation (EC) No 1126/2007 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards Fusarium toxins in maize and maize products.
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 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.
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 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 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.
Mykotoxine werden als sekundäre Metabolite von Schimmelpilzen gebildet und haben diverse schädliche Effekte auf Menschen, Tiere und Pflanzen. Laut FAO sind weltweit bis zu 25 % aller Lebens- und Futtermittel mit Mykotoxinen kontaminiert, wodurch jährlich immense ökonomische Verluste entstehen.
Neben den eigentlichen Mykotoxinen rücken zunehmend deren Transformationsprodukte (TPs) in den Fokus. Diese können als natürliche Stoffwechselprodukte toxischer sein als das eigentliche Mykotoxin und stellen eine potenzielle zusätzliche Gefahr für den Verbraucher dar. TPs werden überwiegend mit in vitro und in vivo Techniken untersucht, welche sowohl zeit- als auch kostenintensiv sind. Ziel war es nun, schnelle und zudem verlässliche Techniken zur Simulation von TPs diverser Mykotoxine zu testen. Die Kopplung von Elektrochemie und Massenspektrometrie, kurz EC/MS, als rein instrumenteller Ansatz ist seit vielen Jahren in der pharmazeutischen Forschung etabliert. Hierbei wird mithilfe einer elektrochemischen Durchflusszelle der oxidative Metabolismus einer Substanz simuliert und zugleich analysiert.
Unter Verwendung von EC/MS, EC/LC/MS und HPLC-MS Techniken (MS/MS und FT-ICR) wurden nun die Mykotoxine Zearalenon, Citrinin und Dihydroergocristin (als Modellsubstanz für die Ergotalkaloide) elektrochemisch bei Potentialen bis zu 2500 mV vs. Pd/H2 oxidiert und deren TPs mit mikrosomalen in vitro Ansätzen verglichen. Hierbei konnten mehrere Übereinstimmungen hydroxylierter Spezies, sowohl elektrochemisch als auch biochemisch generiert, festgestellt werden. Die EC/MS ist somit auch in der Lebensmittelanalytik als nützliches und vielseitiges Werkzeug verwendbar.
Mykotoxine in Lebensmitteln stellen weltweit Probleme dar, Zearalenon in Speiseöl ist ein solches. Neue Hydrazinbasierte Analyseverfahren können jetzt helfen, den EU-Grenzwert besser zu kontrollieren. Aber auch die Strukturaufklärung von Transformationsprodukten und verlässliche Kalibrierstandards sind aktuelle Herausforderungen.