Mycotoxins constitute a very heterogeneous group of toxic substances. The differences in polarity, ultraviolet absorption, fluorescence, and ionic nature as well as the occurrence in different commodities at varying concentration levels have lead to particular extraction, clean-up and detection strategies, which will be discussed in this article. Yet, multimycotoxin methods, which target the determination of several mycotoxins in a single run are highly desirable in order to keep time and costs low. The simultaneous determination of mycotoxins from different families is very complex, but doors have been opened by establishing liquid chromatography with tandem mass spectrometry, allowing accurate determination of currently up to 40 mycotoxins without the need for any cleanup. Besides the already established “classical” methods using a chromatographic system such as high performance liquid chromatography with ultraviolet, mass spectrometry or fluorescence detection or gas chromatography with electron capture detection, flame ionisation detection or mass spectrometry, liquid chromatography with tandem mass spectrometry is coming to the fore-front. A number of ‘rapid’ methods such as enzyme-linked immunosorbent assay or lateral flow devices have already found widespread use in screening of high numbers of samples in the field and also in routine analysis in house. The need to fulfil the requirements of mycotoxins analysis – simple, fast, robust, sensitive, selective, accurate, cost-effective – has also lead to a number of ‘emerging techniques’ such as molecularly imprinted polymers, fluorescence polarisation, infrared technologies, etc. They have shown great potential, but their applicability in routine analysis is yet to be shown. Towering above all methods are certainly the reference methods approved by AOAC International or the European Committee for Standardization to be used in case of official control and surveillance, and in case of dispute. Several measures of quality control such as the use of (certified) reference materials or the participation in proficiency tests are major prerequisites to ensure traceability and comparability of the results in mycotoxin analysis.
On-going research and interdisciplinary networking among scientists and stakeholders are still needed for the development of affordable and practical tools for farmers and food processors to efficiently manage the risk of mycotoxin contamination along food and feed chains. In order to facilitate appropriate actions, thematic interlaboratory projects have been initiated, professional networking-organisations have been founded, dedicated journals have been started and mycotoxin conferences have been organised. The World Mycotoxin Forum (WMF) has established itself as a leading international networking conference series on mycotoxins where food and feed industry representatives meet with representatives from government, food authorities, food and feed industry and with people from universities and other research institutions from around the world. The WMF has gradually moved from food and feed related technological topics to international and regulatory issues and to mitigation strategies based on fungal and plant genetics, big data based decision support tools and sustainable solutions in view of climate change. Over all these years, the conclusions made from each WMF have been summarised by the general conference chair(s) as the ‘top 5 lessons learned’ on the last day of each conference. 50 years after the first international conference on mycotoxins organised by IPUAC in 1972, this article has compiled all ‘top 5 lessons learned’ presented at the 16 editions of the WMF and joint WMF/IUPAC events organised between 2001 and 2022. Since the first international conference on mycotoxins, much progress has been made in the prevention, reduction and control of mycotoxins. However, continuous attention and further efforts are still needed in order to tackle the complex issues of mycotoxin contamination especially in view of climate change and other global challenges on the horizon.
Mycotoxins are secondary metabolites produced by fungi belonging mainly to the Aspergillus, Penicillium and
Fusarium genera. They represent a relevant source of danger to human and animal health causing food- and feedborne
intoxication. One group, produced by Fusarium spp., are the trichothecenes, of which T-2 toxin belongs to
the type-A trichothecenes and deoxynivalenol to the type-B trichothecenes. As these mycotoxins are ubiquitous,
the testing of products is required to keep our food and feed safe. For this purpose, sensitive and reliable tests
are needed to detect contaminations. One detection possibility is an immunoanalytical based test which needs
antibodies as reagents. Cell culture facilities allow cell line selection and production of monoclonal antibodies
for further immunological test development. Especially for mycotoxins antibody development for further use in
immunoassays is a crucial task. T-2 toxin and deoxynivalenol specific monoclonal antibodies were developed and
further characterised to test stability and solvent resistance properties. Especially cross-reactivities were determined
to related mycotoxins also belonging to the trichothecene family, e.g. HT-2 toxin or 3-acetyldeoxynivalenol.
In this paper the stability and degree of epimerisation of six major ergot alkaloids at three different temperature
levels (-20 °C, +4 °C and +20 °C) over periods of 18 hours and six weeks is reported for the first time. The behaviour
of ergometrine, ergocornine, ergocristine, α-ergocryptine, ergosine and ergotamine was thoroughly studied in seven
solvents which are employed for the preparation of calibrants and extraction mixtures, respectively. Moreover, the
stability of the ergot alkaloids was tested in different cereal extracts (rye, wheat, barley, oats) for 1, 2 and 6 days. Of the toxins tested, the ergopeptide-type toxins ergosine, ergotamine, ergocornine, α-ergocryptine and ergocristine showed similar behaviour patterns. The simple lysergic acid derivative ergometrine was more stable and showed
hardly any epimerisation to ergometrinine, with the sum of both epimers remaining constant in all seven solvents.
The ergopeptides tested show variable epimerisation tendencies, and were also less stable during six weeks at
20 °C. Ergosine showed the highest degree of epimerisation (43% after 6 weeks at 20 °C). In general, the order of
epimerisation promotion was methanol/dichloromethane > acetonitrile/buffer > extraction mix > stabilising solution
> acetonitrile >> chloroform. Long-term storage at room temperature can only be carried out in chloroform, which
showed no epimerisation for all toxins even at 20 °C and also kept the sum of R and S forms constant, which indicates
no formation of aci-epimers or other degradation products. Long-term storage of ergot alkaloids in acetonitrile, the
most convenient solvent with respect to HPLC analysis, should be carried out at temperatures of -20 °C or below. The
constant epimer ratio of all ergot alkaloids in the extraction mixture acetonitrile/ammonium carbonate buffer (200
mg/l; 92:8, v/v) during an HPLC run (18 hours) demonstrates the stability of the toxins in this extraction mixture.
After water and tea, beer is the third most popular beverage worldwide. Brewed from malted cereal grains, beer is known to be potentially contaminated with mycotoxins. Some studies have shown that not only the Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-DON (3-ADON), but also the conjugated mycotoxin deoxynivalenol-3-glucoside (D3G) can be found in beer on a regular basis, albeit usually at low concentrations. The aim of this work was to develop the first triple quadrupole LC-MS/MS based method for the determination of DON, D3G and 3-ADON in beer and to perform an in-house validation. The simple sample preparation includes degassing, precipitation of matrix compounds and reconstitution of the dried-down sample in solvent. Since different kinds of beer exist and method performance parameters will likely differ, we categorised the samples into pale, wheat, dark, bock and non-alcoholic beers, as well as shandies, and validated all six matrices. Although three individual beers for each category were spiked at eight levels prior to sample preparation, the repeatability of the overall method was still excellent with relative standard deviations from 4-16% for all analytes and types of beer. Limits of detection were in the sub- or low-μg/kg range. Apparent recoveries of 60-90% for DON, 39-69% for D3G and 96-124% for 3-ADON were obtained for the different types of beer, with dark and bock beers being the most difficult matrices. To prove the applicability of the method, ten beers of each category were analysed. While average concentrations of 6.6 μg/l for DON and D3G were found, no 3-ADON was detected in any of the samples.
Based on the recent scientific opinion of the European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain on the risks to human and animal health related to the presence of T-2 and HT-2 toxins in food and feed that was published by EFSA in the EFSA Journal, this article provides an update on the determination of these Fusarium mycotoxins. After a brief introduction into the chemistry of these toxins, both chromatographic and immuno-analytical methods are discussed for the determination of these type A trichothecenes. During the last decade, liquid chromatography with (tandem) mass spectrometry has become the most frequently used method for the determination of T-2 and HT-2 toxins, often within a multi-analyte approach. However, complex matrices and the resulting signal suppression effects, as observed particularly in electrospray-mass spectrometry methods owing to matrix effects, may require careful optimisation of clean-up, usage of matrix matched standards, or e.g. the use of internal standards. For specific purposes where extremely low limits of quantification are needed, e.g. for the analysis of duplicate diets, a dedicated gas chromatography method with multistage mass spectrometry has become available. Other novel analytical approaches to determine T-2 and HT-2 toxins in food and feed include biosensor-based methods in surface plasmon resonance and electrochemical formats, as well as DNA microchip assays. For rapid screening, several immunochemical methods (mostly ELISAs) have become available and some are sold as commercial test kits. Whereas these methods work fast, cross-reactivities with other trichothecenes can have an undesired effect on their accuracy. While proficiency tests including T-2 and HT-2 toxins have been carried out, none of the chromatographic or immunochemical methods have been formally validated in interlaboratory validation studies. There are no certified reference materials available for T-2 and HT-2 toxins.
Based on the recent scientific opinion published by the EFSA CONTAM panel on the risks to human and animal health related to the presence of nivalenol in food and feed, this article provides an update on the determination of this Fusarium mycotoxin. After a brief introduction into the chemistry of nivalenol, chromatographic methods as well as other approaches are being discussed. Methods for the determination of nivalenol are well established and can be applied for the analysis of cereals, food, feed and biological samples. Accurate quantification of nivalenol is mostly carried out by liquid chromatography coupled with (multi-stage) mass spectrometry (MS) often within a multi-analyte approach. Some novel techniques, such as direct analysis in real time (DART) MS and electrochemical methods, have shown potential to determine nivalenol, but applications for routine measurements are not yet available. None of the currently available analytical methods has been formally validated in interlaboratory validation studies. While a certified calibrant for nivalenol is available, no matrix reference materials have been developed. Due to the scarcity of appropriate antibodies also no rapid immunochemical methods specific for nivalenol have become available.
Azole fungicides have been reported to be the most effective active substances in the control of Fusarium Head Blight (FHB) and in the reduction of the main mycotoxins that occur in cereal grain, such as deoxynivalenol (DON). Four field experiments have been conducted in North West Italy, over a period of 2 growing seasons, in order to evaluate the effect of azole fungicide (prothioconazole) applications on the prevalence of emerging mycotoxins in common winter wheat under naturally-infected field conditions. Wheat samples have been analysed by means of a dilute-and-shoot multi-mycotoxin LC-MS/MS method. Twenty fungal metabolites were detected: enniatins, aurofusarin, moniliformin, equisetin, DON, deoxynivalenol-3-glucoside, culmorin, bikaverin, beauvericin, fumonisins, fusaric acid, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, zearalenone, decalonectrin, butenolide, tentoxin, alternariol and alternariol methyl ether. The most abundant fungal metabolites were DON and culmorin, with an average contamination in the untreated control of 1,360 μg/kg and 875 μg/kg, respectively, in the growing season with the highest disease pressure (2011-2012). On average, the results have shown that the fungicide application significantly reduced the enniatins (from 127 μg/kg to 46 μg/kg), aurofusarin (from 62 μg/kg to 21 μg/kg), moniliformin (from 32 μg/kg to 16 μg/kg), tentoxin (from 5.2 μg/kg to 2.5 μg/kg) and equisetin (from 0.72 μg/kg to 0.06 μg/kg) contents in all the experiments. However, DON, deoxynivalenol-3-glucoside and culmorin were only significantly reduced in the growing season with the highest disease pressure. The other fungal metabolites were mainly found in traces in the untreated plots. These results, which have been obtained in different environmental and agronomic conditions, have underlined for the first time that the fungicide usually applied to control the FHB and DON content, also consistently reduces the main emerging mycotoxins of winter wheat in temperate areas.
This study investigated the diversity of fungal metabolites in maize across four agro-ecological zones of Malawi. A total of 90 maize samples (for human consumption), collected from farmsteads, were analysed for 235 fungal metabolites using liquid chromatography-tandem mass spectrometry. A total of 65 metabolites were found in the samples. 75% of samples from the hottest agro-ecological zone contained either aflatoxins, fumonisins, deoxynivalenol, zearalenone; or a combination thereof in levels exceeding European Union (EU) maximum levels, whereas the related fraction was only 17% in the cool temperature zone. Aflatoxins, citrinin, 3-nitropropionic acid, monocerin and equisetin were most prevalent and in higher levels in samples from hot agro-ecological zones, whereas deoxynivalenol, nivalenol, zearalenone and aurofusarin were most prevalent in cool agro-ecologies. On the basis of per-capita maize consumption, estimated daily intakes for all samples from hot ecologies were well above the JECFA's provisional maximum tolerable daily intake (PMTDI) of 2.0 μg/kg body weight (bw)/day for fumonisins, whereas the PMTDI of 1.0 μg/kg bw/day for deoxynivalenol was exceeded in relatively more (90%) samples from the cool highlands than the other zones. These results demonstrate the influence of micro-climatic conditions on mycotoxin prevalence patterns and underscores the need for development of agro-ecological specific mycotoxin dietary exposure management strategies.
Fumonisin B1 (FB1), first reported in 1988 in South Africa, is the predominant fumonisin among a group of structurally related fumonisins. It occurs in mainly maize and maize-based products. FB1 has been shown to produce a wide range of pathological effects in animals, including leukoencephalomalacia in horses and pulmonary oedema in swine. It also disrupts sphingolipid biosynthetic pathways in animals and plant cells. It is hence understandable that there is considerable interest in the detoxification of FB1. Decontamination using selected micro-organisms with the ability to degrade or biotransform mycotoxins has many advantages. Blackwell et al. (1999) showed that the fungus Exophiala spinifera produces enzymes that metabolise fumonisins. This fumonisin-metabolism consists of de-esterification of fumonisins tricarballylic acid esters followed by oxidative deamination of the resulting amine alcohol backbone resulting in a 2-oxo-12,16-dimethyl-3,5,10,14,15-icosanepentol (2-OP1) that undergoes internal cyclisation to form a hemiketal. Since hydrolysed fumonisin B1 (HFB1) can easily be adsorbed and is as toxic as or even more toxic than FB1, a study on other micro-organisms with the capability to detoxify FB1 and HFB1 was conducted. As a first step, reported here, samples were analysed after degradation with E. spinifera. Using LC-MS/MS for characterisation of the samples, we could reconstruct the transformation of FB1 over partially hydrolysed FB1 to completely hydrolysed FB1, which was then further metabolised. This metabolism led to a degradation product HFB1, which showed a mass spectrum with similar molecular ion signal and fragmentation pattern like 2-OP1. To characterise the product we performed full scan and also product ion scan experiments in both, positive and negative ionisation mode. Detoxification of the degraded samples was also demonstrated in a bioassay.