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The main objective of this study was to determine the presence of Fusarium metabolites in maize samples collected from different regions of Northern Serbia (Backa, Banat and Srem) during a period o...

Monitoring of food contaminants and residues has undergone a significant improvement in recent years and is now performed in an intensive manner. Achievements in the area of chromatography-mass spectrometry coupling techniques enabled the development of quantitative multi-target approaches covering several hundred analytes. Although the majority of methods are focusing on the analysis of one specific group of substances, such as pesticides, mycotoxins, or veterinary drugs, current trends are going towards the simultaneous determination of multiclass compounds from several families of contaminants and residues. This work provides an overview of relevant multiclass concepts based on LC-MS/MS and LC-HRMS instruments. Merits and shortcomings will be critically discussed based on current performance characteristics of the EU legislation system. In addition, the discussion of a recently developed multiclass approach covering >1000 substances is presented as a case study to illustrate the current developments in this area.

Affordable and practical tools for farmers and food processors along the chain are required to efficiently reduce the risk of mycotoxin contamination of crops, feeds and foods. Developing new tools and enhancing existing ones was the mission of MyToolBox—a four-year EU-project that included important Chinese partners and joint research efforts. To identify future directions in mycotoxin research and management in China and their role in China–EU relations, a unique stakeholder workshop including group discussions was organized in Beijing. Six related topics: biocontrol, forecasting, sampling and analysis, silo management, detoxification, and the development of safe use options for contaminated materials were covered. The discussions clearly identified a critical need for smart, integrated strategies to address mycotoxin issues to attain safer food and feed, and to minimize losses and export rejections. Managing data on when, where and the size of mycotoxin contamination events and identifying the institution(s) to manage them are complex issues in China. Studies of microbes and novel, genetically-altered enzymes to limit pre-harvest contamination and to manage post-harvest product detoxification and alternate uses of contaminated materials are in the early stages in China. Further efforts are needed to increase the visibility of mycotoxin problems beyond the scientific and research communities.

This work provides a proposal for proper determination of matrix effects and extraction efficiencies as an integral part of full validation of liquid chromatography coupled to tandem mass spectrometry-based multiclass methods for complex feedstuff. Analytical performance data have been determined for 100 selected analytes in three compound feed matrices and twelve single feed ingredients using seven individual samples per matrix type. Apparent recoveries ranged from 60-140% for 52-89% of all compounds in single feed materials and 51-72% in complex compound feed. Regarding extraction efficiencies, 84-97% of all analytes ranged within 70-120% in all tested feed materials, implying that signal suppression due to matrix effects is the main source for the deviation from 100% of the expected target deriving from external calibration. However, the comparison between compound feed and single feed materials shows great variances regarding the apparent recoveries and matrix effects. Therefore, model compound feed formulas for cattle, pig, and chicken were prepared in-house in order to circumvent the issue of the lack of a true blank sample material and to simulate compositional uncertainties. The results of this work highlight that compound feed modeling enables a more realistic estimation of the method performance and therefore should be implemented in future validation guidelines.

Highlight Barley HvUGT13248 catalyzes conversion of nivalenol (NIV) to its non-toxic form 3-O-glucoside, and in transgenic wheat confers resistance to NIV-producing Fusarium graminearum, Fusarium Head Blight is a disease of cereal crops that causes severe yield losses and mycotoxin contamination of grain. The main causal pathogen, Fusarium graminearum, produces the trichothecene toxins deoxynivalenol or nivalenol as virulence factors. Nivalenol-producing isolates are most prevalent in Asia but co-exist with deoxynivalenol producers in lower frequency in North America and Europe. Previous studies identified a barley UDP-glucosyltransferase, HvUGT13248, that efficiently detoxifies deoxynivalenol, and when expressed in transgenic wheat results in high levels of type II resistance against deoxynivalenol-producing F. graminearum. Here we show that HvUGT13248 is also capable of converting nivalenol into the non-toxic nivalenol-3-O-β-d-glucoside. We describe the enzymatic preparation of a nivalenol-glucoside standard and its use in development of an analytical method to detect the nivalenol-glucoside conjugate. Recombinant Escherichia coli expressing HvUGT13248 glycosylates nivalenol more efficiently than deoxynivalenol. Overexpression in yeast, Arabidopsis thaliana, and wheat leads to increased nivalenol resistance. Increased ability to convert nivalenol to nivalenol-glucoside was observed in transgenic wheat, which also exhibits type II resistance to a nivalenol-producing F. graminearum strain. Our results demonstrate the HvUGT13248 can act to detoxify deoxynivalenol and nivalenol and provide resistance to deoxynivalenol- and nivalenol-producing Fusarium.

Many analytical methods for antioxidant determination in foodstuffs and raw materials based on various principles have been published so far. However, not all of them are applicable to barley and malt. The results of total antioxidant capacity (TEAC) of barley and malt obtained with methods based on ability to eliminate radicals of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) were compared and statistically evaluated. The average TEAC of barley determined using ABTS and DPPH ranged from 2.1 to 2.5 μmol g −1 and from 1.2 to 1.7 μmol g −1 , respectively. The TEAC ranges in malt were 2.7–3.0 μmol g −1 (ABTS) and 1.8–2.6 μmol g −1 (DPPH). TEAC of barley and malt were affected by the weather conditions (year), variety and application of Zn 2+ fertilizer. The ABTS and the DPPH methods represent an effective tool for the assessment of antioxidant capacity of spring barley and malt and the factors having an impact on it.

A simple and reliable method for the simultaneous determination of major type B trichothecene mycotoxins, deoxynivalenol (DON) and nivalenol (NIV), along with their 3-β-d-glucosides (DON-3-glucoside (DON3G) and NIV-3-glucoside (NIV3G)) in baby formula and Korean rice wine was validated in the present study. The method was based on immunoaffinity cleanup followed by analysis using an HPLC-UV technique. The method was validated in-house for two matrices as follows: linearity (R2 > 0.99) was established in the range of 20–1000 μg kg–1; accuracy (expressed as recovery) ranged from 78.7 to 106.5% for all the analytes; good intermediate precision (relative standard deviation < 12%), and adequate detection and quantitation limits (< 4.4 and < 13.3 μg kg–1, respectively) were achieved. Furthermore, the estimated measurement expanded uncertainty was determined to be 4–24%. The validated method was successfully applied to the analysis of 31 baby formulas and Korean rice wines marketed in Korea.

Crossover animal trials were performed with intravenous and oral administration of deoxynivalenol-3-β-D-glucoside (DON3G) and deoxynivalenol (DON) to broiler chickens and pigs. Systemic plasma concentrations of DON, DON3G and de-epoxy-DON were quantified using liquid chromatography-tandem mass spectrometry. Liquid chromatography coupled to high-resolution mass spectrometry was used to unravel phase II metabolism of DON. Additionally for pigs, portal plasma was analysed to study presystemic hydrolysis and metabolism. Data were processed via tailor-made compartmental toxicokinetic models. The results in broiler chickens indicate that DON3G is not hydrolysed to DON in vivo. Furthermore, the absolute oral bioavailability of DON3G in broiler chickens was low (3.79 ± 2.68 %) and comparable to that of DON (5.56 ± 2.05 %). After PO DON3G administration to pigs, only DON was detected in plasma, indicating a complete presystemic hydrolysis of the absorbed fraction of DON3G. However, the absorbed fraction of DON3G, recovered as DON, was approximately 5 times lower than after PO DON administration, 16.1 ± 5.4 compared with 81.3 ± 17.4 %. Analysis of phase II metabolites revealed that biotransformation of DON and DON3G in pigs mainly consists of glucuronidation, whereas in chickens predominantly conjugation with sulphate occurred. The extent of phase II metabolism is notably higher for chickens than for pigs, which might explain the differences in sensitivity of these species to DON. Although in vitro studies demonstrate a decreased toxicity of DON3G compared with DON, the species-dependent toxicokinetic data and in vivo hydrolysis to DON illustrate the toxicological relevance and consequently the need for further research to establish a tolerable daily intake.

The first quantitative multiclass approach enabling the accurate quantification of >1200 biotoxins, pesticides and veterinary drugs in complex feed using liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed. Optimization of HPLC/UHPLC (chromatographic column, flow rate and injection volume) and MS/MS conditions (dwell time and cycle time) were carried out in order to allow the combination of five major substance classes and the high number of target analytes with different physico-chemical properties. Cycle times and retention windows were carefully optimized and ensured appropriate dwell times reducing the overall measurement error. Validation was carried out in two compound feed matrices according to the EU SANTE validation guideline. Apparent recoveries matching the acceptable range of 60-140% accounted 60% and 79% for all analytes in cattle and chicken feed, respectively. High extraction efficiencies were obtained for all analyte/matrix combinations and revealed matrix effects as the main source for deviation of the targeted performance criteria. Concerning the methods repeatability 99% of all analytes in chicken and 96% in cattle feed complied with the acceptable RSD ≤ 20% criterion. Limits of quantification were between 1-10 µg/kg for the vast majority of compounds. Finally, the methods applicability was tested in >130 real compound feed samples and provides first insights into co-exposure of agro-contaminants in animal feed.

The main objective of this study was to screen, for the first time, the natural occurrence of non-regulated fungal metabolites in 204 maize samples harvested in Serbia in maize growing seasons with extreme drought (2012), extreme precipitation and flood (2014) and moderate drought conditions (2013 and 2015). In total, 109 non-regulated fungal metabolites were detected in examined samples, whereby each sample was contaminated between 13 and 55 non-regulated fungal metabolites. Moniliformin and beauvericin occurred in all samples collected from each year. In samples from year 2012, oxaline, questiomycin A, cyclo ( l -Pro- l -Val), cyclo ( l -Pro- l -Tyr), bikaverin, kojic acid and 3-nitropropionic acid were the most predominant (98.0–100%). All samples from 2014 were contaminated with 7-hydroxypestalotin, 15-hydroxyculmorin, culmorin, butenolid and aurofusarin. Bikaverin and oxaline were quantified in 100% samples from 2013 and 2015, while 3-nitropropionic acid additionally occurred in 100% samples from 2015.

The main objective of this study was to apply a liquid chromatography-tandem mass spectrometric method to investigate the presence of 20 mycotoxins in 204 maize samples harvested in Northern Serbia in the period 2012-2015, including seasons with extreme drought (2012), hot and dry conditions (2013 and 2015) and extreme precipitation (2014). Between 2 and 20 mycotoxins contaminated examined samples. In samples collected from each year, all of six examined fumonisins were detected with very high prevalence (from 76% to 100%). Aflatoxin B1 was detected in 94% and 90% maize samples from 2012 and 2015, respectively. In samples from year 2014, deoxynivalenol, zearalenone and its derivatives were detected in 100% of samples. Furthermore, ochratoxin A (25%) was the most predominant in samples from 2012. The obtained results indicate that changes in weather conditions, recorded in the period of four years, had significant influence on the occurrence of examined mycotoxins in maize.

Trichothecene toxins are confirmed or suspected virulence factors of various plant-pathogenic Fusarium species. Plants can detoxify these to a variable extent by glucosylation, a reaction catalyzed by UDP-glucosyltransferases (UGTs). Due to the unavailability of analytical standards for many trichothecene-glucoconjugates, information on such compounds is limited. Here, the previously identified deoxynivalenol-conjugating UGTs HvUGT13248 (barley), OsUGT79 (rice) and Bradi5g03300 (Brachypodium), were expressed in E. coli, affinity purified, and characterized towards their abilities to glucosylate the most relevant type A and B trichothecenes. HvUGT13248, which prefers nivalenol over deoxynivalenol, is also able to conjugate C-4 acetylated trichothecenes (e.g., T-2 toxin) to some degree while OsUGT79 and Bradi5g03300 are completely inactive with C-4 acetylated derivatives. The type A trichothecenes HT-2 toxin and T-2 triol are the kinetically preferred substrates in the case of HvUGT13248 and Bradi5g03300. We glucosylated several trichothecenes with OsUGT79 (HT-2 toxin, T-2 triol) and HvUGT13248 (T-2 toxin, neosolaniol, 4,15-diacetoxyscirpenol, fusarenon X) in the preparative scale. NMR analysis of the purified glucosides showed that exclusively β-D-glucosides were formed regio-selectively at position C-3-OH of the trichothecenes. These synthesized standards can be used to investigate the occurrence and toxicological properties of these modified mycotoxins.

Liquid chromatography (LC) coupled with mass spectrometry (MS) is widely used for the determination of mycotoxins in cereals and cereal-based products. In addition to the regulated mycotoxins, for which official control is required, LC–MS is often used for the screening of a large range of mycotoxins and/or for the identification and characterization of novel metabolites. This review provides insight into the LC–MS methods used for the determination of co-occurring mycotoxins with special emphasis on multiple-analyte applications. The first part of the review is focused on targeted LC–MS approaches using cleanup methods such as solid-phase extraction and immunoaffinity chromatography, as well as on methods based on minimum cleanup (quick, easy, cheap, effective, rugged, and safe; QuEChERS) and dilute and shoot. The second part of the review deals with the untargeted determination of mycotoxins by LC coupled with high-resolution MS, which includes also metabolomics techniques to study the fate of mycotoxins in plants. Electronic supplementary material The online version of this article (10.1007/s00216-017-0750-7) contains supplementary material, which is available to authorized users.

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.

A new type A trichothecene mycotoxin, NX-2, was previously reported to be produced by North American isolates of the cereal pathogen Fusarium graminearum. Here we describe the isolation and structural characterization of a rearrangement product, called NX2-M1, and related compounds with different acetylation patterns (NX3-M1 and NX4-M1). In the NX-M1 derivatives, the epoxide ring is opened, and a covalent bridge between C-10 and C-12 of the trichothecene backbone is formed. In vitro translation assays showed that NX3-M1 is less toxic for eukaryotic ribosomes than NX-3. NX3-M1 also has a greatly reduced cytotoxic potential on two tested human colon cell lines. Formation of NX3-M1 can therefore be regarded as a detoxification reaction. The related F. graminearum mycotoxin deoxynivalenol (DON), which is frequently occurring worldwide, is very stable during food processing. Testing NX-3 at different pH-values and temperature conditions, as well as under conditions that simulate the storage of infected grains and bread-making process, revealed a strongly reduced stability of NX-3 and concurrent formation of NX3-M1. Although the NX-3 formed in planta is as toxic as DON, the extensive formation of the non-toxic rearrangement product should be taken into account for risk assessment of this emerging food contaminant.

Fusarium verticillioides causes ear rot in maize and contaminates the kernels with the fumonisin mycotoxins. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defence against pathogens and that the host-pathogen lipid cross-talk influences the pathogenesis. The expression profiles of fifteen genes of the LOX pathway were studied in kernels of resistant and susceptible maize lines, grown in field condition, at 3, 7 and 14 days post inoculation (dpi) with F. verticillioides. Plant defence responses were correlated with the pathogen growth, the expression profiles of fungal FUM genes for fumonisin biosynthesis and fumonisin content in the kernels. The resistant genotype limited fungal growth and fumonisin accumulation between 7 and 14 dpi. Pathogen growth became exponential in the susceptible line after 7 dpi, in correspondence with massive transcription of FUM genes and fumonisins augmented exponentially at 14 dpi. LOX pathway genes resulted strongly induced after pathogen inoculation in the resistant line at 3 and 7 dpi, whilst in the susceptible line the induction was reduced or delayed at 14 dpi. In addition, all genes resulted overexpressed before infection in kernels of the resistant genotype already at 3 dpi. The results suggest that resistance in maize may depend on an earlier activation of LOX genes and genes for jasmonic acid biosynthesis.

In the scientific field, there is a progressive awareness about the potential implications of food processing on mycotoxins especially concerning thermal treatments. High temperatures may cause, in fact, transformation or degradation of these compounds. This work is aimed to study the fate of mycotoxins during bakery processing, focusing on deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON3Glc), along the chain of industrial rusk production. Starting from naturally contaminated bran, we studied how concentrations of DON and DON3Glc are influenced by modifying ingredients and operative conditions. The experiments were performed using statistical Design of Experiment (DoE) schemes to synergistically explore the relationship between mycotoxin reduction and the indicated processing transformation parameters. All samples collected during pilot plant experiments were analyzed with an LC-MS/MS multimycotoxin method. The obtained model shows a good fitting, giving back relevant information in terms of optimization of the industrial production process, in particular suggesting that time and temperature in baking and toasting steps are highly relevant for minimizing mycotoxin level in rusks. A reduction up to 30% for DON and DON3Glc content in the finished product was observed within an acceptable technological range.

Fumonisin B 1 (FB 1 ) is a Fusarium mycotoxin frequently occurring in maize-based food and feed. Alkaline processing like nixtamalisation of maize generates partially and fully hydrolysed FB 1 (pHFB 1 and HFB 1 ) and thermal treatment in the presence of reducing sugars leads to formation of N- (1-deoxy-D-fructos-1-yl) fumonisin B 1 (NDF). The toxicity of these metabolites, in particular their effect on the sphingolipid metabolism, is either unknown or discussed controversially. We produced high purity FB 1 , pHFB 1 a+b, HFB 1 and NDF and fed them to male Sprague Dawley rats for three weeks. Once a week, urine and faeces samples were collected over 24 h and analysed for fumonisin metabolites as well as for the sphinganine (Sa) to sphingosine (So) ratio by validated LC–MS/MS based methods. While the latter was significantly increased in the FB 1 positive control group, the Sa/So ratios of the partially and fully hydrolysed fumonisins were indifferent from the negative control group. Although NDF was partly cleaved during digestion, the liberated amounts of FB 1 did not raise the Sa/So ratio. These results show that the investigated alkaline and thermal processing products of FB 1 were, at the tested concentrations, non-toxic for rats, and suggest that according food processing can reduce fumonisin toxicity for humans.

Přehledove sděleni shrnuje strucný přehled analytických metod použivaných pro stanoveni deoxynivalenolu a jeho derivatů deoxynivalenol-3-β-D-glukosidu, 3-acetyldeoxynivalenolu a 15-acetyldeoxynivalenolu v pivu. Teprve nedavno byly v pivu identifikovany take oligoglykosidy deoxynivalenolu. Mezi analytickými metodami jsou uvedeny jak plynova chromatografie ve spojeni s plamenoionizacni detekci, detekci elektronoveho zachytu ci hmotnostni spektrometrii, tak kapalinova chromatografie se spektrofotometrickou detekci, a v neposledni řadě take kapalinova chromatografie ve spojeni s hmotnostni spektrometrii. Diskuse je převažně zaměřena na připravu vzorků před vlastni analýzou. Zavěrecna cast clanku je věnovana imunochemickým metodam. Nejrozsiřenějsi imunochemickou metodou je tzv. ELISA (Enzyme-Linked Immunosorbent Assay), ktera představuje velice efektivni nastroj pro rychlou analýzu diky sve jednoduchosti a přime aplikaci tekutých vzorků bez předchoziho přecistěni.

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.

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.

Increasing frequencies of 3-acetyl-deoxynivalenol (3-ADON)-producing strains of Fusarium graminearum (3-ADON chemotype) have been reported in North America and Asia. 3-ADON is nearly nontoxic at the level of the ribosomal target and has to be deacetylated to cause inhibition of protein biosynthesis. Plant cells can efficiently remove the acetyl groups of 3-ADON, but the underlying genes are yet unknown. We therefore performed a study of the family of candidate carboxylesterases (CXE) genes of the monocot model plant Brachypodium distachyon. We report the identification and characterization of the first plant enzymes responsible for deacetylation of trichothecene toxins. The product of the BdCXE29 gene efficiently deacetylates T-2 toxin to HT-2 toxin, NX-2 to NX-3, both 3-ADON and 15-acetyl-deoxynivalenol (15-ADON) into deoxynivalenol and, to a lesser degree, also fusarenon X into nivalenol. The BdCXE52 esterase showed lower activity than BdCXE29 when expressed in yeast and accepts 3-ADON, NX-2, 15-ADON and, to a limited extent, fusarenon X as substrates. Expression of these Brachypodium genes in yeast increases the toxicity of 3-ADON, suggesting that highly similar genes existing in crop plants may act as susceptibility factors in Fusarium head blight disease.

Fusarium toxins, Alternaria toxins, and ergot alkaloids represent common groups of mycotoxins that can be found in cereals grown under temperate climatic conditions. Because most of them are chemically and thermally stable, these toxic fungal secondary metabolites might be transferred from grains into the final products. To get information on the commensurate contamination of various cereal-based products collected from the Czech retail market in 2010, the occurrence of "traditional" mycotoxins such as groups of A and B trichothecenes and zearalenone, less routinely determined Alternaria toxins (alternariol, alternariol monomethyl ether and altenuene), ergot alkaloids (ergosine, ergocryptine, ergocristine, and ergocornine) and "emerging" mycotoxins (enniatins A, A1, B, and B1 and beauvericin) were monitored. In a total 116 samples derived from white flour and mixed flour, breakfast cereals, snacks, and flour, only trichothecenes A and B and enniatins were found. Deoxynivalenol was detected in 75% of samples with concentrations ranging from 13 to 594 μg/kg, but its masked form, deoxynivalenol-3-β-d-glucoside, has an even higher incidence of 80% of samples, and concentrations ranging between 5 and 72 μg/kg were detected. Nivalenol was found only in three samples at levels of 30 μg/kg. For enniatins, all of the samples investigated were contaminated with at least one of four target enniatins. Enniatin A was detected in 97% of samples (concentration range of 20-2532 μg/kg) followed by enniatin B with an incidence in 91% of the samples (concentration range of 13-941 μg/kg) and enniatin B1 with an incidence of 80% in the samples tested (concentration range of 8-785 μg/kg). Enniatin A1 was found only in 44% of samples at levels ranging between 8 and 851 μg/kg.

The objective of this study was to determine the occurrence of trichothecenes of both the A-type and B-type, masked mycotoxin derived from DON - deoxynivalenol-3-glucoside (DON-3-Glc), 3- and 15-acetyldeoxynivalenol (ADONs), fusarenon-X (FUS-X) and nivalenol (NIV)) as well as zearalenone (ZON) in winter wheat. Total of 54 samples were collected during the harvest of 2007 representing the most important Serbian wheat-growing regions. The samples were prepared by one-step simple method and analyzed by high performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The obtained recoveries proved that the used method could be successfully applied for multi-component analysis of Fusarium mycotoxins. DON, DON-3-Glc and HT-2 contents were detected approximately in 28%, 13% and 6% of the total number of samples, respectively. The amount of these toxins ranged from 17 μg/kg for DON-3-Glc to 309 μg/kg for DON. ADONs, FUS-X, NIV, T-2 toxin as well as ZON were below the limit of detection. Different susceptibility of wheat cultivars towards detected mycotoxins was observed. The results were compared to the EC Regulative and with available the literature data concerning the neighboring countries. This is first report on the simultaneous presence of 8 Fusarium mycotoxins in the wheat cultivated in the Balkan Countries region.

The objective of the presented study was to develop and optimize a simple, high-throughput method for the control of 32 mycotoxins (Fusarium and Alternaria toxins, aflatoxins, ergot alkaloids, ochratoxins, and sterigmatocystin) in beer. Due to the broad range of their physicochemical properties, the sample preparation step was simplified as much as possible to avoid analyte losses. The addition of acetonitrile to beer samples enabled precipitation of abundant matrix components. Theclean-upefficiencywas controlledbyambientmassspectrometryemployingadirectanalysis inreal time (DART) ion source. For determination of analytes, ultra-high-performance liquid chromatography hyphenated with high-resolution mass spectrometry utilizing an orbitrap (U-HPLC‐orbitrapMS) or time-of-flight (TOFMS) technology was used. Because of significantly better detection capabilities of the orbitrap technology, the U-HPLC‐orbitrapMS method was chosen as a determinative step and fully validated. To compensate matrix effects, matrix-matched calibration was employed. The lowest calibration levels for most of the target mycotoxins ranged from 1 to 8 mgL � 1 beer and the recoveries of analytes were in range from 86 to 124%. Copyright # 2010 John Wiley & Sons, Ltd. Mycotoxins are toxic secondary metabolites produced by many species of microscopic filamentous fungi occurring on field cereals, including barley. The most abundant fungal genera affecting the malting barley are Alternaria, Aspergillus, Penicillium, and Fusarium, which simultaneously showed relatively high producing potential for a wide range of mycotoxins (almost 30% of Alternaria, 20% of Aspergillus, and 88% of Fusarium fungi isolated from barley grains was able to produce alternaria toxins, aflatoxins, ochratoxin A, deoxynivalenol and zearalenone). 1 Additionally to the relatively common micromycetes mentioned above, also the Clavicepspurpureacausing the ergot disease belongs to frequent barley pathogens. 2

BACKGROUND:Fusarium toxins, secondary metabolites of toxinogenic Fusarium species, are found in a range of cereal grains. In this study the occurrence of the most commonest Fusarium toxins, namely nivalenol (NIV), deoxynivalenol (DON), deoxynivalenol-3-glucoside, fusarenon-X, 3- and 15-acetyldeoxynivalenol, HT-2 and T-2 toxins and zearalenone, in various barley cultivars harvested in 2005–2008 was monitored. The impact of weather, locality, fungicide treatment and barley cultivar (hulless or covered) on contamination was evaluated. The transfer of these mycotoxins into malt was assessed. RESULTS: The most prevalent toxin was DON, which was found in 83% of samples (maximum level 180 µg kg−1), while HT-2 was detected in 62% of samples (maximum level 716 µg kg−1). Using analysis of covariance, weather was found to be the key factor in all years (P < 0.001). A relationship between cultivar and contamination was confirmed only for HT-2 (P < 0.001) and T-2 (P = 0.037), with higher levels of these toxins being observed in hulless cultivars. With the exception of NIV (P = 0.008), no significant relationship was found between fungicide treatment and contamination. No distinct trend regarding DON levels in malt was found, with both decreases and increases occurring. CONCLUSION: The results show an inter-annual variation in mycotoxin occurrence in barley cultivars as well as differences in contamination of malt produced from fungicide-treated and untreated barley. Copyright © 2010 Society of Chemical Industry

Rapid, simple and cost-effective analytical methods with performance characteristics matching regulatory requirements are needed for effective control of occurrence of Fusarium toxins in cereals and cereal-based products to which they might be transferred during processing. Within this study, two alternative approaches enabling retrospective data analysis and identification of unknown signals in sample extracts have been implemented and validated for determination of 11 major Fusarium toxins. In both cases, ultra-high performance liquid chromatography (U-HPLC) coupled with high resolution mass spectrometry (HR MS) was employed. 13C isotopically labeled surrogates as well as matrix-matched standards were employed for quantification. As far as time of flight mass analyzer (TOF-MS) was a detection tool, the use of modified QuEChERS (quick easy cheap effective rugged and safe) sample preparation procedure, widely employed in multi-pesticides residue analysis, was shown as an optimal approach to obtain low detection limits. The second challenging alternative, enabling direct analysis of crude extract, was the use of mass analyzer based on Orbitrap technology. In addition to demonstration of full compliance of the new methods with Commission Regulation (EC) No. 401/2006, also their potential to be used for confirmatory purposes according to Commission Decision 2002/657/EC has been critically assessed.

Fusariove mykotoxiny jsou toxicke sekundarni metabolity mikroskopických vlaknitých hub rodu Fusarium. Prezentovana studie shrnuje ziskane poznatky o výskytu sirokeho spektra těchto mykotoxinů v produkcnim řetězci jecmen - slad ve 12 vybraných odrůdach/liniich jecmene jarniho pěstovaných low-input a konvencnim přistupem v letech 2005-2008 na lokalitě Žabcice a Kroměřiž.

An LC–MS/MS “dilute and shoot” method for the determination of 295 fungal and bacterial metabolites was optimized and validated according to the guidelines established in the Directorate General for Health and Consumer Affairs of the European Commission (SANCO) document No. 12495/2011. Four different types of food matrices were chosen for validation: apple puree for infants (high water content), hazelnuts (high fat content), maize (high starch and low fat content) and green pepper (difficult or unique matrix). Method accuracy and precision was evaluated using spiked samples in five replicates at two concentration levels. Method trueness was demonstrated through participation in various proficiency tests. Although the method covers a total number of 331 analytes, validation data were acquired only for 295 analytes, either due to the non-availability of analytical standards or due other reasons described in this paper. Concerning the apparent recovery, the percentage of 295 analytes matching the acceptable recovery range of 70–120% lied down by SANCO varied from 21% in green pepper to 74% in apple puree at the highest spiking level. At the levels close to limit of quantification only 20–58% of the analytes fulfilled this criterion. The extent of matrix effects was strongly dependent on the analyte/matrix combination. In general, the lowest matrix effects were observed in apple puree (59% of analytes were not influenced by enhancement/suppression at all at the highest validation level). The highest matrix effects were observed in green pepper, where only 10% of analytes did not suffer from signal suppression/enhancement. The repeatability of the method was acceptable (RSD ≤ 20) for 97% of all analytes in apple puree and hazelnuts, for 95% in maize and for 89% in green pepper. Concerning the trueness of the method, Z -scores were generally between −2 and 2, despite a broad variety of different matrices. Based on these results it can be concluded that quantitative determination of mycotoxins by LC–MS/MS based on a “dilute and shoot” approach is also feasible in case of complex matrices.

An extensive study of the metabolism of the type A trichothecene mycotoxins HT-2 toxin and T-2 toxin in barley using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) is reported. A recently developed untargeted approach based on stable isotopic labelling, LC-Orbitrap-MS analysis with fast polarity switching and data processing by MetExtract software was combined with targeted LC-Q-TOF-MS(/MS) analysis for metabolite structure elucidation and quantification. In total, 9 HT-2 toxin and 13 T-2 toxin metabolites plus tentative isomers were detected, which were successfully annotated by calculation of elemental formulas and further LC-HRMS/MS measurements as well as partly identified with authentic standards. As a result, glucosylated forms of the toxins, malonylglucosides, and acetyl and feruloyl conjugates were elucidated. Additionally, time courses of metabolite formation and mass balances were established. For absolute quantification of those compounds for which standards were available, the method was validated by determining apparent recovery, signal suppression, or enhancement and extraction recovery. Most importantly, T-2 toxin was rapidly metabolised to HT-2 toxin and for both parent toxins HT-2 toxin-3-O-β-glucoside was identified (confirmed by authentic standard) as the main metabolite, which reached its maximum already 1 day after toxin treatment. Graphical Abstract Isotope-assisted untargeted screening of HT-2 toxin and T-2 toxin metabolites in barley Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8975-9) contains supplementary material, which is available to authorized users.

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3- O -β- d -glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial ( Lactobacillus brevis and Bifidobacterium adolescentis ) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis ( Ba Bgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3- O -β- d -glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A K m of 5.4 mM and a V max of 16 μmol min −1 mg −1 were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.

In this study, a total of nine different biotransformation products of the Fusarium mycotoxin deoxynivalenol (DON) formed in wheat during detoxification of the toxin are characterized by liquid chromatography—high resolution mass spectrometry (LC-HRMS). The detected metabolites suggest that DON is conjugated to endogenous metabolites via two major metabolism routes, namely 1) glucosylation (DON-3-glucoside, DON-di-hexoside, 15-acetyl-DON-3-glucoside, DON-malonylglucoside) and 2) glutathione conjugation (DON-S-glutathione, “DON-2H”-S-glutathione, DON-S-cysteinyl-glycine and DON-S-cysteine). Furthermore, conjugation of DON to a putative sugar alcohol (hexitol) was found. A molar mass balance for the cultivar ‘Remus’ treated with 1 mg DON revealed that under the test conditions approximately 15% of the added DON were transformed into DON-3-glucoside and another 19% were transformed to the remaining eight biotransformation products or irreversibly bound to the plant matrix. Additionally, metabolite abundance was monitored as a function of time for each DON derivative and was established for six DON treated wheat lines (1 mg/ear) differing in resistance quantitative trait loci (QTL) Fhb1 and/or Qfhs.ifa-5A. All cultivars carrying QTL Fhb1 showed similar metabolism kinetics: Formation of DON-Glc was faster, while DON-GSH production was less efficient compared to cultivars which lacked the resistance QTL Fhb1. Moreover, all wheat lines harboring Fhb1 showed significantly elevated D3G/DON abundance ratios.