Mycotoxin contamination in moldy slices of bread is mostly limited to the immediate vicinity of the visible infestation.

Bread is an important staple food that is susceptible to spoilage, making it one of the most wasted foods. To determine the safety of partially moldy bread, five types of bread were inoculated with common mold species. After incubation, the metabolite profile was determined in and under the inoculation spot, as well as at a lateral distance of 3 cm from the moldy spot. The result showed that the metabolites were exclusively concentrated in the inoculation area and directly below the inoculation area. The only exception was citrinin, a mycotoxin produced by Penicillia such as Penicillium citrinum, which was detected in almost all tested bread areas when inoculated with the corresponding strains. The results of our study suggest that the removal of moldy parts may be a solution to reduce food waste if the remaining bread is to be used, for example for insect farming to produce animal feed.

Extention and interlaboratory comparison of an LC-MS/MS multi-class method for the determination of 15 different classes of veterinary drug residues in milk and poultry feed.

An HPLC-MS/MS multi-class method for quantitation of 15 different classes of veterinary drug residues (>140 analytes) in milk and poultry feed was developed and validated. Accuracy criteria for routine laboratories were met for the majority of analytes, > 83 % in milk and between 50 and 60 % in chicken feed, with an apparent recovery of 60-140 %. Extraction efficiency criteria were met for >95 % of the analytes for milk and > 80 % for chicken feed. Intermediate precision meets the SANTE criterion of RSD < 20 % for 80-90 % of the analytes in both matrices. For all analytes with an existing MRL in milk, the LOQ was below the related MRL. Twenty-nine samples of commercial milk and chicken feed were analyzed within the interlaboratory comparison. No residues of veterinary drugs were found in the milk samples. However, the feed samples exhibited high levels of nicarbazin, salinomycin, and decoquinate.

Introduction to This Special Issue of Toxins: Reduction and Control of Mycotoxins along Entire Food and Feed Chain.

Contamination of food and feed by mycotoxins is considered a significant issue in food and feed safety worldwide [...].

An Interlaboratory Comparison Study of Regulated and Emerging Mycotoxins Using Liquid Chromatography Mass Spectrometry: Challenges and Future Directions of Routine Multi-Mycotoxin Analysis including Emerging Mycotoxins.

The present interlaboratory comparison study involved nine laboratories located throughout the world that tested for 24 regulated and non-regulated mycotoxins by applying their in-house LC-MS/MS multi-toxin method to 10 individual lots of 4 matrix commodities, including complex chicken and swine feed, soy and corn gluten. In total, more than 6000 data points were collected and analyzed statistically by calculating a consensus value in combination with a target standard deviation following a modified Horwitz equation. The performance of each participant was evaluated by a z-score assessment with a satisfying range of ±2, leading to an overall success rate of 70% for all tested compounds. Equal performance for both regulated and emerging mycotoxins indicates that participating routine laboratories have successfully expanded their analytical portfolio in view of potentially new regulations. In addition, the study design proved to be fit for the purpose of providing future certified reference materials, which surpass current analyte matrix combinations and exceed the typical scope of the regulatory framework.

Fusarium metabolites in maize from regions of Northern Serbia in 2016-2017.

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 of two years (2016-2017). A total of 458 maize samples were analysed by liquid chromatography-tandem mass spectrometry. A total of 40 metabolites were detected, where 94% of the samples contained at least 5 metabolites. Fumonisins (including B1, B2, B3 and B4), moniliformin and bikaverin were the most frequent (80-98%) Fusarium metabolites in both years. Furthermore, in samples from 2016, fumonisin A1 and A2, deoxynivalenol, deoxynivalenol-3-glucoside, zearalenone, culmorin, 15-hydroxyculmorin, fusapyron, fusaproliferin and aurofusarin were detected with frequencies of 58-80%. Levels of certain Fusarium metabolites in 2016 were higher on average due to increased humidity when compared to 2017, which was characterised by warm and dry conditions.

Challenges and future directions in LC-MS-based multiclass method development for the quantification of food contaminants.

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.

The MyToolbox EU-China Partnership-Progress and Future Directions in Mycotoxin Research and Management.

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.

Realizing the simultaneous liquid chromatography-tandem mass spectrometry based quantification of >1200 biotoxins, pesticides and veterinary drugs in complex feed.

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.

Evaluation of Matrix Effects and Extraction Efficiencies of LC-MS/MS Methods as the Essential Part for Proper Validation of Multiclass Contaminants in Complex Feed.

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.

Mycotoxins in maize harvested in Serbia in the period 2012-2015. Part 2: Non-regulated mycotoxins and other fungal metabolites.

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.

Mycotoxins in maize harvested in Republic of Serbia in the period 2012-2015. Part 1: Regulated mycotoxins and its derivatives.

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.

Simple validated method for simultaneous determination of deoxynivalenol, nivalenol, and their 3-ß-D-glucosides in baby formula and Korean rice wine via HPLC-UV with immunoaffinity cleanup.

A simple and reliable method for the simultaneous determination of major type B trichothecene mycotoxins, deoxynivalenol (DON) and nivalenol (NIV), along with their 3-ß-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.

UDP-Glucosyltransferases from Rice, Brachypodium, and Barley: Substrate Specificities and Synthesis of Type A and B Trichothecene-3-O-ß-d-glucosides.

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 ß-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.

Advanced LC-MS-based methods to study the co-occurrence and metabolization of multiple mycotoxins in cereals and cereal-based food.

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.

Less-toxic rearrangement products of NX-toxins are formed during storage and food processing.

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.

Determinants and Expansion of Specificity in a Trichothecene UDP-Glucosyltransferase from Oryza sativa.

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 barley UDP-glucosyltransferase inactivates nivalenol and provides Fusarium Head Blight resistance in transgenic wheat.

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-ß-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.

Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/ß-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077.

Zearalenone (ZEN) is an estrogenic mycotoxin occurring in Fusarium-infected cereals. Glucosylation is an important plant defense mechanism and generally reduces the acute toxicity of mycotoxins to humans and animals. Toxicological information about ZEN-glucosides is limited due to the unavailability of larger amounts required for animal studies. HvUGT14077, a recently-validated ZEN-conjugating barley UDP-glucosyltransferase was expressed in Escherichia coli, affinity purified, and characterized. HvUGT14077 possesses high affinity (Km = 3 µM) and catalytic efficiency (kcat/Km = 190 s-1·mM-1) with ZEN. It also efficiently glucosylates the phase-I ZEN-metabolites α-zearalenol and ß-zearalenol, with kcat/Km of 40 and 74 s-1·mM-1, respectively. HvUGT14077 catalyzes O-glucosylation at C-14 and C-16 with preference of 14-glucoside synthesis. Furthermore, relatively slow consecutive formation of 14,16-di-glucosides was observed; their structures were tentatively identified by mass spectrometry and for ZEN-14,16-di-glucoside confirmed by nuclear magnetic resonance spectroscopy. Recombinant HvUGT14077 allowed efficient preparative synthesis of ZEN-glucosides, yielding about 90% ZEN-14-glucoside and 10% ZEN-16-glucoside. The yield of ZEN-16-glucoside could be increased to 85% by co-incubation with a ß-glucosidase highly selective for ZEN-14-glucoside. Depletion of the co-substrate UDP-glucose was counteracted by a sucrose synthase based regeneration system. This strategy could also be of interest to increase the yield of minor glucosides synthesized by other glucosyltransferases.