Occurrence of modified mycotoxins in Latin America: an up-to-date review.

The Latin America region has a considerable extent of varied climate conditions: from tropical, subtropical, and warm temperate to temperate. Among the surface territory, different agricultural products are produced, making them an important food source for human consumption. Fungal species commonly colonize those important agricultural products and often contaminate them with mycotoxins that have a major impact on health, welfare, and productivity. Nowadays, special attention is paid to modified mycotoxins, which are those that cannot be detected by conventional analytical methods. However, little data about their natural occurrence in food and feed is available, especially in Latin American countries, where, among all the countries in this region, only a few of them are working on this subject. Thus, the present review summarizes the published information available in order to determine the possible human exposure risk to these toxins.

Screening and dietary exposure assessment of T-2 toxin and its modified forms in commercial cereals and cereal-based products in Shanghai.

A reliable and sensitive UPLC-MS/MS method coupled with HLB-SPE was developed for simultaneous determination of T-2 and its modified forms (HT-2, NEO, T-2-triol, T-2-tetraol, T-2-3G, and HT-2-3G) in cereals and cereal-based products. Acceptable linearity (R2 ≥ 0.99), limits of quantitation (0.5-10.0 µg/kg), intra-day precision (RSD < 12.8 %), inter-day precision (RSD ≤ 15.8 %), and recovery (76.8 %-115.2 %) were obtained for all analytes in all matrices investigated. 107 commercial foodstuffs were analyzed, and T-2 was detected in 29.0 % of maize and maize flour samples (0.51 to 56.61 µg/kg) and in 10-33.3 % of wheat flour and barley samples (1.27 to 78.51 µg/kg). Moreover, 66.7 % of the positive samples were simultaneously contaminated with two or more T-2 forms. The possible health risk related to T-2 and its modified forms in cereals and cereal-based products was evaluated using a probabilistic dietary exposure assessment. The 95th percentile dietary exposure values of the sum of T-2 forms ranged from 0.16 to 1.70 ng/kg b.w./day for lower bound (LB), and 0.17 to 7.59 ng/kg b.w./day for upper bound (UB). Results strongly suggested that the presence of T-2 and its modified forms in cereals and cereal-based products warrants greater attention and investigation, although probabilistic dietary exposure values currently remain below the tolerable daily intake (TDI) value of 20 ng/kg b.w./day.

Metabolite Analysis of Alternaria Mycotoxins by LC-MS/MS and Multiple Tools.

Alternaria fungi are widely distributed plant pathogens that invade crop products, causing significant economic damage. In addition, toxic secondary metabolites produced by the fungi can also endanger consumers. Many of these secondary metabolites are chemically characterized as mycotoxins. In this study, Q Exactive Orbitrap mass spectrometry was used for the non-targeted analysis of the metabolome of seven Alternaria isolates cultured on Potato Carrot Agar (PCA), Potato Dextrose Agar (PDA) and Potato Sucrose Agar (PSA) medium. Due to the difficulty of detecting modified toxins, an analytical strategy with multiple visual analysis tools was also used to determine the presence of sulfate conjugated toxins, as well as to visualize the molecular network of Alternaria toxins. The results show that PSA medium exhibits more advantageous properties for the culture of Alternaria, with more toxigenic species and quantities and more obvious metabolic pathways. Based on high-resolution tandem mass spectrometry (MS/MS) data, the mycotoxins and their metabolites were mainly clustered into four groups: alternariol (AOH)/alternariol monomethyl ether (AME)/altenusin (ALU)/altenuene (ALT)/dehydroaltenusin (DHA)/Desmethyldehydroaltenusin (DMDA) families, Altertoxin-I (ATX-I) family, tentoxin (TEN) family and tenuazonic acid (TeA) family. Moreover, the PSA medium is more suitable for the accumulation of AOH, AME, ALU, ALT, DHA and DMDA, while the PDA medium is more suitable for the accumulation of ATX-I, TEN and TeA. This research may provide theoretical support for the metabolomics study of Alternaria.

Temperature-dependent mycotoxins production investigation in Alternaria infected cherry by ultra-high performance liquid chromatography and Orbitrap high resolution mass spectrometry.

For temperature-dependent Alternaria mycotoxins production analysis, cherry samples were inoculated with Alternaria sp. and incubated at two different temperatures (4 °C and 25 °C). Six Alternaria mycotoxins, including altenuene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), altertoxin-I (ATX-I), tenuazonic acid (TeA), and tentoxin (TEN), in cherries were detected with integrated visible data-processing tools. Maximum concentration of these mycotoxins reached 71,862.2 µg/kg at 25 °C. Notably, considerable amount of TeA (290.4 µg/kg) was detected at 4 °C, which indicated that low temperature is not a safe storage condition for fruits. A total of 102 compounds were detected with a neutral loss of 162.0528 Da, and TeA-glucose was identified in this work. Based on MS/MS cosine similarity, products were verified and annotated with feature based molecular networking (FBMN) in global natural products social networking (GNPS). The results showed Alternaria mycotoxins in cherry samples were mainly demethylation, hydrogenation, and dehydration. This work revealed the production of Alternaria mycotoxins in cherries under different storage temperature, which will provide theoretical basis for the control of mycotoxin contamination in food commodities.

Recent Research on Fusarium Mycotoxins in Maize-A Review.

Maize (Zea mays L.) is one of the most susceptible crops to pathogenic fungal infections, and in particular to the Fusarium species. Secondary metabolites of Fusarium spp.-mycotoxins are not only phytotoxic, but also harmful to humans and animals. They can cause acute or chronic diseases with various toxic effects. The European Union member states apply standards and legal regulations on the permissible levels of mycotoxins in food and feed. This review summarises the most recent knowledge on the occurrence of toxic secondary metabolites of Fusarium in maize, taking into account modified forms of mycotoxins, the progress in research related to the health effects of consuming food or feed contaminated with mycotoxins, and also the development of biological methods for limiting and/or eliminating the presence of the same in the food chain and in compound feed.

In Vitro Effects of Lemon Balm Extracts in Reducing the Growth and Mycotoxins Biosynthesis of Fusarium culmorum and F. proliferatum.

The objectives of this research were to obtain the extracts of lemon balm (Melissa officinalis) using supercritical CO2 (SC-CO2) and methanol as co-solvent and evaluate the antifungal activity of those extracts against two selected strains of Fusarium species (Fusarium culmorum and Fusarium proliferatum). The extraction conditions were set at 40 and 60 °C and 250 bar. The obtained extracts were characterized in terms of antifungal activity on potato dextrose agar media (PDA). The results showed that the extraction parameters had different effects on mycelium growth and mycotoxins biosynthesis reduction. All studied lemon balm extracts (1, 2.5, 5, 7.5, and 10%) inhibited the growth of F. proliferatum and F. culmorum mycelia compared to the control. The lemon balm extracts significantly reduced ergosterol content and synthesized mycotoxins in both tested strains. These findings support the antifungal activity of lemon balm extracts against F. proliferatum and F. culmorum. However, more research on other Fusarium species is needed, as well as in vivo applications, before considering lemon balm extracts as a natural alternative to synthetic fungicides.

Dynamics of Deoxynivalenol and Nivalenol Glucosylation in Wheat Cultivars Infected with Fusarium culmorum in Field Conditions─A 3 Year Study (2018-2020).

Fusarium head blight (FHB) caused by pathogenic species of Fusarium fungi is one of the most important diseases of cereal plants and a factor contributing to losses in plant production. The growth of FHB-associated species is often accompanied by biosynthesis of secondary metabolites─mycotoxins, which serve as a virulence factor. The aim of the study was to evaluate the ratios between deoxynivalenol (DON) and nivalenol (NIV) and their derivatives in the ears of six cultivars of winter wheat with varying resistance to FHB, taking into account a range of factors (weather conditions, location, cultivar, and year) after inoculation with Fusarium culmorum, during a 3 year field experiment, 2018-2020. The presence of toxins in the ears was measured within 21 days of inoculation. The toxins were found in the ears as soon as on the third day from the start of the experiment, whereas relative humidity higher than 80% was a decisive factor for FHB incidence. All wheat cultivars showed the ability to biotransform DON and NIV present in the ears to glucosides, that is, deoxynivalenol-3-glucoside (DON-3G) and nivalenol-3-glucoside (NIV-3G). The levels of these metabolites showed significant correlation with the levels of their basic analogues. In most cases, higher levels of DON and NIV in wheat ears and higher levels of their metabolites were observed, but the relative levels of DON-3G/DON and NIV-3G/NIV at relatively high levels of toxins were lower compared to the ear samples with relatively low toxin levels. The presented results are the first studies, which systematically correlate a variety of wheat cultivars with their extent to glucosylate trichothecenes.

Transformation of Selected Fusarium Toxins and Their Masked Forms during Malting of Various Cultivars of Wheat.

This study investigated the impact of malting of six wheat cultivars inoculated with Fusarium culmorum on the dynamics of content changes of selected Fusarium toxins. The grains of all the tested cultivars showed a high content of deoxynivalenol (DON), zearalenone (ZEN), and their derivatives, whereas nivalenol (NIV) and its glucoside were found only in the Legenda cultivar. Our experiments confirmed that the malting process of wheat grain enables the secondary growth of Fusarium, and mycotoxin biosynthesis. The levels of toxins in malt were few-fold higher than those in grain; an especially high increase was noted in the case of ZEN and its sulfate as the optimal temperature and pH conditions for the biosynthesis of these toxins by the pathogen are similar to those used in the grain malting process. This is the first paper reporting that during the malting process, biosynthesis of ZEN sulfate occurs, instead of glycosylation, which is a typical modification of mycotoxins by plant detoxication enzymes.

Updated Review of the Toxicity of Selected Fusarium Toxins and Their Modified Forms.

Mycotoxins are one of the most dangerous food and feed contaminants, hence they have significant influence on human and animal health. This study reviews the information reported over the last few years on the toxic effects of the most relevant and studied Fusarium toxins and their modified forms. Deoxynivalenol (DON) and its metabolites can induce intracellular oxidative stress, resulting in DNA damage. Recent studies have also revealed the capability of DON and its metabolites to disturb the cell cycle and alter amino acid expression. Several studies have attempted to explore the mechanism of action of T-2 and HT-2 toxins in anorexia induction. Among other findings, two neurotransmitters associated with this process have been identified, namely substance P and serotonin (5-hydroxytryptamine). For zearalenone (ZEN) and its metabolites, the literature points out that, in addition to their generally acknowledged estrogenic and oxidative potentials, they can also modify DNA by altering methylation patterns and histone acetylation. The ability of the compounds to induce alterations in the expression of major metabolic genes suggests that these compounds can contribute to the development of numerous metabolic diseases, including type 2 diabetes.

Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modified Forms in Maize.

The Fusarium mycotoxin deoxynivalenol (DON) is a common contaminant of cereals and is often co-occurring with its modified forms DON-3-glucoside (D3G), 3-acetyl-DON (3ADON) or 15-acetyl-DON (15ADON). A stable-isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method for their determination in cereals was developed and validated for maize. Therefore, 13C-labelled D3G was enzymatically produced using 13C-DON and [13C6Glc]-sucrose and used as an internal standard (IS) for D3G, while uniformly 13C labelled IS was used for the other mycotoxins. Baseline separation was achieved for the critical peak pair DON/D3G, while 3ADON/15ADON could not be fully baseline separated after testing various reversed phase, fluorinated phase and chiral LC columns. After grinding, weighing and extracting the cereal samples, the raw extract was centrifuged and a mixture of the four 13C-labelled ISs was added directly in a microinsert vial. The subsequent analytical run took 7 min, followed by negative electrospray ionization and selected reaction monitoring on a triple quadrupole MS. Maize was used as a complex cereal model matrix for validation. The use of the IS corrected the occurring matrix effects efficiently from 76 to 98% for D3G, from 86 to 103% for DON, from 68 to 100% for 15ADON and from 63 to 96% for 3ADON.

Integrated Mycotoxin Management System in the Feed Supply Chain: Innovative Approaches.

Exposure to mycotoxins is a worldwide concern as their occurrence is unavoidable and varies among geographical regions. Mycotoxins can affect the performance and quality of livestock production and act as carriers putting human health at risk. Feed can be contaminated by various fungal species, and mycotoxins co-occurrence, and modified and emerging mycotoxins are at the centre of modern mycotoxin research. Preventing mould and mycotoxin contamination is almost impossible; it is necessary for producers to implement a comprehensive mycotoxin management program to moderate these risks along the animal feed supply chain in an HACCP perspective. The objective of this paper is to suggest an innovative integrated system for handling mycotoxins in the feed chain, with an emphasis on novel strategies for mycotoxin control. Specific and selected technologies, such as nanotechnologies, and management protocols are reported as promising and sustainable options for implementing mycotoxins control, prevention, and management. Further research should be concentrated on methods to determine multi-contaminated samples, and emerging and modified mycotoxins.

Controlled Production of Zearalenone-Glucopyranoside Standards with Cunninghamella Strains Using Sulphate-Depleted Media.

In recent years, conjugated mycotoxins have gained increasing interest in food safety, as their hydrolysis in human and animal intestines leads to an increase in toxicity. For the production of zearalenone (ZEN) glycosides reference standards, we applied Cunninghamellaelegans and Cunninghamella echinulata fungal strains. A sulphate-depleted medium was designed for the preferred production of ZEN glycosides. Both Cunninghamella strains were able to produce zearalenone-14-ß-D-glucopyranoside (Z14G), zearalenone-16-ß-D-glucopyranoside (Z16G) and zearalenone-14-sulphate (Z14S). In a rich medium, Cunninghamellaelegans preferably produced Z14S, while Cunninghamellaechinulata preferably produced Z14G. In the sulphate-depleted medium a dramatic change was observed for Cunninghamellaelegans, showing preferred production of Z14G and Z16G. From 2 mg of ZEN in sulphate-depleted medium, 1.94 mg of Z14G and 0.45 mg of Z16G were produced. Following preparative Liquid Chromatography-Mass Spectrometry (LC-MS) purification, both fractions were submitted to 1H and 13C NMR and High-Resolution Mass Spectrometry (HRMS). These analyses confirmed that the purified fractions were indeed Z14G and Z16G. In conclusion, the presented research shows that a single Cunninghamella strain can be an effective and efficient tool for the controlled biotransformation of ZEN glycosides and other ZEN metabolites. Additionally, the biotransformation method was extended to zearalanone, ß-zearalenol and other mycotoxins.

Transformation of Selected Trichothecenes during the Wheat Malting Production.

The transformation of deoxynivalenol (DON), nivalenol (NIV), and their glucosides (DON3G and NIV3G) during the malting of grains of two wheat varieties was studied. The concentration of DON3G and NIV3G started to increase significantly before the concentration of DON and NIV increased. This may reflect the transformation of the parent mycotoxin forms into their glucosides due to xenobiotic detoxification reactions. After a sharp rise during the last 2 days of the process (day 6 and 7), the DON concentration reached 3010 ± 338 µg/kg in the Legenda wheat-based malt and 4678 ± 963 µg/kg in the Pokusa wheat-based malt. The NIV concentration, at 691 ± 65 µg/kg, remained the same as that in the dry grain. The concentration of DON3G in the Legenda and Pokusa wheat-based malt was five and three times higher, respectively, than that in the steeped grain. The concentration of NIV3G in the Legenda wheat-based malt was more than twice as high as that in the steeped grain. The sharp increase in the concentration of DON at the end of the malting process reflected the high pathogen activity. We set aside some samples to study a batch that was left undisturbed without turning and aeration, for the entire period of malting. The concentration of DON in the malt produced from the latter batch was 135% and 337% higher, for Legenda and Pokusa, respectively, than that in the malt produced from the batch that was turned and aerated. The NIV concentration was 22% higher in the latter batch.

Development, validation, and application of a multi-method for the determination of mycotoxins, plant growth regulators, tropane alkaloids, and pesticides in cereals by two-dimensional liquid chromatography tandem mass spectrometry.

Mycotoxins and pesticides regularly co-occur in agricultural products worldwide. Thus, humans can be exposed to both toxic contaminants and pesticides simultaneously, and multi-methods assessing the occurrence of various food contaminants and residues in a single method are necessary. A two-dimensional high performance liquid chromatography tandem mass spectrometry method for the analysis of 40 (modified) mycotoxins, two plant growth regulators, two tropane alkaloids, and 334 pesticides in cereals was developed. After an acetonitrile/water/formic acid (79:20:1, v/v/v) multi-analyte extraction procedure, extracts were injected into the two-dimensional setup, and an online clean-up was performed. The method was validated according to Commission Decision (EC) no. 657/2002 and document N° SANTE/12682/2019. Good linearity (R2 > 0.96), recovery data between 70-120%, repeatability and reproducibility values < 20%, and expanded measurement uncertainties < 50% were obtained for a wide range of analytes, including very polar substances like deoxynivalenol-3-glucoside and methamidophos. However, results for fumonisins, zearalenone-14,16-disulfate, acid-labile pesticides, and carbamates were unsatisfying. Limits of quantification meeting maximum (residue) limits were achieved for most analytes. Matrix effects varied highly (-85 to +1574%) and were mainly observed for analytes eluting in the first dimension and early-eluting analytes in the second dimension. The application of the method demonstrated the co-occurrence of different types of cereals with 28 toxins and pesticides. Overall, 86% of the samples showed positive findings with at least one mycotoxin, plant growth regulator, or pesticide.

Occurrence of Fusarium Mycotoxins and Their Modified Forms in Forage Maize Cultivars.

Forage maize is often infected by mycotoxin-producing Fusarium fungi during plant growth, which represent a serious health risk to exposed animals. Deoxynivalenol (DON) and zearalenone (ZEN) are among the most important Fusarium mycotoxins, but little is known about the occurrence of their modified forms in forage maize. To assess the mycotoxin contamination in Northern Germany, 120 natural contaminated forage maize samples of four cultivars from several locations were analysed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) for DON and ZEN and their modified forms deoxynivalenol-3-glucoside (DON3G), the sum of 3- and 15-acetyl-deoxynivalenol (3+15-AcDON), α- and ß-zearalenol (α-ZEL, ß-ZEL). DON and ZEN occurred with high incidences (100 and 96%) and a wide range of concentrations, reaching levels up to 10,972 and 3910 µg/kg, respectively. Almost half of the samples (46%) exceeded the guidance value in complementary and complete feeding stuffs for ZEN (500 µg/kg), and 9% for DON (5000 µg/kg). The DON related mycotoxins DON3G and 3+15-AcDON were also present in almost all samples (100 and 97%) with amounts of up to 3038 and 2237 µg/kg and a wide range of concentrations. For the ZEN metabolites α- and ß-ZEL lower incidences were detected (59 and 32%) with concentrations of up to 423 and 203 µg/kg, respectively. Forage maize samples were contaminated with at least three co-occurring mycotoxins, whereby 95% of all samples contained four or more mycotoxins with DON, DON3G, 3+15-AcDON, and ZEN co-occurring in 93%, together with α-ZEL in 57% of all samples. Positive correlations were established between concentrations of the co-occurring mycotoxins, especially between DON and its modified forms. Averaged over all samples, ratios of DON3G/DON and 3+15-AcDON/DON were similar, 20.2 and 20.5 mol%; cultivar-specific mean ratios ranged from 14.6 to 24.3 mol% and 15.8 to 24.0 mol%, respectively. In total, 40.7 mol% of the measured DON concentration was present in the modified forms DON3G and 3+15-AcDON. The α-ZEL/ZEN ratio was 6.2 mol%, ranging from 5.2 to 8.6 mol% between cultivars. These results demonstrate that modified mycotoxins contribute substantially to the overall mycotoxin contamination in forage maize. To avoid a considerable underestimation, it is necessary to analyse modified mycotoxins in future mycotoxin monitoring programs together with their parent forms.

The fate of mycotoxins during secondary food processing of maize for human consumption.

Mycotoxins are naturally occurring fungal metabolites that are associated with health hazards and are widespread in cereals including maize. The most common mycotoxins in maize that occur at relatively high levels are fumonisins (FBs), zearalenone, and aflatoxins; furthermore, other mycotoxins such as deoxynivalenol and ochratoxin A are frequently present in maize. For these toxins, maximum levels are laid down in the European Union (EU) for maize raw materials and maize-based foods. The current review article gives a comprehensive overview on the different mycotoxins (including mycotoxins not regulated by EU law) and their fate during secondary processing of maize, based on the data published in the scientific literature. Furthermore, potential compliance with the EU maximum levels is discussed where appropriate. In general, secondary processing can impact mycotoxins in various ways. Besides changes in mycotoxin levels due to fractionation, dilution, and/or concentration, mycotoxins can be affected in their chemical structure (causing degradation or modification) or be released from or bound to matrix components. In the current review, a special focus is set on the effect on mycotoxins caused by different heat treatments, namely, baking, roasting, frying, (pressure) cooking, and extrusion cooking. Production processes involving multiple heat treatments are exemplified with the cornflakes production. For that, potential compliance with FB maximum levels was assessed. Moreover, effects of fermentation of maize matrices and production of maize germ oil are covered by this review.

Development and validation of a liquid chromatography tandem mass spectrometry multi-method for the determination of 41 free and modified mycotoxins in beer.

A fast high performance liquid chromatography tandem mass spectrometry multi-method based on an ACN-precipitation extraction was developed for the analysis of 41 (modified) mycotoxins in beer. Validation according to the performance criteria defined by the European Commission (EC) in Commission Decision no. 657/2002 revealed good linearity (R2 > 0.99), repeatability (RSDr < 15%), reproducibility (RSDR < 15%), and recovery (79-100%). Limits of quantification ranging from 0.04 to 75 µg/L were obtained. Matrix effects varied from -67 to +319% and were compensated for using standard addition. In total, 87 beer samples, produced worldwide, were analyzed for the presence of mycotoxins with a focus on modified mycotoxins, whereof 76% of the samples were contaminated with at least one mycotoxin. The most prevalent mycotoxins were deoxynivalenol-3-glucoside (63%), HT-2 toxin (15%), and tenuazonic acid (13%). Exposure estimates of deoxynivalenol and its metabolites for German beer revealed no significant contribution to intake of deoxynivalenol.

Transformations of Selected Fusarium Toxins and Their Modified Forms During Malt Loaf Production.

An increasing number of studies have found that modified mycotoxins, such as free mycotoxins, naturally occur in food, and severely impact food safety. The present study investigated concentrations of trichothecenes nivalenol (NIV), deoxynivalenol (DON), and zearalenone (ZEN), together with their modified forms, nivalenol-3-glucoside (NIV-3G), deoxynivalenol-3-glucoside (DON-3G), and zearalenone-14-glucoside (ZEN-14G) and zearalenone-14-sulfate (ZEN-14S), respectively, at successive stages of malt loaf production (flour, dough kneading/fermentation, loaf baking). Toxins in bakery products originate in flour produced from wheat grain that is naturally contaminated with Fusarium culmorum. Mycotoxin concentrations were determined using high-performance liquid chromatography-high resolution mass spectrometry, and did not significantly change during the successive stages of bread production. After the dough kneading/fermentation stage, concentrations of NIV-3G and DON-3G were slightly increased, whereas those of ZEN and ZEN-14S were slightly decreased. The largest average decrease (21%) was found in ZEN-14G. After the baking stage, the average concentrations of NIV-3G, DON-3G, ZEN-14S, and ZEN-14G in the loaf crumb and crust decreased by 23%, 28%, 27%, and 20%, respectively, compared with those in the dough. During this technical process, the concentration of ZEN-14G in loaf crumb significantly decreased by an average of 48%, and those of ZEN, ZEN-14S, and ZEN-14G in loaf crust decreased by an average of 29%, 42%, and 48%, respectively. Considering the possibility of modified mycotoxins degradation to free forms, as well as the ability to synthesize them from free forms during technological processes, it would be prudent to consider them together during analysis.

Simultaneous Determination of Deoxynivalenol, Its Modified Forms, Nivalenol and Fusarenone-X in Feedstuffs by the Liquid Hromatography-Tandem Mass Spectrometry Method.

A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3Ac-DON), 15-acetyldeoxynivalenol (15Ac-DON), DON-3-glucoside (DON-3Glc) nivalenol and fusarenone-X in feedstuffs. Different techniques of sample preparation were tested: solid-liquid-extraction, QuEChERS, solid phase extraction with OASIS HLB columns or immunoaffinity columns and a Mycosep 225 Trich column. None of the six immunoaffinity columns tested showed cross-reactivity to all of the mycotoxins. Surprisingly, the results show that if the immunoaffinity columns bound 3Ac-DON, then they did not bind 15Ac-DON. The most efficient sample preparation was achieved with a Mycosep 225 Trich column clean-up. The chromatography was optimised to obtain full separation of all analytes (including 3Ac-DON and 15Ac-DON isomeric form). The validation results show the relative standard deviations for repeatability and reproducibility varied from 4% to 24%. The apparent recovery ranged between 92% and 97%, and the limit of quantification described a 1.30 to 50 µg/kg range. The method trueness was satisfactory, as assessed by a proficiency test and analysis of reference material. A total of 99 feed samples were analysed by the developed method, revealing the presence of DON and DON-3Glc in 85% and 86% of examined animal feeds, respectively at concentrations between 1.70 and 1709 µg/kg. The ratios DON-3Glc to DON in the surveyed feedstuffs were from a low of 3% to high of 59%.

Enzymatic Synthesis of Modified Alternaria Mycotoxins Using a Whole-Cell Biotransformation System.

Reference standards for Alternaria mycotoxins are rarely available, especially the modified mycotoxins alternariol-3-glucoside (AOH-3-G), alternariol-9-glucoside (AOH-9-G), and alternariol monomethylether-3-glucoside (AME-3-G). To obtain these three glucosides as analytical standards for method development and method validation, alternariol and alternariol monomethylether were enzymatically glycosylated in a whole-cell biotransformation system using a glycosyltransferase from strawberry (Fragaria x ananassa), namely UGT71A44, expressed in Escherichia coli (E. coli). The formed glucosides were isolated, purified, and structurally characterized. The exact amount of the isolated compounds was determined using high-performance liquid chromatography with UV-detection (HPLC-UV) and quantitative nuclear resonance spectroscopy (qNMR). This method has proved to be highly effective with biotransformation rates of 58% for AOH-3-G, 5% for AOH-9-G, and 24% for AME-3-G.