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A QuEChERS (quick, easy, cheap, effective, rugged, and safe)-based multi-mycotoxin method was developed, analyzing 24 (17 free and 7 modified) Alternaria and Fusarium toxins in cereals via ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A modified QuEChERS approach was optimized for sample preparation. Quantification was conducted using a combination of stable isotope dilution analysis (SIDA) for nine toxins and matrix-matched calibration for ten toxins. Quantification via a structurally similar internal standard was conducted for four analytes. Alternariol-9-sulfate (AOH-9-S) was measured qualitatively. Limits of detection (LODs) were between 0.004 µg/kg for enniatin A1 (ENN A1) and 3.16 µg/kg for nivalenol (NIV), while the limits of quantification were between 0.013 and 11.8 µg/kg, respectively. The method was successfully applied to analyze 136 cereals and cereal-based foods, including 28 cereal-based infant food products. The analyzed samples were frequently contaminated with Alternaria toxins, proving their ubiquitous occurrence. Interestingly, in many of those samples, some modified Alternaria toxins occurred, mainly alternariol-3-sulfate (AOH-3-S) and alternariol monomethyl ether-3-sulfate (AME-3-S), thus highlighting the importance of including modified mycotoxins in the routine analysis as they may significantly add to the total exposure of their parent toxins. Over 95% of the analyzed samples were contaminated with at least one toxin. Despite the general contamination, no maximum or indicative levels were exceeded., (© 2024. The Author(s).)

Fungi of the genus Alternaria are ubiquitous in the environment. Their mycotoxins can leach out of contaminated plants or crop debris into the soil entering the plant via the roots. We aim to evaluate the importance of this entry pathway and its contribution to the overall content of Alternaria toxins (ATs) in wheat plants to better understand the soil-plant-phytopathogen system. A hydroponic cultivation system was established and wheat plants were cultivated for up to two weeks under optimal climate conditions. One half of the plants was treated with a nutrient solution spiked with alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA), whereas the other half of the plants was cultivated without mycotoxins. Plants were harvested after 1 and 2 weeks and analyzed using a QuEChERS-based extraction and an in-house validated LC-MS/MS method for quantification of the ATs in roots, crowns, and leaves separately. ATs were taken up by the roots and transported throughout the plant up to the leaves after 1 as well as 2 weeks of cultivation with the roots showing the highest ATs levels followed by the crowns and the leaves. In addition, numerous AOH and AME conjugates like glucosides, malonyl glucosides, sulfates, and di/trihexosides were detected in different plant compartments and identified by high-resolution mass spectrometry. This is the first study demonstrating the uptake of ATs in vivo using a hydroponic system and whole wheat plants examining both the distribution of ATs within the plant compartments and the modification of ATs by the wheat plants., (© 2023. The Author(s).)

After ingestion of food commodities, the gastrointestinal tract (GIT) poses the first barrier against xenobiotics and pathogens. Therefore, it is regularly confronted with external stressors potentially affecting the inflammatory response and the epithelial barrier. Alternaria mycotoxins such as alternariol (AOH) and altertoxin II (ATX-II) are frequently occurring food and feed contaminants that are described for their immunomodulatory capacities. Hence, this study aimed at exploring the effect of AOH and ATX-II as single compounds or binary mixtures on the immune response and epithelial homeostasis in noncancerous colon epithelial cells HCEC-1CT. Both toxins suppressed mRNA levels of proinflammatory mediators interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and secretion of IL-8, as well as mRNA levels of the matrix metallopeptidase 2 (MMP-2). Binary combinations of AOH and ATX-II reduced the response of the single toxins. Additionally, AOH and ATX-II modified immunolocalization of transmembrane proteins such as integrin β1, zona occludens 1 (ZO-1), claudin 4 (Cldn 4), and occludin (Ocln), which support colonic tissue homeostasis and intestinal barrier function. Moreover, the cellular distribution of ZO-1 was affected by ATX-II. Mechanistically, these effects could be traced back to the involvement of several transcription factors. AOH activated the nuclear translocation of the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid 2-related factor 2 (Nrf2), governing cell metabolic competence and structural integrity. This was accompanied by altered distribution of the NF-κB p65 protein, an important regulator of inflammatory response. ATX-II also induced AhR and Nrf2 translocation, albeit failing to substantiate the effect of AOH on the colonic epithelium. Hence, both toxins coherently repress the intestinal immune response on the cytokine transcriptional and protein levels. Furthermore, both mycotoxins affected the colonic epithelial integrity by altering the cell architecture.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) multi-mycotoxin method was developed for the analysis of the Alternaria toxins alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), altertoxin I (ATX I), altertoxin II (ATX II), alterperylenol (ALTP), and altenuene (ALT), as well as the modified toxins AOH-3-glucoside (AOH-3-G), AOH-9-glucoside (AOH-9-G), AME-3-glucoside (AME-3-G), AOH-3-sulfate (AOH-3-S), and AME-3-sulfate (AME-3-S) in barley and malt. The toxin tenuazonic acid (TeA) was analyzed separately as it could not be included into the multi-mycotoxin method. Quantitation was conducted by using a combination of stable isotope dilution analysis (SIDA) for AOH, AME, and TeA, and matrix-matched calibration for all other toxins. Limits of detection were between 0.05 µg/kg (AME) and 2.45 µg/kg (ALT), whereas limits of quantitation ranged from 0.16 µg/kg (AME) to 8.75 µg/kg (ALT). Recoveries between 96 and 107% were obtained for the analytes when SIDA was applied, while recoveries between 84 and 112% were found for analytes quantified by matrix-matched calibration. The method was applied for the analysis of 50 barley samples and their respective malts from the harvest years 2016-2020 for their mycotoxin content, showing the overall potential of toxin formation during the malting process. The toxins ALTP and ATX I were mainly found in the malt samples, but not in barley., (© 2022. The Author(s).)

Epidemiology of Fusarium Head Blight (FHB) of spring barley is relatively little understood. In a five-year study, we assessed quantitative resistance to FHB in an assortment of 17 spring barley genotypes in the field in southern Germany. To this end, we used soil and spray inoculation of plants with F. culmorum and F. avenaceum . This increased disease pressure and provoked genotypic differentiation. To normalize effects of variable weather conditions across consecutive seasons, we used a disease ranking of the genotypes based on quantification of fungal DNA contents and multiple Fusarium toxins in harvested grain. Together, this allowed for assessment of stable quantitative FHB resistance of barley in several genotypes. Fungal DNA contents were positively associated with species-specific Fusarium toxins in single years and over several years in plots with soil inoculation. In those plots, plant height limited FHB; however, this was not observed after spray inoculation. A multiple linear regression model of recorded weather parameter and fungal DNA contents over five years identified time periods during the reproductive phase of barley, in which weather strongly influenced fungal colonization measured in mature barley grain. Environmental conditions before heading and late after anthesis showed strongest associations with F. culmorum DNA in all genotypes, whereas for F. avenaceum , this was less consistent where we observed weather-dependent associations, depending on the genotype. Based on this study, we discuss aspects of practical resistance breeding in barley relevant to improve quantitative resistance to FHB and associated mycotoxin contaminations.

Malting and brewing have previously been demonstrated to be risky procedures in terms of mycotoxins contamination. The goal of the study was to describe the fate of less investigated Fusarium and Alternaria mycotoxins, together with their conjugates, during these processes. The Pilsner malt producing process, together with double-mash brewing, were performed in a pilot-scale malting and brewery plants to simulate production of lager - the most popular type of central European beer. In addition, changes in temperature during barley germination were investigated to assess the influence of this critical step. QuEChERS-like extraction followed by UHPLC-HRMS/MS were utilized to quantify the mass balance of 13 mycotoxins and four of their conjugates. The results confirmed germination as the most determining malting step, followed by mashing of malt during brewing. Occurrence of type A trichothecenes, Alternaria mycotoxins and their conjugates in the final beer product indicates the need to take mitigation measures., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A multi-mycotoxin LC-MS/MS method was developed to quantify 13 free and modified Alternaria toxins in different beer types by applying a combination of stable-isotope dilution assays (SIDAs) and matrix-matched calibration. With limits of detection (LODs) between 0.03 µg/L (alternariol monomethyl ether, AME) and 5.48 µg/L (altenuene, ALT), limits of quantitation (LOQs) between 0.09 µg/L (AME) and 16.24 µg/L (ALT), and recoveries between 72 and 113%, we obtained a sensitive and reliable method, which also covers the emerging toxins alternariol-3-glucoside (AOH-3-G), alternariol-9-glucoside (AOH-9-G), alternariol monomethyl ether-3-glucoside (AME-3-G) and alternariol-3-sulfate (AOH-3-S) and alternariol monomethylether-3-sulfate (AME-3-S). Furthermore, 50 different beer samples were analyzed, showing no contamination with Alternaria toxins apart from tenuazonic acid (TeA) in concentrations between 0.69 µg/L and 16.5 µg/L. According to this study, the exposure towards TeA through beer consumption can be considered as relatively low, as the threshold of toxicological concern (TTC) value of 1500 ng/kg body weight per day might not be reached when consuming reasonable amounts of beer.

Alternaria molds simultaneously produce a large variety of mycotoxins, of which several were previously reported to induce enzymes of phase I metabolism through aryl hydrocarbon receptor activation. Thus, we investigated the potential of naturally occurring Alternaria toxin mixtures to induce Cytochrome P450 (CYP) 1A1/1A2/1B1 activity. Two variants of an extract from cultured Alternaria alternata, as well as the toxins alternariol (AOH), alternariol monomethyl ether (AME), altertoxin I (ATX-I), and altertoxin II (ATX-II), were tested singularly and in binary mixtures applying the 7-ethoxy-resorufin- O -deethylase (EROD) assay in MCF-7 breast cancer cells. Sub-cytotoxic concentrations of the two toxin mixtures, as well as ATX-I, ATX-II and AOH, exhibited dose-dependent enhancements of CYP 1 activity. ATX-I and ATX-II interacted synergistically in this respect, demonstrating the two perylene quinones as major contributors to the extract's potential. Binary mixtures between AOH and the two altertoxins respectively exhibited concentration-dependent antagonistic as well as synergistic combinatory effects. Notably, AME showed no efficacy towards EROD enzyme activity or impact on other toxins' efficacy. Hence, this study provides insights into synergistic and other combinatory effects of Alternaria toxins in natural co-occurrence scenarios in the context of AhR signalling pathway activation in breast cancer cells.

Ruminal microbiota of cattle are not able to detoxify all mycotoxins. In addition, detoxification can be hampered by adverse ruminal conditions (e.g., low ruminal pH). Hence, in the cattle husbandry, mycotoxin binders and modifiers could be used to prevent animal exposure to mycotoxins. In this study, an in vitro rumen model, including feed matrix, was established as first screening tool to test the efficacy of five products claiming to detoxify mycotoxins. The detoxifiers had different modes of action: (a) binding (three products); (b) enzymatic detoxification of zearalenone (ZEN; one product, ZenA); and (c) bacterial transformation of trichothecenes (one product, BBSH 797). For the mycotoxin binders, the binding to the mycotoxins enniatin B (ENN B), roquefortine C (ROQ-C), mycophenolic acid (MPA), deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) were tested at a dose recommended by the manufacturers. The in vitro model demonstrated that all binders adsorbed ENN B to a certain extent, while only one of the binders also partially adsorbed ROQ-C. The binders did not change the concentrations of the other mycotoxins in the ruminal fluid. The enzyme ZenA detoxified ZEN very quickly and prevented the formation of the more toxic metabolite α-zearalenol (α-ZEL), both at normal (6.8) and low ruminal pH (5.8). The addition of BBSH 797 enhanced detoxification of DON and NIV, both at normal and low ruminal pH. The in vitro rumen model demonstrated that the addition of ZenA seems to be a very promising strategy to prevent estrogenic effects of ZEN contaminated feed, and BBSH 797 is efficient in the detoxification of trichothecenes.

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.

Scope: Alternaria fungi are widely distributed plant pathogens infecting grains and vegetables and causing major harvest losses in the field and during postharvest storage. Besides, consumers are endangered by the formation of toxic secondary metabolites. Some of these secondary metabolites are chemically characterized as mycotoxins, but the majority of the Alternaria mycobolome still remains unknown., Methods and Results: Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) and LC-MS/MS are combined for the non-targeted and targeted analysis of the metabolome of three A. alternata isolates and one A. solani isolate. Due to the ultra-high resolution of FTICR-MS, unique molecular formulae are assigned to measured m/z signals. The molecular formulae are matched to entries of the databases Antibase and Kyoto Encyclopedia of Genes and Genomes. The non-targeted analysis of the fungal extracts reveals variations in the secondary metabolite profile of A. alternata and A. solani. Differences in the biosynthesis of dibenzo-α-pyrones, perylene quinones, tentoxin, and tenuazonic acid of the A. alternata and A. solani isolates are determined applying targeted LC-MS/MS., Conclusion: FTICR-MS analyses reveal clear differences in the metabolic profile of the A. solani and the A. alternata isolates., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ruminants are less susceptible to the effects of mycotoxins than monogastric animals as their rumen microbiota are claimed to degrade and/or deactivate at least some of these toxic compounds. However, the mycotoxin degradation is not well-known yet. For this, a sensitive, specific, and accurate analytical method is needed to determine mycotoxins in the rumen fluid. This study aims to develop and thoroughly validate an ultra-performance liquid chromatography tandem mass spectrometry method for the quantitative determination in the rumen fluid of some of the most relevant mycotoxins found in maize silage in Western Europe: deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), mycophenolic acid (MPA), roquefortine C (ROQ-C) and enniatin B (ENN B), as well as their metabolites deepoxy-deoxynivalenol (DOM-1), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL). As feed is often present in the rumen fluid samples, the potential interaction of feed particles with the mycotoxin extraction and analysis was investigated. Extraction recovery and matrix effects were determined in the rumen fluid with and without maize silage. Differences in those parameters between rumen fluid alone and rumen fluid with maize silage highlight the importance of using matrix-matched calibration curves for the quantification of mycotoxins in rumen fluid samples. A cross-validation of the method with rumen fluid and maize silage demonstrates that this analytical method can be applied in research on rumen fluid samples to investigate the degradation of the reported mycotoxins by rumen microbiota if matrix-matched calibration is performed.

A reliable and practical multi-method was developed for the quantification of mycotoxins in plasma, urine, and feces of pigs, and plasma and excreta of broiler chickens using liquid chromatography⁻tandem mass spectrometry. The targeted mycotoxins belong to the regulated groups, i.e., aflatoxins, ochratoxin A and Fusarium mycotoxins, and to two groups of emerging mycotoxins, i.e., Alternaria mycotoxins and enniatins. In addition, the developed method was transferred to a LC-high resolution mass spectrometry instrument to qualitatively determine phase I and II metabolites, for which analytical standards are not always commercially available. Sample preparation of plasma was simple and generic and was accomplished by precipitation of proteins alone (pig) or in combination with removal of phospholipids (chicken). A more intensive sample clean-up of the other matrices was needed and consisted of a pH-dependent liquid⁻liquid extraction (LLE) using ethyl acetate (pig urine), methanol/ethyl acetate/formic acid (75/24/1, v / v / v ) (pig feces) or acetonitrile (chicken excreta). For the extraction of pig feces, additionally a combination of LLE using acetone and filtration of the supernatant on a HybridSPE-phospholipid cartridge was applied. The LC-MS/MS method was in-house validated according to guidelines defined by the European and international community. Finally, the multi-methods were successfully applied in a specific toxicokinetic study and a screening study to monitor the exposure of individual animals.

A comprehensive definition introducing the term "modified mycotoxins" to encompass all possible forms in which mycotoxins and their modifications can occur was recently proposed and has rapidly gained wide acceptance within the scientific community. It is becoming increasingly evident that exposure to such modified mycotoxins due to their presence in food and feed has the potential to pose a substantial additional risk to human and animal health. Zearalenone (ZEN) is a well-characterized Fusarium toxin. Considering the diversity of modified forms of ZEN occurring in food and feed, the toxicologically relevant endocrine activity of many of these metabolites, and the fact that modified forms add to a dietary exposure which approaches the tolerable daily intake by free ZEN alone, modified forms of ZEN present an ideal case study for critical evaluation of modified mycotoxins in food safety. Following a summary of recent scientific opinions of EFSA dealing with health risk assessment of ZEN alone or in combination with its modified forms, uncertainties and data gaps are highlighted. Issues essential for evaluation and prioritization of modified mycotoxins in health risk assessment are identified and discussed, including opportunities to improve exposure assessment using biomonitoring data. Further issues such as future consideration of combinatory effects of the parent toxin with its modified forms and also other compounds co-occurring in food and feed are addressed. With a particular focus on ZEN, the most pressing challenges associated with health risk assessment of modified mycotoxins are identified and recommendations for further research to fill data gaps and reduce uncertainties are made.

There is a critical need to better understand the patterns, levels and combinatory effects of exposures we are facing through our diet and environment. Mycotoxin mixtures are of particular concern due to chronic low dose exposures caused by naturally contaminated food. To facilitate new insights into their role in chronic disease, mycotoxins and their metabolites are quantified in bio-fluids as biomarkers of exposure. Here, we describe a highly sensitive urinary assay based on ultra-high performance liquid chromatography - tandem mass spectrometer (UHPLC-MS/MS) and 13 C-labelled or deuterated internal standards covering the most relevant regulated and emerging mycotoxins. Utilizing enzymatic pre-treatment, solid phase extraction and UHPLC separation, the sensitivity of the method was significantly higher (10-160x lower LODs) than in a previously described method used for comparison purpose, and stable isotopes provided compensation for challenging matrix effects. This method was in-house validated and applied to re-assess mycotoxin exposure in urine samples obtained from Nigerian children, adolescent and adults, naturally exposed through their regular diet. Owing to the methods high sensitivity, biomarkers were detected in all samples. The mycoestrogen zearalenone was the most frequently detected contaminant (82%) but also ochratoxin A (76%), aflatoxin M 1 (73%) and fumonisin B 1 (71%) were quantified in a large share of urines. Overall, 57% of 120 urines were contaminated with both, aflatoxin M 1 and fumonisin B 1 , and other co-exposures were frequent. These results clearly demonstrate the advanced performance of the method to assess lowest background exposures (pg mL -1 range) using a single, highly robust assay that will allow for the systematic investigation of low dose effects on human health., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Molecularly imprinted porous polymer microspheres selective to Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME), were synthesized and applied to the extraction of both mycotoxins in food samples. The polymer was prepared using 4-vinylpiridine (VIPY) and methacrylamide (MAM) as functional monomers, ethylene glycol dimethacrylate (EDMA) as cross-linker and 3,8,9-trihydroxy-6H-dibenzo[b,d]pyran-6-one (S2) as AOH surrogate template. A molecularly imprinted solid phase extraction (MISPE) method has been optimized for the selective isolation of the mycotoxins from aqueous samples coupled to HPLC with fluorescence (λ ex =258nm; λ em =440nm) or MS/MS analysis. The MISPE method was validated by UPLC-MS/MS for the determination of AOH and AME in tomato juice and sesame oil based on the European Commission Decision 2002/657/EC. Method performance was satisfactory with recoveries from 92.5% to 106.2% and limits of quantification within the 1.1-2.8µgkg -1 range in both samples., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A stable isotope dilution LC-MS/MS multi-mycotoxin method was developed for 12 different Fusarium toxins including modified mycotoxins in beer (deoxynivalenol-3-glucoside, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyl-deoxynivalenol, HT2-toxin, T2-toxin, enniatin B, B1, A1, A, beauvericin and zearalenone). As sample preparation and purification of beer a combined solid phase extraction for trichothecenes, enniatins, beauvericin and zearalenone was firstly developed. The validation of the new method gave satisfying results: intra-day and inter-day precision and recoveries were 1-5%, 2-8% and 72-117%, respectively. In total, 61 different organic and conventional beer samples from Germany and all over the world were analyzed by using the newly developed multi-mycotoxin method. In summary, deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivaleneol and enniatin B were quantified in rather low contents in the investigated beer samples. None of the other monitored Fusarium toxins like 15-acetyldeoxynivalenol, HT2- and T2-toxin, zearalenone, enniatin B1, A1, A or beauvericin were detectable., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

An enzyme-linked immunosorbent assay (ELISA) for the Alternaria mycotoxin tenuazonic acid (TeA) was evaluated by comparative analysis of naturally contaminated sorghum grains and sorghum-based infant food, using a stable isotope dilution LC-MS assay (SIDA; limit of detection (LOD) 1.0 μg/kg) as the reference method. LODs of the ELISA were 30 μg/kg in sorghum grains and 220 μg/kg in sorghum-based infant cereals. With SIDA, 100% of the samples (n = 28) had been positive for TeA in a concentration range of 6-584 μg/kg (mean 113 μg/kg). The ELISA consistently detected TeA in all naturally contaminated samples at cut-off levels of 30-60 μg/kg (sorghum) and 200-300 μg/kg (infant cereals), as based on corresponding to SIDA values. Although the ELISA was much less sensitive than the SIDA method, it may be useful as a screening method for sorghum and sorghum-based infant foods and can be employed to identify samples containing elevated concentrations of TeA in food, well below the proposed level of concern (500 μg/kg).

Some information is available about the fate of Fusarium toxins during the brewing process, but only little is known about the single processing steps in detail. In our study we produced beer from two different barley cultivars inoculated with three different Fusarium species, namely, Fusarium culmorum, Fusarium sporotrichioides, and Fusarium avenaceum, producing a wide range of mycotoxins such as type B trichothecenes, type A trichothecenes, and enniatins. By the use of multi-mycotoxin LC-MS/MS stable isotope dilution methods we were able to follow the fate of Fusarium toxins during the entire brewing process. In particular, the type B trichothecenes deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol showed similar behaviors. Between 35 and 52% of those toxins remained in the beer after filtration. The contents of the potentially hazardous deoxynivalenol-3-glucoside and the type A trichothecenes increased during mashing, but a rapid decrease of deoxynivalenol-3-glucoside content was found during the following steps of lautering and wort boiling. The concentration of enniatins greatly decreased with the discarding of spent grains or finally with the hot break. The results of our study show the retention of diverse Fusarium toxins during the brewing process and allow for assessing the food safety of beer regarding the monitored Fusarium mycotoxins.

Alternaria (A.) is a genus of widespread fungi capable of producing numerous, possibly health-endangering Alternaria toxins (ATs), which are usually not the focus of attention. The formation of ATs depends on the species and complex interactions of various environmental factors and is not fully understood. In this study the influence of temperature (7 °C, 25 °C), substrate (rice, wheat kernels) and incubation time (4, 7, and 14 days) on the production of thirteen ATs and three sulfoconjugated ATs by three different Alternaria isolates from the species groups A. tenuissima and A. infectoria was determined. High-performance liquid chromatography coupled with tandem mass spectrometry was used for quantification. Under nearly all conditions, tenuazonic acid was the most extensively produced toxin. At 25 °C and with increasing incubation time all toxins were formed in high amounts by the two A. tenuissima strains on both substrates with comparable mycotoxin profiles. However, for some of the toxins, stagnation or a decrease in production was observed from day 7 to 14. As opposed to the A. tenuissima strains, the A. infectoria strain only produced low amounts of ATs, but high concentrations of stemphyltoxin III. The results provide an essential insight into the quantitative in vitro AT formation under different environmental conditions, potentially transferable to different field and storage conditions., Competing Interests: The authors declare no conflict of interest.

An analytical method based on high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) detection for the simultaneous quantification of 12 Alternaria toxins in wine, vegetable juices and fruit juices was developed. Excellent chromatographic performance was demonstrated for tenuazonic acid (TeA) in a multi-analyte method. This comprehensive study is also the first to report the determination of TeA, alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN) and altenuene (ALT), altertoxin I (ATX-I), altertoxin II (ATX-II), altenuisol (ATL), iso-altenuene (isoALT), altenuic acid III (AA-III) and the AAL toxins TB1 und TB2 in samples from the German market. Several types of HPLC columns were tested for the liquid chromatographic separation of the toxins of interest that widely differ in their polarities. The focus was on gaining suitable retention while avoiding derivatization steps especially for TeA and AA-III. Three atmospheric pressure ionization techniques used with liquid chromatography (electrospray, chemical and photo ionization) were tested to obtain the best selectivity and sensitivity. Samples were diluted with sodium hydrogen carbonate buffer and extracted on a diatomaceous earth solid phase extraction cartridge. Method validation was carried out by using tomato juice, citrus juice and white wine as blank matrices. Limits of detection ranged from 0.10 to 0.59μgL(-1) and limits of quantification ranged from 0.4-3.1μgL(-1) depending on the toxin and matrix. Recoveries were around 100±9% for all toxins except stemphyltoxin III (STTX-III) and altenusin (ALS) due to instability during sample clean up. Matrix-induced effects leading to ion suppression especially for ATX-I, ATX-II and AA-III were investigated. Relative standard deviations of repeatability (RSDr) and intermediate reproducibility (RSDR) were ≤9.3 and ≤17.1, respectively, for the toxins in different matrices at levels of 5 and 30μgL(-1). Finally, 103 commercially obtained wine and juice samples from the German market in 2015 were analysed. TeA was found most frequently (68% of all analysed samples) in concentrations of up to 60.0μgL(-1). AOH, AME and TEN were detected in fewer samples (37%, 16% and 30%) at lower concentrations of up to 8.2, 1.5 and 10.3μgL(-1), respectively. AA-III and ATL were detected for the first time in 3% and 17% of food all samples, in concentrations of up to 6.0μgL(-1) and 5.9μgL(-1), respectively., (Copyright © 2016. Published by Elsevier B.V.)

Ultraperformance liquid chromatography tandem mass spectrometry and Quick, Easy, Cheap, Effective, Rugged, and Safe based analytical methodologies to quantitate both free (alternariol (1), alternariol monomethyl ether (2), tenuazonic acid (3), tentoxin (4), altenuene (5), altertoxin-I (6)) and conjugated (sulfates and glucosides of 1 and 2) Alternaria toxins in fruit and vegetable juices and tomato products were developed and validated. Acceptable limits of quantitation (0.7-5.7 μg/kg), repeatability (RSDr < 15.7%), reproducibility (RSDR < 17.9%), and apparent recovery (87.0-110.6%) were obtained for all analytes in all matrices investigated. 129 commercial foodstuffs were analyzed, and 3 was detected in 100% of tomato product samples (

Little is known about the fate of Fusarium mycotoxins during the barley malting process. To determine the fungal DNA and mycotoxin concentrations during malting, we used barley grain harvested from field plots that we had inoculated with Fusarium species that produce type A or type B trichothecenes or enniatins. Using a recently developed multimycotoxin liquid chromatography-tandem mass stable isotope dilution method, we identified Fusarium-species-specific behaviors of mycotoxins in grain and malt extracts and compared toxin concentrations to amounts of fungal DNA in the same samples. In particular, the type B trichothecenes and Fusarium culmorum DNA contents were increased dramatically up to 5400% after kilning. By contrast, the concentrations of type A trichothecenes and Fusarium sporotrichioides DNA decreased during the malting process. These data suggest that specific Fusarium species that contaminate the raw grain material might have different impacts on malt quality.

A multi-mycotoxin stable isotope dilution LC-MS/MS method was developed for 14 Fusarium toxins including modified mycotoxins in cereals (deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, HT2-toxin, T2-toxin, enniatin B, enniatin B1, enniatin A1, enniatin A, beauvericin, fusarenone X, nivalenol, deoxynivalenol-3-glucoside, and zearalenone). The chromatographic separation of the toxins with particular focus on deoxynivalenol and deoxynivalenol-3-glucoside was achieved using a C18-hydrosphere column. An expedient sample preparation method was developed that uses solid-phase extraction for the purification of trichothecenes combined with zearalenone, enniatins, and beauvericin and provides excellent validation data. Linearity, intra-day precision, inter-day precision, and recoveries were ≥0.9982, 1-6%, 5-12%, and 79-117%, respectively. Method accuracy was verified by analyzing certified reference materials for deoxynivalenol, HT2-toxin, and T2-toxin with deviations below 7%. The results of this method found barley malt samples from 2012, 2013, and 2014 frequently contaminated with high concentrations of enniatin B, deoxynivalenol, and its modified mycotoxin deoxynivalenol-3-glucoside. Samples from 2012 were especially contaminated. Fusarenone X was not detected in any of the analyzed samples.

Stable isotope dilution assays (SIDAs) are becoming ever commoner in mycotoxin analysis, and the number of synthesized or commercially available isotopically labelled compounds has greatly increased in the 7 years since our last review dealing with this topic. Thus, this review is conceived as an update for new applications or improvements of SIDAs for compounds discussed earlier, but the main focus is on newly introduced labelled substances and the development of SIDAs for, for example, fusarin C, moniliformin or the enniatins. Mycotoxin research has concentrated on the emerging group of Alternaria toxins in recent years, and a series of SIDAs have been developed, including ones for tenuazonic acid, alternariol, altertoxins and tentoxin that are discussed in detail in this review. Information about synthetic routes, isotopic purity and mass-spectrometric characterization of labelled compounds is given, as well as about the development and validation of SIDAs and their application to foods, feeds or biological samples. As the number of commercially available labelled standards is increasing continuously, a general tendency for the use of analytical methods based on liquid chromatography coupled with mass spectrometry capable of identifying a series of mycotoxins simultaneously ("multimethods") and using one or more labelled internal standards can be observed. An overview of these applications is given, thus demonstrating that SIDAs are increasingly being used in routine analysis.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantitate tenuazonic acid (TeA) in pig and broiler chicken plasma was successfully developed and validated. Linear matrix-matched calibration curves ranged between 5 and 200 ng/mL. Correlation coefficients, goodness-of-fit coefficients, and within-day and between-day precision and accuracy fell well within the acceptance criteria. The limit of quantitation was 5.0 ng/mL in both pig and broiler chicken plasma. The LC-MS/MS method was applied in a comparative toxicokinetic study in both pigs and broiler chickens. TeA was completely bioavailable after oral administration in both animal species. However, absorption was deemed to be slower in broiler chickens (mean tmax 0.32 h in pigs vs 2.60 h in chickens). TeA was more slowly eliminated in broiler chickens (mean t1/2el 0.55 h in pigs vs 2.45 h in chickens after oral administration), mainly due to the significantly lower total body clearance (mean Cl 446.1 mL/h/kg in pigs vs 59.2 mL/h/kg in chickens after oral administration). Tissue residue studies and further research to elucidate the biotransformation and excretion processes of TeA in pigs, broiler chickens, and other animal species are imperative.

An unprecedented stable isotope dilution assay for the genotoxic altertoxins along with exposure data of consumers is presented to enable a first risk assessment of these Alternaria toxins in foods. Altertoxins were produced as the most abundant Alternaria toxins in a modified Czapek-Dox medium with a low level of glucose as the carbon source and ammonium sulfate as the sole nitrogen source. Labeled altertoxins were synthesized in the same way using [(13)C6]glucose. Moreover, labeled alternariol, alternariol methyl ether, altenuene, and alternuisol were biosynthesized in another modified medium containing [(13)C6]glucose and sodium [(13)C2]acetate. A stable isotope dilution LC-MS/MS method was developed and used for food analysis. For altertoxin I, altertoxin II, alterperylenol, alternariol, and alternariol methyl ether, the limits of detection ranged from 0.09 to 0.53 μg kg(-1). The inter-/intra-day (n = 3 × 6) relative standard deviations of the method were below 13%, and the recoveries ranged between 96 and 109%. Among the various commercial food samples, some of the organic whole grains revealed low-level contamination with altertoxin I and alterperylenol, and paprika powder, which was heavily loaded with alternariol, alternariol methyl ether, and tentoxin, showed higher contamination level of altertoxin I and alterperylenol. Altertoxin II and III and stemphyltoxin III were not detectable. In addition, if the food was contaminated with altertoxins, it was likely to be co-contaminated with the other Alternaria toxins, but not necessarily vice versa. Maximum concentrations of altertoxin I and alterperylenol were detected in sorghum feed samples containing 43 and 58 μg kg(-1), respectively. This was significantly higher than that in the measured food samples.

As the term "masked mycotoxins" encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, "modified mycotoxins" into "biologically modified" and "chemically modified" with all variations of metabolites of the former and dividing the latter into "thermally formed" and "non-thermally formed" ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term "modified mycotoxins" should be used in the future and the term "masked mycotoxins" to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.

A total of 60 Chinese medicinal herbs were examined for contamination of the emerging Fusarium mycotoxins enniatins (ENNs) A, A1, B, B1 and beauvericin (BEA). The herbs under study are commonly used in China as both medicines and food. The dried samples of herbs were randomly collected from traditional Chinese medicine stores in Zhejiang province, China. Sample preparation was achieved by methanol extraction, followed by a simple membrane filtration step; no tedious clean-ups were involved. ENNs A, A1, B, B1 and BEA were analysed by the recently developed stable isotope dilution assays, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With limits of detection ranging between 0.8 and 1.2 µg kg(-1) for the analytes under study, 25% of all analysed samples were contaminated with at least one of the ENNs and BEA. BEA was the most frequently detected toxin with a 20% incidence in all samples. The percentages of ENN-positive samples were lower: each single ENN was detected in 6.7-11.7% of all samples. Considering the total amounts of the five mycotoxins in single samples, values between 2.5 and 751 µg kg(-1) were found. The mean total amount in positive samples was 126 µg kg(-1). Regarding ginger, the frequent occurrence of ENNs and BEA in dried ginger could be confirmed in samples from Germany. However, in fresh ginger root the toxins were not detectable. This is the first report on the presence of ENNs and BEA in Chinese medicinal herbs.

Analogues of the Alternaria mycotoxin tenuazonic acid (TA, biosynthesized by the fungus from the amino acid isoleucine) derived from valine (ValTA), leucine (LeuTA), alanine (AlaTA) and phenylalanine (PheTA) were synthesized and characterized by mass spectrometry (MS) and (1)H nuclear magnetic resonance (NMR) spectroscopy. Concentrations of stock solutions were determined by quantitative (1)H NMR (qHNMR). Two analytical methods based on high performance liquid chromatography (HPLC) and MS detection were developed, one with derivatization with 2,4-dinitrophenylhydrazine (DNPH) and one without derivatization. Limits of detection (LODs) were between 1-3 μg/kg (with derivatization) and 50-80 μg/kg (without derivatization). Respective limits of quantitation (LOQs) were about three times higher. Beside TA, the analogues LeuTA (about 4% of TA content) and ValTA (about 10% of TA content) were found in highly contaminated sorghum infant cereals and sorghum grains. Other analogues were not detected. Quantification of LeuTA and ValTA was performed using [(13)C6,(15)N]-TA as internal standard and matrix matched calibration. Recovery was between 95±11% and 102±10% for both compounds. Precision (relative standard deviation of triplicate sorghum cereal analyses three times during 3 weeks) was 7% for TA (912±60 μg/kg), 17% for LeuTA (43±8 μg/kg) and 19% for ValTA (118±22 μg/kg). These results indicate that several TA-like compounds, which are not yet characterized in aspects of their toxic properties, were detected in sorghum based infant food highly contaminated with TA, already., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The content of tenuazonic acid in human urine was determined by a stable isotope dilution assay (SIDA) that was recently developed for the analysis of food commodities and extensively re-validated for urine matrix in this study. Linearity of the response curve was proven between molar ratios n(labeled standard)/n(analyte) of 0.02-100. The limits of detection and determination were 0.2 and 0.6 μg/L, respectively. The mean recovery of the stable isotope dilution assay was 102 ± 3 % in the range between 1.0 and 100 μg/L. Interassay precision was 6.7 % (relative standard deviation of three triplicate analyses of a human urine sample during 3 weeks). The method was applied to two studies dealing with urinary excretion of tenuazonic acid: In the first study, tenuazonic acid was quantified in the 24-h urine of six volunteers from Germany (three female, three male) in a concentration range of 1.3-17.3 μg/L or 2.3-10.3 ng/mg(-1) creatinine, respectively. In the second study, two volunteers (one female, one male) ingested 30 μg tenuazonic acid by consumption of naturally contaminated whole meal sorghum infant cereals and tomato juice, respectively. The urinary excretion of the ingested tenuazonic acid was 54-81 % after 6 h, depending on matrix and volunteer. After 24 h, 87-93 % of the ingested amount of tenuazonic acid was excreted, but the fate of the remaining about 10 % is open. Thus, it is not possible to exclude potential health hazards for the consumer, completely.

The Alternaria mycotoxin tenuazonic acid (TA) was quantified in fruit juices (n = 50), cereals (n = 12) and spices (n = 38) using a recently developed stable isotope dilution assay (SIDA). [(13) C6,(15) N]-TA was used as the internal standard. Method validation revealed low limits of detection (LODs) of 0.15 μg/kg (fruit juices), 1.0 μg/kg (cereals) and 17 μg/kg (spices). The respective limits of quantitation were about three times higher. Recovery was about 100% for all matrices. The precision (relative standard deviation of replicate analyses of naturally contaminated samples) was 4.2% (grape juice; 1.7 μg/kg), 3.5% (whole wheat flour; 36 μg/kg) and 0.9% (curry powder; 215 μg/kg). The median content of TA in the analyzed samples was 1.8 μg/kg (fruit juices), 16 μg/kg (cereals) and 500 μg/kg (spices). Positive samples amounted to 86% (fruit juices), 92% (cereals) and 87% (spices).

The principle and applications of stable isotope dilution assays (SIDAs) in mycotoxin analysis are critically reviewed. The general section includes historical aspects of SIDAs, the prerequisites and limitations of the use of stable isotopically labelled internal standards, and possible calibration procedures. In the application section actual SIDAs for the analysis of trichothecenes, zearalenone, fumonisins, patulin, and ochratoxin A are presented. The syntheses and availability of labelled mycotoxins for use as internal standards is reviewed and specific advances in food analysis and toxicology are demonstrated. The review indicates that LC-MS applications, in particular, require the use of stable isotopically labelled standards to compensate for losses during clean-up and for discrimination due to ion suppression. As the commercial availability of these compounds continues to increase, SIDAs can be expected to find expanding use in mycotoxin analysis.

A stable isotope dilution assay (SIDA) was developed for quantification of the mycotoxin ochratoxin A (OTA) by using [2H5]-OTA as internal standard. The synthesis of labelled OTA was accomplished by acid hydrolysis of unlabelled OTA and subsequent coupling one of the products, ochratoxin alpha, to [2H5]-L-phenylalanine. The mycotoxin was quantified in foods by LC-tandem MS after extraction with buffers containing [2H5]-OTA and clean-up by immuno affinity chromatography or by solid phase extraction on silica. The method showed a sufficient sensitivity with a low detection and quantification limit of 0.5 and 1.4 microg/kg, respectively, and good precision in inter-assay studies showing a CV (n = 3) of 3.6%. The analysis of certified reference materials resulted in a low bias of 2.1% from the certified values and revealed excellent accuracy of the new method. To prove the suitability of SIDA. OTA was quantified in a number of food samples and resulted mainly in not detectable OTA contents. However, three samples of raisins exceeded the legal limit of 10 microg/kg and highlighted the need for further controlling the contamination with the mycotoxin.