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There is limited and inconsistent evidence, primarily from cross-sectional studies, linking mycotoxins to adverse birth outcomes. This study investigates the potential role of maternal dietary exposure to multiple mycotoxins in the development of several adverse pregnancy and birth outcomes. We analyzed data from 436 singleton pregnancies enrolled in a prospective cohort study in the rural Habiganj district, Bangladesh, between July 2018 and November 2019. Thirty-five urinary mycotoxin biomarkers were quantified using liquid chromatography coupled with tandem mass spectrometry and used to estimate dietary mycotoxin exposure. Multivariable regression models, adjusted for potential confounding and clustering, were fitted to assess the associations between maternal exposure to frequently occurring mycotoxins (ochratoxin A-OTA, citrinin- CIT, and Deoxynivalenol- DON) and pregnancy loss, preterm birth (PTB), low birth weight (LBW), born small-for-gestational-age (SGA) and small-vulnerable newborn. The results indicate that only in 16 of 436 pregnancies (4%) were urine samples free from all investigated mycotoxins. Biomarkers for six major mycotoxins were detected in the urine samples. OTA (95%), CIT (61%), and DON (6%) were most frequently detected, with at least two mycotoxins co-occurring in the majority of women (63%). There was evidence that maternal dietary intake of OTA was associated with higher odds of having an LBW baby, with the odds increasing in a dose-dependent manner. We found no evidence of associations between pregnancy loss, PTB, SGA, small-vulnerable newborns, and maternal dietary exposure to OTA, CIT, and DON, albeit with large confidence intervals, so findings are consistent with protective as well as large harmful effects. Exposure to multiple mycotoxins during pregnancy is widespread in this rural community and represents a health risk for mothers and babies. Tailored public health policies and interventions must be implemented to reduce mycotoxin exposure to the lowest possible level.

The mycotoxin ochratoxin A (OTA) is a contaminant in food that causes nephrotoxicity and to a minor degree hepatotoxicity. Recently, we observed that OTA induces liver damage preferentially to the cytochrome P450 (CYP)-expressing pericentral lobular zone, similar to hepatotoxic substances known to be metabolically toxified by CYP, such as acetaminophen or carbon tetrachloride. To investigate whether CYP influences OTA toxicity, we used a single dose of OTA (7.5 mg/kg; intravenous) with and without pre-treatment with the pan CYP-inhibitor 1-aminobenzotriazole (ABT) 2 h before OTA administration. Blood, urine, as well as liver and kidney tissue samples were collected 24 h after OTA administration for biochemical and histopathological analyses. Inhibition of CYPs by ABT strongly increased the nephro- and hepatotoxicity of OTA. The urinary kidney damage biomarkers kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) were increased > 126-fold and > 20-fold, respectively, in mice treated with ABT and OTA compared to those receiving OTA alone. The blood biomarkers of liver damage, alanine transaminase (ALT) and aspartate transaminase (AST) both increased > 21- and 30-fold, respectively, when OTA was administered to ABT pre-treated mice compared to the effect of OTA alone. Histological analysis of the liver revealed a pericentral lobular damage induced by OTA despite CYP-inhibition by ABT. Administration of ABT alone caused no hepato- or nephrotoxicity. Overall, the results presented are compatible with a scenario where CYPs mediate the detoxification of OTA, yet the mechanisms responsible for the pericental liver damage pattern still remain to be elucidated.

Local accumulation of xenobiotics in human and animal tissues may cause adverse effects. Large differences in their concentrations may exist between individual cell types, often due to the expression of specific uptake and export carriers. Here we established a two-photon microscopy-based technique for spatio-temporal detection of the distribution of mycotoxins in intact kidneys and livers of anesthetized mice with subcellular resolution. The mycotoxins ochratoxin A (OTA, 10 mg/kg b.w.) and aflatoxin B1 (AFB1, 1.5 mg/kg b.w.), which both show blue auto-fluorescence, were analyzed after intravenous bolus injections. Within seconds after administration, OTA was filtered by glomeruli, and enriched in distal tubular epithelial cells (dTEC). A striking feature of AFB1 toxicokinetics was its very rapid uptake from sinusoidal blood into hepatocytes (t1/2 ~ 4 min) and excretion into bile canaliculi. Interestingly, AFB1 was enriched in the nuclei of hepatocytes with zonal differences in clearance. In the cytoplasm of pericentral hepatocytes, the half-life (t1/2~ 63 min) was much longer compared to periportal hepatocytes of the same lobules (t1/2 ~ 9 min). In addition, nuclear AFB1 from periportal hepatocytes cleared faster compared to the pericentral region. These local differences in AFB1 clearance may be due to the pericentral expression of cytochrome P450 enzymes that activate AFB1 to protein- and DNA-binding metabolites. In conclusion, the present study shows that large spatio-temporal concentration differences exist within the same tissues and its analysis may provide valuable additional information to conventional toxicokinetic studies.

Hypoalbuminemia (HA) is frequently observed in systemic inflammatory diseases and in liver disease. However, the influence of HA on the pharmacokinetics and toxicity of compounds with high plasma albumin binding remained insufficiently studied. The ‘lack-of-delivery-concept’ postulates that HA leads to less carrier mediated uptake of albumin bound substances into hepatocytes and to less glomerular filtration; in contrast, the ‘concept-of-higher-free-fraction’ argues that increased concentrations of non-albumin bound compounds facilitate hepatocellular uptake and enhance glomerular filtration. To address this question, we performed intravital imaging on livers and kidneys of anesthetized mice to quantify the spatio-temporal tissue distribution of the mycotoxin ochratoxin A (OTA) based on its auto-fluorescence in albumin knockout and wild-type mice. HA strongly enhanced the uptake of OTA from the sinusoidal blood into hepatocytes, followed by faster secretion into bile canaliculi. These toxicokinetic changes were associated with increased hepatotoxicity in heterozygous albumin knockout mice for which serum albumin was reduced to a similar extent as in patients with severe hypoalbuminemia. HA also led to a shorter half-life of OTA in renal capillaries, increased glomerular filtration, and to enhanced uptake of OTA into tubular epithelial cells. In conclusion, the results favor the ‘concept-of-higher-free-fraction’ in HA; accordingly, HA causes an increased tissue uptake of compounds with high albumin binding and increased organ toxicity. It should be studied if this concept can be generalized to all compounds with high plasma albumin binding that are substrates of hepatocyte and renal tubular epithelial cell carriers.

A simple and effective approach for HPLC-MS/MS based multi-mycotoxin analysis in human urine samples was developed by application of dried urine spots (DUS) as alternative on-site sampling strategy. The newly developed method enables the detection and quantitation of 14 relevant mycotoxins and mycotoxin metabolites, including citrinin (CIT), dihydrocitrinone (DH-CIT), deoxynivalenol (DON), fumonisin B1 (FB1), T-2 Toxin (T-2), HT-2 Toxin (HT-2), ochratoxin A (OTA), 2′R-ochratoxin A (2′R-OTA), ochratoxin α (OTα), tenuazonic acid and allo-tenuazonic acid (TeA + allo-TeA), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), and β-zearalenol (β-ZEL). Besides the spotting procedure, sample preparation includes enzymatic cleavage of glucuronic acid conjugates and stable isotope dilution analysis. Method validation revealed low limits of detection in the range of pg/mL urine and excellent apparent recovery rates for most analytes. Stability investigation of DUS displayed no or only slight decrease of the analyte concentration over a period of 28 days at room temperature. The new method was applied to the analysis of a set of urine samples (n = 91) from a Swedish cohort. The four analytes, DH-CIT, DON, OTA, and TeA + allo-TeA, could be detected and quantified in amounts ranging from 0.06 to 0.97 ng/mL, 3.03 to 136 ng/mL, 0.013 to 0.434 ng/mL and from 0.36 to 47 ng/mL in 38.5%, 70.3%, 68.1%, and 94.5% of the samples, respectively. Additional analysis of these urine samples with an established dilute and shoot (DaS) approach displayed a high consistency of the results obtained with both methods. However, due to higher sensitivity, a larger number of positive samples were observed using the DUS method consequently providing a suitable approach for human biomonitoring of mycotoxin exposure.

This review summarises developments on the analysis of various matrices for mycotoxins published in the period from mid-2019 to mid-2020. Notable developments in all aspects of mycotoxin analysis, from sampling and quality assurance/quality control of analytical results, to the various detection and quantitation technologies ranging from single mycotoxin biosensors to comprehensive instrumental methods are presented and discussed. Aside from sampling and quality control, discussion of this past year’s developments is organised by detection and quantitation technology and covers chromatography with targeted or non-targeted high resolution mass spectrometry, tandem mass spectrometry, detection other than mass spectrometry, biosensors, as well as assays that use alternatives to antibodies. This critical review aims to briefly present the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies.

α-Asarone and β-asarone are reported as bioactive constituents of Acorus calamus. Phase I metabolism of asarone isomers results in a multiple spectrum of genotoxic metabolites. Thus, the question arises whether structural analogues of the known phase I metabolites also naturally occur in A. calamus-based food products. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for three product classes, herbal infusions, alcoholic beverages, and food supplements. High asarone contents were detected in herbal infusions (total mean 9.13 mg/kg, n = 8) and food supplements (total mean 14.52 mg/kg, n = 6); hence, these food products can highly contribute to human exposure to genotoxic asarone derivatives. Also, the occurrence of asarone oxidation products found in food and food supplements has to be taken under consideration because data on toxicity is limited so far.

Health issues of residents of mold-infested housing are reported on a regular basis, and reasons for the arising impairments can be manifold. One possible cause are the toxic secondary metabolite produced by indoor microfungi (mycotoxins). To enable a more thorough characterization of the exposure to mycotoxins in indoor environments, data on occurrence and quantities of mycotoxins is essential. In the presented study, 51 naturally mold-infested building material samples were analyzed applying a previously developed method based on ultra-high performance liquid chromatography (UHPLC) separation in combination with triple-quadrupole mass spectrometry (TQMS) detection. A total of 38 secondary metabolites derived from different indoor mold genera like Aspergillus, Fusarium, Penicillium, and Stachybotrys were analyzed, of which 16 were detectable in 28 samples. As both the spectrum of target analytes and the investigated sample matrices showed high chemical varieties, an alternative calibration approach was applied complementary to identify potentially emerging matrix effects during ionization and mass spectrometric detection. Overall, strong alterations of analyte signals were rare, and compensation of considerable matrix suppression/enhancement only had to be performed for certain samples. Besides mycotoxin determination and quantification, the presence of 18 different mold species was confirmed applying microbiological approaches in combination with macro- and microscopic identification according to DIN ISO 16000–17:2010–06. These results additionally highlight the diversity of mycotoxins potentially arising in indoor environments and leads to the assumption that indoor mycotoxin exposure stays an emerging topic of research, which has only just commenced.

The analysis of (trace) contaminants in environmental samples represents an important tool for exposure assessment and for the evaluation of potential risks to human health. Currently, mass spectrometric detection using triple quadrupole (TQMS) systems is the established method of choice. However, screening methods using high resolution mass spectrometry (HRMS) find increasing application as they provide advantages such as enhanced selectivity. A complex composition of environmental samples is known to have enormous effects on mass analyzers. The present work therefore compares the impact of a highly matrix-loaded sample material like house-dust on the performance of mass spectrometric detection of the emerging indoor contaminant group of mycotoxins by quadrupole time-of-flight (QTOF) and TQMS after ultrahigh-performance liquid chromatographic separation. Furthermore, the role of ionization efficiencies of different ion sources in instrument sensitivity was compared using an electrospray ionization source and a newly developed heated electrospray ion source (Bruker VIP-HESI) during QTOF experiments. Finally, it was evaluated whether an additional dimension of separation enables increased sensitivity in QTOF-HRMS detection by applying mycotoxins in house-dust to an (trapped) ion mobility spectrometry instrument. The sensitivity of the QTOF detection was positively influenced by the application of the VIP-HESI ion source, and overall HRMS instruments provided enhanced selectivity resulting in simplified data evaluation compared to the TQMS. However, all performed experiments revealed strong signal suppression due to matrix components. QTOF results showed more severe effects, enabling a more sensitive detection of mycotoxins in house-dust by applying TQMS detection.

This review summarises developments on the analysis of various matrices for mycotoxins that have been published in the period from mid-2018 to mid-2019. Analytical methods to determine aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes, and zearalenone are covered in individual sections. Advances in sampling strategies are also discussed in a dedicated section. In addition, developments in multi-mycotoxin methods – including comprehensive mass spectrometric-based methods as well as simple immunoassays – are also reviewed. This critical review aims to briefly present the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies.

Aflatoxins (AFs), ochratoxin A (OTA), citrinin (CIT), fumonisin B1 (FB1), zearalenone (ZEN), and deoxynivalenol (DON) are mycotoxins that may contaminate diets, especially in low-income settings, with potentially severe health consequences. This study investigates the exposure of 439 pregnant women in rural Bangladesh to 35 mycotoxins and their corresponding health risks and links their exposure to certain foods and local stimulants. Overall, 447 first-morning urine samples were collected from pregnant women between July 2018 and November 2019. Mycotoxin biomarkers were quantified by DaS-HPLC–MS/MS. Urinary concentration of frequently occurring mycotoxins was used to estimate dietary mycotoxin exposure. Median regression analyses were performed to investigate the association between the consumption of certain foods and local stimulants, and urinary concentration of frequently occurring mycotoxins. Only in 17 of 447 urine samples (4%) were none of the investigated mycotoxins detected. Biomarkers for six major mycotoxins (AFs, CIT, DON, FB1, OTA, and ZEN) were detected in the urine samples. OTA (95%), CIT (61%), and DON (6%) were most frequently detected, with multiple mycotoxins co-occurring in 281/447 (63%) of urine samples. Under the lowest exposure scenario, dietary exposure to OTA, CIT, and DON was of public health concern in 95%, 16%, and 1% of the pregnant women, respectively. Consumption of specific foods and local stimulants—betel nut, betel leaf, and chewing tobacco—were associated with OTA, CIT, and DON urine levels. In conclusion, exposure to multiple mycotoxins during early pregnancy is widespread in this rural community and represents a potential health risk for mothers and their offspring.

The exposure to mycotoxins of Swedish adolescents is currently unknown. The aim of the present study was to investigate the exposure to mycotoxins and their association with food intake, and background characteristics in adolescents of a national dietary survey. About 3000 school students (1000 from the 5th, 8th and 11th school years) were recruited for the survey. The participants completed Web-based questionnaires on food propensity, sociodemography and health, and a Web-based dietary recall. Spot urine and blood samples were collected from 1105 of the participants for mycotoxin biomarker analysis. Mycotoxins were analysed with multibiomarker methods in urine (HPLC-MS/MS) and serum (HPLC-MS/MS). Of the 35 different analytes in urine, the frequency of positive samples were the following: deoxynivalenol (DON, 4.8%), DON-15-β-D-O-glucuronide (DON-15GlcA, 9.1%), dihydro-citrinone (DH-CIT, 0.5%), HT-2-glucuronide (HT-2-3-GlcA, 0.1%) and ochratoxin A (OTA, 0.1%). Of the 27 different analytes in serum, OTA was detected in all samples, while 2’R-ochratoxin A (2’R-OTA) was found in 8.3% and enniatin B (EnB) in 99.2% of the samples. Exposure assessment calculations were performed on OTA from the serum concentration and on DON equivalents (DON eqv) from the urine concentration. All probable daily intake (PDI) estimates were below tolerable daily intakes, except for 1.6% of the participants for DON. The maximum PDI was 4.3 μg DON eqv/kg body weight and day. Consumption of cereal grain commodities was associated with levels of DON, EnB or OTA in biofluids. Serum OTA was also associated with intakes of raisins and coffee. Furthermore, coffee consumption correlated well with 2’R-OTA concentration in serum. In conclusion, exposure to mycotoxins in Swedish adolescents is common, but fortunately, high exposure was rare. Electronic supplementary material The online version of this article (10.1007/s12550-019-00381-9) contains supplementary material, which is available to authorized users.

Vegans might have a higher exposure to mycotoxins due to their heightened consumption of typical mycotoxin containing food sources. Yet, data on internal exposure among vegans in comparison to omnivores are currently lacking.This cross-sectional study includes 36 vegans and 36 omnivores (50% females, 30-60 years). A set of 28 and 27 mycotoxins is analyzed in 24-h urine and serum samples, respectively, by validated multi-mycotoxin methods (HPLC-MS/MS). Ochratoxin A (OTA), 2'R-OTA, and enniatin B in serum as well as deoxynivalenol-glucuronide in 24-h urine are quantified in 57-100% of the samples. Serum OTA levels are twofold higher in vegans than in omnivores (median 0.24 ng mLThe results indicate a higher exposure of vegans to OTA, but not to other mycotoxins. However, larger studies with repeated measurements are required to better evaluate the exposure to mycotoxins from plant-based diets.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin with nephrocarcinogenic potential found in a broad spectrum of food commodities. The mode of action of this compound, as well as its metabolism, is still not fully understood. To determine whether the conjugation of OTA with glutathione plays an important role in human OTA metabolism, an ochratoxin-glutathione conjugate (OTB-GSH), as well as the corresponding urinary metabolite ochratoxin-N-acetyl-l-cysteine (OTB-NAC), were synthesized and their structures confirmed by NMR spectroscopy. By means of synthesized stable isotope-labeled d5-OTB-GSH and d5-OTB-NAC references, a sensitive HPLC-MS/MS method has been developed and applied for the screening of human urine samples. OTB-NAC could be detected in 11 of the analyzed 18 urine samples and was quantified in 5 urine samples in the range between 0.023 and 0.176 ng mg−1 creatinine. OTB-GSH has not been detected in the urine samples. In OTB-NAC positive samples, this metabolite contributed to a comparable concentration range to the total OTA excretion as the parent compound. This is the first direct analysis of an OTA phase 2 metabolite in humans. Electronic supplementary material The online version of this article (10.1007/s12550-019-00360-0) contains supplementary material, which is available to authorized users.

This study deals with the influence of food matrix components on the degradation of the mycotoxins T-2 toxin (T-2) and HT-2 toxin (HT-2) and with the binding of T-2 to starch during thermal food processing. Both mycotoxins were heated in a simulated food environment and subsequently analyzed via HPLC-HRMS to generate degradation curves and to draw conclusions regarding the thermal degradation under food processing conditions. Thermal degradation increased generally with increasing time and temperature with a maximum degradation rate of 93% (T-2) and 99% (HT-2). Furthermore, HRMS data were exploited to screen the samples for degradation products. In model heating experiments, T-2 was bound to 1-O-methyl-α-D-glucopyranoside, a model compound that was used to simulate starch. The formed reaction products were isolated and identified by NMR, giving detailed insights into a potential binding of T-2 to starch. In the next step, further model heating experiments were performed, which proved the covalent binding of T-2 to starch. Finally, the amount of matrix-bound T-2 was estimated roughly in a semi-quantitative approach in the model heating experiments as well as during cookie-making via GC-MS analysis of the isovaleric acid ester moiety of T-2, released after alkaline hydrolysis.

This review summarises developments that have been published in the period from mid-2017 to mid-2018 on the analysis of various matrices for mycotoxins. Analytical methods to determine aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes, and zearalenone are covered in individual sections. Advances in sampling strategies are discussed in a dedicated section, as are methods used to analyse botanicals and spices, and newly developed comprehensive liquid chromatographic-mass spectrometric based multi-mycotoxin methods. This critical review aims to briefly discuss the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies.

α-Asarone and β-asarone are reported as bioactive constituents of

The mycotoxins T-2 and HT-2 toxin are frequently occurring food contaminants which are produced by Fusarium species. Humans and animals are mainly exposed to these substances by the consumption of contaminated oats, maize and wheat. For the production of crunchy muesli, bread and bakery products, these cereals undergo multiple processing steps, including baking, roasting and extrusion cooking. However, the influence of food processing on T-2 and HT-2 toxin levels is to date poorly understood. Thus, the effects of baking and roasting on both mycotoxins were evaluated during biscuit-, crunchy muesli- and toasted oat flakes-production under precise variation of various parameters: heating time and temperature as well as recipe formulation were varied in the range they are applied in the food processing industry. Therefore, oatmeal or flaked oats were artificially contaminated individually with both toxins and processed at the laboratory scale. T-2 toxin generally showed a higher degradation rate than HT-2 toxin. During biscuit-making up to 45% of T-2 toxin and 20% of HT-2 toxin were thermally degraded, showing a dependency on water content, baking time and temperature. The preparation of crunchy muesli yielded no significant toxin degradation which is probably due to the low temperatures applied. Roasting led to a degradation of 32% of T-2 toxin and 24% of HT-2 toxin. Taken together, both mycotoxins are partially degraded during thermal food processing; the degradation rates are influenced by the food composition and processing parameters.

Plant-derived phase II metabolites of T-2 toxin (T2) and HT-2 toxin (HT2) were first described in 2011 and further characterized in the following years. Since then, some efforts have been made to understand their biosynthesis, occurrence, toxicity, toxicokinetics, and finally relevance for consumers. Thus, the probably most important question is whether and how these metabolites contribute to toxicity upon hydrolysis either during food processing or the gastrointestinal passage. To answer this question, firstly, knowledge on the correct stereochemistry of T2 and HT2 glucosides is important as this affects hydrolysis and chemical behavior. So far, contradictory results have been published concerning the number and anomericity of occurring glucosides. For this reason, we set up different strategies for the synthesis of mg-amounts of T2, HT2, and T2 triol glucosides in both α and ß configuration. All synthesized glucosides were fully characterized by NMR spectroscopy as well as mass spectrometry and used as references for the analysis of naturally contaminated food samples to validate or invalidate their natural occurrence. Generally, 3-O-glucosylation was observed with two anomers of HT2 glucoside being present in contaminated oats. In contrast, only one anomer of T2 glucoside was found. The second aspect of this study addresses the stability of the glucosides during thermal food processing. Oat flour was artificially contaminated with T2 and HT2 glucosides individually and extruded at varying initial moisture content and temperature. All four glucosides appear to be more stable during food extrusion than the parent compounds with the glucosidic bond not being hydrolyzed.

This review summarises developments in the determination of mycotoxins over a period between mid-2016 and mid-2017. Analytical methods to determine aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes and zearalenone are covered in individual sections. Advances in proper sampling strategies are discussed in a dedicated section, as are methods used to analyse botanicals and spices and newly developed LC-MS based multi-mycotoxin methods. This critical review aims to briefly discuss the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies.

The analysis of the nephrotoxic mycotoxin citrinin in food, feed, and physiological samples is still challenging. Nowadays, liquid chromatography coupled with mass spectrometry is the method of choice for achieving low limits of detection. But matrix effects can present impairments for this method. Stable isotope dilution analysis can prevent some of these problems. Therefore, a stable isotopically labeled standard of citrinin for use in stable isotope dilution analysis was synthesized on large scale. The improved diastereoselective total synthetic strategy offered the possibility to introduce three 13C-labels in two steps by ortho-toluate anion chemistry. This led to a mass difference of 3 Da, sufficient for preventing spectral overlap. Additionally, a stable isotopically labeled form of dihydrocitrinone, the main urinary metabolite of citrinin, was synthesized with the same mass difference. This was achieved by a sequence of cyclisation, oxidation, deprotection, and carboxylation reactions starting from a protected intermediate of the labeled citrinin synthesis. Thus, this method also offers a complete way to synthesize dihydrocitrinone from citrinin on large scale.

Penitrems are fungal indole diterpene-derived tremorgenic secondary metabolites, which are mainly produced by Penicillium spp. Several cases of intoxications with penitrems and subsequent occurrences of penitrem A in foodstuff underline the need for reliable quantitation methods for the detection of these mycotoxins in food. In this study, a simple and fast high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the quantitative analysis of penitrems A-F in cheese was developed. Therefore, penitrems A-F were isolated from Penicillium crustosum as analytical reference standards. The analysis of 60 cheese samples from the European single market (EU) revealed the occurrence of penitrem A in 10% of the analyzed samples with an average concentration of 28.4 μg/kg and a maximum concentration of 429 μg/kg. In addition to penitrem A, other members of the group of penitrems, namely, penitrems B, C, D, E, and F, were for the first time quantitatively detected in food samples, although in lower concentrations and with lower incidence in comparison to penitrem A. Moreover, we report cytotoxic effects of all penitrems on two cell lines (HepG2 and CCF-STTG1). This clearly underlines their relevance and the importance to analyze food samples in order to get insights into the human exposure toward these mycotoxins.

In the course of assessing the human exposure to mycotoxins, biomarker-based approaches have proven to be important tools. Low concentration levels, complex matrix compositions, structurally diverse analytes, and the large size of sample cohorts are the main challenges of analytical procedures. For that reason, an online solid phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry (online SPE-UHPLC-MS/MS) method was developed, allowing for the sensitive, robust, and rapid analysis of 11 relevant mycotoxins and mycotoxin metabolites in human urine. The included spectrum of analytes comprises aflatoxin M1 (AFM1), altenuene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), citrinin (CIT) and its metabolite dihydrocitrinone (DH-CIT), fumonisin B1 (FB1), ochratoxin A (OTA), and zearalenone (ZEN) as well as α- and β-zearalenol (α- and β-ZEL). Reliable quantitation was achieved by means of stable isotope dilution, except for ALT, AME and AOH using matrix calibrations. The evaluation of method performance displayed low limits of detection in the range of pg/mL urine, satisfactory apparent recovery rates as well as high accuracy and precision during intra- and interday repeatability. Within the analysis of Zimbabwean urine samples (n = 50), the applicability of the newly developed method was shown. In addition to FB1 being quantifiable in all analyzed samples, six other mycotoxin biomarkers were detected. Compared to the occurrence rates obtained after analyzing the same sample set using an established dilute and shoot (DaS) approach, a considerably higher number of positive samples was observed when applying the online SPE method. Owing to the increased sensitivity, less need of sample handling, and low time effort, the herein presented online SPE approach provides a valuable contribution to human biomonitoring of mycotoxin exposure.

The fungus Claviceps purpurea produces highly toxic ergot alkaloids and accumulates these in the hardened bodies of fungal mycelium. These so-called sclerotia, or ergot bodies, replace the crop seed of infected plants, which can include numerous important food- and feedstuff such as rye and wheat. While several studies have explored details of the infection process and development of ergot bodies, little information is available on the spatial distribution of the mycotoxins in the sclerotia. Here we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at a lateral resolution of 35 μm to visualize the distribution of two representative alkaloids, ergocristine and ergometrine, produced by Ecc93 and Gal 310 variants of C. purpurea, respectively, after infection of rye. To improve cryosectioning of this fragile biological material tissue with complex texture, we developed a practical embedding protocol based on cellulose polymers. The MALDI-MS images recorded from the so produced intact tissues sections revealed that ergometrine exhibited a relatively homogeneous distribution throughout the ergot body, whereas ergocristine was found to be enriched in the proximal region. This finding can be correlated to the morphological development of sclerotia as ergot alkaloids are only produced in the sphacelial stage. The ability to localize toxins and other secondary metabolites in intact sections of crop-infecting fungi with high lateral resolution renders MALDI-MSI a powerful tool for investigating biosynthetic pathways and for obtaining a deeper understanding of the parasite-host interaction. Graphical abstract Workflow for identification and spatial localization of ergot alkaloids in infected rye grains.

Deoxynivalenol (DON), the most commonly occurring trichothecene in nature, may affect animal and human health through causing diarrhea, vomiting, gastrointestinal inflammation, and immunomodulation. DON-3-glucoside (DON-3G) as a major plant metabolite of the mycotoxin is another "emerging" food safety issue in recent years. Humans may experience potential health risks by consuming DON-contaminated food products. Thus, it is crucial for human and animal health to study also the degradation of DON and DON-3G during thermal food processing. Baking, boiling, steaming, frying, and extrusion cooking are commonly used during thermal food processing and have promising effects on the reduction of mycotoxins in food. For DON, however, the observed effects of these methods, as reported in numerous studies, are ambiguous and do not present a clear picture with regard to reduction or transformation. This review summarized the influence of thermal processing on the stability of DON and the formation of degradation/conversion products. Besides this, also a release of DON and DON-3G from food matrix as well as the release of DON from DON-3G during processing is discussed. In addition, some conflicting findings as reported from the studies on thermal processing as well as cause-effect relationships of the different thermal procedures are explored. Finally, the potential toxic profiles of DON degradation products are discussed as well when data are available.

Ochratoxin A (OTA), a mycotoxin with nephrotoxic and carcinogenic properties, is an important contaminant of food and feed. Analysis of OTA in human biological fluids (blood, urine, or breast milk) has documented frequent exposure to this mycotoxin, yet at quite variable levels in different population groups across the world. Urine is the preferred matrix in biomonitoring since sample collection is non-invasive and better accepted by study participants. As only a small fraction of the ingested OTA is excreted in urine, determination of urinary OTA requires sensitive analytical techniques, and phase-II-metabolites should be also considered as biomarkers of exposure. Yet, data published so far on the presence of OTA-glucuronide/sulfate in human urine have been contradictory. In this study, urines (n = 38) from two groups of breastfed infants (German and Turkish) and from German adults were now analysed for the presence of OTA glucuronides or sulfates by an indirect method, i.e. by comparing the levels of OTA (aglycone) in urines without and after enzymatic hydrolysis with ß-glucuronidase/arylsulfatase. Additionally, ochratoxin A-8-β-glucuronide and open lactone ochratoxin A-8-β-glucuronide were synthesized to serve as reference materials for metabolite analysis. Attempts for definitive confirmation of glucuronides of OTA via direct identification in LC-MS/MS analysis were hampered by the lower ionizability of the conjugates compared to the parent compound. Considerable increases in OTA levels were found after enzymatic hydrolysis in several (not all) urine samples and provide clear evidence for the excretion of OTA-conjugates. The latter observation is of importance, since OTA phase-II-metabolites may escape detection when direct methods are applied for urinary biomarker analysis. In conclusion, enzymatic hydrolysis of urine samples is highly advisable in order to avoid an underestimation of the OTA-exposure.

The genus Stachybotrys belongs to filamentous fungi found in indoor environment, mostly on cellulose-rich substrates after water-damage. The major purpose of this study was to investigate the influence of different building materials in case of mold infestation on the mycotoxin production of Stachybotrys species. Fifteen Stachybotrys mycotoxins including satratoxins, phenylspirodrimanes, and recently discovered stachybotrychromenes were in the focus of the investigations. Artificial and natural infestations were compared to determine whether environmental factors, for example, time of growth, temperature, humidity, and material additives have an influence on the observed mycotoxin profiles. It turned out that mycotoxin profiles from Stachybotrys spp. on building materials can be influenced by cellulose, paints, and paste of the materials. The total toxin levels of artificially and naturally contaminated gypsum board samples ranged up to 30 µg/cm2 , whereas wallpaper samples showed total toxin levels in the range of 20-66 µg/cm2 . A naturally infested sample disclosed the conversion of the dialdehyde components to the corresponding lactone isomers under the influence of light.

Food raw materials can contain the mycotoxin ochratoxin A (OTA). Thermal processing of these materials may result in decreased OTA levels but also in the formation of the thermal isomerization product 2&prime, R-ochratoxin A (2&prime, R-OTA). So far, only 2&prime, R-OTA levels reported from 15 coffee samples in 2008 are known, which is little when compared to the importance of coffee as a food and trading good. Herein, we present results from a set of model experiments studying the effect of temperatures between 120 &deg, C and 270 &deg, C on the isomerization of OTA to 2&prime, R-OTA. It is shown that isomerization of OTA starts at temperatures as low as 120 &deg, C. At 210 &deg, C and above, the formation of 25% 2&prime, R-OTA is observed in less than one minute. Furthermore, 51 coffee samples from France, Germany, and Guatemala were analyzed by HPLC-MS/MS for the presence of OTA and 2&prime, R-OTA. OTA was quantified in 96% of the samples, while 2&prime, R-OTA was quantifiable in 35% of the samples. The highest OTA and 2&prime, R-OTA levels of 28.4 &micro, g/kg and 3.9 &micro, g/kg, respectively, were detected in coffee from Guatemala. The OTA:2&prime, R-OTA ratio in the samples ranged between 2.5:1 and 10:1 and was on average 5.5:1. Besides coffee, 2&prime, R-OTA was also for the first time detected in a bread sample and malt coffee powder.

The genus Stachybotrys produces a broad diversity of secondary metabolites, including macrocyclic trichothecenes, atranones, and phenylspirodrimanes. Although the class of the phenylspirodrimanes is the major one and consists of a multitude of metabolites bearing various structural modifications, few investigations have been carried out. Thus, the presented study deals with the quantitative determination of several secondary metabolites produced by distinct Stachybotrys species for comparison of their metabolite profiles. For that purpose, 15 of the primarily produced secondary metabolites were isolated from fungal cultures and structurally characterized in order to be used as analytical standards for the development of an LC-MS/MS multimethod. The developed method was applied to the analysis of micro-scale extracts from 5 different Stachybotrys strains, which were cultured on different media. In that process, spontaneous dialdehyde/lactone isomerization was observed for some of the isolated secondary metabolites, and novel stachybotrychromenes were quantitatively investigated for the first time. The metabolite profiles of Stachybotrys species are considerably influenced by time of growth and substrate availability, as well as the individual biosynthetic potential of the respective species. Regarding the reported adverse effects associated with Stachybotrys growth in building environments, combinatory effects of the investigated secondary metabolites should be addressed and the role of the phenylspirodrimanes re-evaluated in future research.

Mycotoxins are secondary metabolites of filamentous fungi, which are able to contaminate food and feed worldwide. As mycotoxins are toxic to humans and animals, their presence in food and feed may pose a potential health risk. Besides their occurrence as parent compounds in food and feed, mycotoxins can also be modified in various chemical and/or biochemical reactions leading to changes in their chemical structure. Such modified mycotoxins can be formed either during biotransformation by microorganisms, infested plants or the mammalian organism or by chemical reactions e.g. during food processing. As modified mycotoxins are in many cases not detectable with routine analytical methods their occurrence might lead to an underestimation of the potential health risk of mycotoxins for humans or animals. Currently there are many open questions and for this reason research activities dealing with the occurrence, analysis, formation and toxicity of modified mycotoxins have to be intensified in the near future.

Rationale Mycotoxins are toxic secondary metabolites produced by various fungi. Their distribution within contaminated material is of high interest to obtain insight into infection mechanisms and the possibility of reducing contamination during food processing. Methods Various vegetable foodstuffs were infected with fungi of the genera Fusarium and Aspergillus. The localization of the produced mycotoxins was studied by matrix-assisted laser desorption/ionization time-of flight mass spectrometry imaging (MALDI-MSI) of cryosections obtained from infected material. The results were confirmed by high-performance liquid chromatography/electrospray ionization triple quadrupole mass spectrometry (HPLC/MS/MS). Results The mycotoxins ochratoxin A (OTA) and fumonisins of the B- and C-series (FB1, FB2, FB3, FB4, FC1, FC2/3, and FC4) as well as partially hydrolyzed fumonisins (pHFB1, pHFB2, pHFB3, pHFC1, and pHFC2/3) could successfully be detected by MALDI-MSI in mold-infested foodstuffs. The toxins are distributed differently in the material: OTA is co-localized with visible fungal spoilage while fumonisins could be detected throughout the whole sample. Conclusions This work shows the applicability of MALDI-MSI to mycotoxin analysis. It has been demonstrated that the analyzed mycotoxins are differently distributed within moldy foodstuffs. These findings show the potential of MALDI-MSI for the localization of these hazardous compounds in various plant tissues. Copyright © 2016 John Wiley & Sons, Ltd.

Tenuazonic acid (1) is a mycotoxin produced mainly by fungi of the genus Alternaria. It occurs in a variety of agricultural products. allo-Tenuazonic acid (2) is an isomer of 1 that is not chromatographically separated from 1 in most analytical methods. Therefore, both isomers are quantitated as a sum parameter. In this study a QuEChERS (quick, easy, cheap, effective, rugged and safe) based stable isotope dilution HPLC-MS/MS method including the chromatographic separation of both isomers was developed and applied to 20 tomato products from the German market. All products showed contamination with both toxins. 1 was found in a range from 5.3 ± 0.1 to 550 ± 15 μg/kg (average = 120 μg/kg) and 2 in a range from 1.5 ± 0.4- to 270 ± 0.8 μg/kg (average = 58 μg/kg). 2 represents 7.0-44% of the sum of both isomers (average = 29%). This is the first reported occurrence of 2 in food samples. To evaluate and compare the cytotoxicities of 1 and 2, both compounds were isolated from a synthetic racemic mixture. 1 showed moderate cytotoxic effects on HT-29 cells starting at 100 μM, whereas 2 exhibited no activity. 2 was not produced in liquid cultures of Alternaria alternata in yeast extract sucrose (YES) medium, but could be detected in small amounts in tomato puree inoculated with the fungus.

Scope The aim of this study is to obtain a deeper knowledge of the kinetics of 2'R-ochratoxin A (2'R-OTA), the thermal degradation product of the mycotoxin ochratoxin A (OTA). To investigate the correlation between the amount of this compound in roasted coffee and human blood samples, a human study is performed. Methods and results An 18-week human study is carried out. During the first eight weeks, all known 2'R-OTA-containing food sources are excluded from the diet and the reduction of 2'R-OTA in venous blood is analyzed. Afterwards, participants are allowed to consume coffee with known OTA and 2'R-OTA concentrations. On a biweekly scale, 2'R-OTA and OTA blood levels are determined. After eight weeks of fasting on 2'R-OTA-containing foods, the 2'R-OTA blood concentration decreased by about 10%. Based on this, a long biological half-life of over seven months is estimated. In the 24 h urine samples collected before and after the coffee fasting period, only traces of 2'R-OTA are detected. Conclusion Results show that 2'R-OTA has a more than seven-fold higher biological half-life in human blood compared to OTA (approx. 35 days). The reason for the long persistence of 2'R-OTA in human blood is still unclear and further research is needed.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. Roasting of OTA-contaminated coffee results in the formation of 2&prime, R-ochratoxin A (2&prime, R-OTA), which appears in the blood of coffee drinkers. Human serum albumin (HSA) binds 2&prime, R-OTA (and OTA) with high affinity, therefore, albumin may influence the tissue uptake and elimination of ochratoxins. We aimed to investigate the binding site of 2&prime, R-OTA (verses OTA) in HSA and the displacing effects of site markers to explore which molecules can interfere with its albumin-binding. Affinity of 2&prime, R-OTA toward albumins from various species (human, bovine, porcine and rat) was tested to evaluate the interspecies differences regarding 2&prime, R-OTA-albumin interaction. Thermodynamic studies were performed to give a deeper insight into the molecular background of the complex formation. Besides fluorescence spectroscopic and modeling studies, effects of HSA, and fetal bovine serum on the cytotoxicity of 2&prime, R-OTA and OTA were tested in MDCK kidney cell line in order to demonstrate the influence of albumin-binding on the cellular uptake of ochratoxins. Site markers displaced more effectively 2&prime, R-OTA than OTA from HSA. Fluorescence and binding constants of 2&prime, R-OTA-albumin and OTA-albumin complexes showed different tendencies. Albumin significantly decreased the cytotoxicity of ochratoxins. 2&prime, R-OTA, even at sub-toxic concentrations, increased the toxic action of OTA.

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus. 2&prime, R-Ochratoxin A (2&prime, R-OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 &micro, g/L with 2&prime, R-OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2&prime, R-OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2&prime, R-OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2&prime, R-OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a logK of 7.0&ndash, 7.6 was calculated, while for its thermally produced isomer 2&prime, R-OTA, a lower, but still high, logK of 6.2&ndash, 6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2&prime, R-OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2&prime, R-OTA found in human blood samples.