A new approach using micro HPLC-MS/MS for multi-mycotoxin analysis in maize samples.

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 possible contamination by aflatoxins (AFs), ochratoxin A (OTA), fumonisin B1 (FB1), fusaric acid (FA), and beauvericin (BEA), was investigated in 55 samples of dried figs and dried figs‐based products purchased from the South Italy (Calabria) market. A total of 41 samples showed contamination by at least one of the mycotoxins investigated. Aflatoxin B1 was found in six samples (0.19 — 8.41 μg/kg) total aflatoxins were found in 13 samples (0.5–17.12 μg/kg), OTA was found in 21 samples (<LOQ—158.58 μg/kg), FB1 was found in eight samples (153.81–5,412.96 μg/kg), BEA was found in 12 samples (<LOQ—5,708.49 μg/kg), and FA was found in 28 samples (<LOQ—74,520.20 μg/kg). The analyzed samples were contaminated with one (41.8%), two (7.3%), three (5.5%), four (10.9%), and five (5.5%) mycotoxins. To the best of our knowledge, the incidence of contamination by FA and BEA has been shown for the first time in dried figs.Practical applicationsThe present work was focused on mycotoxin mixtures contamination levels of dried figs and dried figs‐based products. It is known that the co‐occurrence of mycotoxins leads to additive or synergistic effects. For some analyzed samples the AFB1, aflatoxins, and ochratoxin A (OTA) levels exceed the European Union countries legal limit. In addition, the simultaneous presence of FB1, FA, and BEA can be a potential threat to the health of consumers. Although different studies examined AFs, OTA, and Fusarium toxins as single mycotoxins in dried figs, to our knowledge, the co‐occurrence of AFs, OTA, FB1, FA, and BEA in dried figs and in dried figs‐based products samples has not been reported previously. Therefore, to prevent mycotoxins contamination, the employment of good practices in all the processing steps is necessary. Furthermore, the quality control of the ingredients used in figs preparations (spices, other dried fruits, cocoa, etc.), usually not adequately, is essential. Finally, it is of importance to establish more stringent rules to ensure food safety, even if no legal limit was still set for Fusarium toxins in dried fruit.

Background Bentonite, a mineral authorized in Europe as feed additive for reducing mycotoxin contamination in feed (EU Reg. No. 1060/2013), is selective in adsorbing aflatoxins AFB1, but ineffective for other mycotoxins of zootechnical interest. We develop and assess the efficacy of an innovative bentonite-based material (bio-organoclay) acting as a multi-mycotoxin binder, by performing in vitro and in vivo experiments. Methods The efficacy of the bio-organoclay in sequestering aflatoxins B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA), and zearalenone (ZEA) was firstly tested in vitro. Then, its efficacy in reducing the urinary excretion of mycotoxins was tested in rats by using the biomarker approach. For each toxin, two groups of rats (0.3 kg initial body weight) were considered: controls received the mycotoxins without the detoxifier, and treated rats received the mycotoxins with the bio-organoclay at 0.5% w/w of feed consumption. Mycotoxins were administered by a intragastric oral bolus. Samples of urine were collected at different time points (4-72 h for AFB1, ZEA and FB1; 4-320 h for OTA). AFB1, ZEA and its metabolites of phase I biotransformation (α-ZOL, β-ZOL and β-ZAL), OTA and FB1 were analyzed in urine by in-house validated UPLC methods. Mycotoxin content in urine was normalized to urinary creatinine concentration. Toxicokinetic parameters were calculated. Results A bentonite containing Na-smectite as major mineral was modified and functionalized. Acid-activation and functionalization processes were optimized at lab level, and optimal conditions were identified. In in vitro studies, at low dosages (0.25-0.5% w/v), the new produced bio-organoclay sequestered more than 95% of AFB1, FB1, OTA, and ZEA, in a large range of pH values (3-9). Mycotoxin adsorption occurred simultaneously with high capacity and affinity as determined by equilibrium isotherms. In rats, the bio-organoclay reduced urinary excretion of AFM1, ZEA, FB1 and OTA respect to the control group, by reducing the AUC and Cmax values. AUC0à72 was significantly reduced by -94% for AFM1, -54% for OTA, and -40% for FB1 (p<0.05). Cmax value of ZEA was reduced by -65% (p<0.05). Conclusion The use of the bio-organoclay as a feed additive can be considered a valid approach to reduce mycotoxins bioavailability in animals exposed to the main mycotoxins, answering to the great demand of these kind of products by animal feed producers. This product can be considered safe, as it has been obtained using reagents that are listed in the E. U. Register of Feed Additives.

A survey for aflatoxin B1 (AFB1), fumonisin B1 and ochratoxin A (OTA) was conducted on 127 samples that included 30 food-grade barley, 32 barley foods, 18 food-grade corn and 47 corn foods, randomly collected during 1998–99 in Seoul, Korea. The presence of mycotoxins was analysed by direct competitive enzyme-linked immunosorbent assay (ELISA), and most of the positive samples from ELISA were confirmed using high-performance liquid chromatography (HPLC). Recoveries of AFB1 and OTA spiked at 10 ng g-1 and FB1 spiked at 50 ng g-1 were 106, 87 and 105a% by ELISA, whereas those by HPLC were 80, 79 and 84%, respectively. Detection limits by ELISA for AFB1, FB1 and OTA were 1, 5 and 5 ng g-1, and those by HPLC were 0.6, 35 and 1 ng g-1. Naturally occurring AFB1, FB1 and OTA were found in 4/32 (12%), 2/32(6%) and 4/32 (12%) samples of barley foods with an average of 26, 16 and 9 ng g-1, respectively. AFB1 and FB1 in corn foods were detected in 4/47 (8%) and 9/47 (19%) samples with the average being 20 and 74 ng g-1, while no OTA was found in any corn foods samples. No AFB1, FB1 or OTA was detected in any of food-grade barley and corn samples. This is the first report on the natural co-occurrence of AFB1 and FB1 in barley and corn foods as well as on surveillance of OTA in Korea.

A survey was undertaken of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), ochratoxin A (OTA), and fumonisin B1 (FB1), B2 (FB2) and B3 (FB3) contamination of various retail foods in Japan during 2004–05. The mycotoxins were analysed by high-performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry (LC/MS) or high-performance thin-layer chromatography (HPTLC). Aflatoxins (AFs) were detected in ten of 21 peanut butter and in 22 of 44 bitter chocolate samples; the highest level of AFB1, 2.59 µg kg−1, was found in peanut butter. Aflatoxin contamination was not observed in corn products (n = 55), corn (n = 110), peanuts (n = 120), buckwheat flour (n = 23), dried buckwheat noodles (n = 59), rice (n = 83) or sesame oil (n = 20). OTA was detected in 120 out of 192 samples of oatmeal, wheat flour, rye, buckwheat flour, raw coffee, roasted coffee, raisin, beer, wine and bitter chocolate, but not in rice or corn products. OTA levels in the positive samples were below 13 µg kg−1. AFs and OTA intakes through the consumption of foods containing cacao were estimated using the data for mycotoxin contamination in bitter chocolate and those for the consumption of foods containing cacao in Japan.

This study has examined the pattern of mycotoxin contamination of maize destined for animal feed in different global regions over a period of 3 years (2018–2020) with up to 1000+ samples analysed in each year. Overall, >75% of samples in each of the survey years were contaminated with multiple mycotoxins regardless of the global region (Europe, Africa, Asia, South Americas countries). Using LC-MS/MS, it was possible to quantify the relative contamination present in the samples in each year from the different regions of eight different mycotoxins including aflatoxin B1 (AFB1), ochratoxin A (OTA) deoxynivalenol (DON), fumonisin B1 (FB1) and B2, zearalenone (ZEA), T-2 and HT-2 toxins. The trends in mycotoxin contamination showed that there was a consistent contamination with DON in the 3 sampling years in all four regions. Interestingly, AFB1 contamination was prevalent in all regions in 2018, but more predominant in Europe and in 2019. In contrast, in 2020 it was found to be the major contaminant in Africa only. However, FB1 contamination of maize which was prevalent in Europe in 2018, became more prevalent in Asia and LATAM countries in 2019 and even in African maize in 2020. Comparisons of contamination with different mycotoxins in each of the years globally showed significant differences for AFB1, FB1, DON and ZEA between the different years. These results are discussed in relation to the trends of contamination of maize with mixtures of mycotoxins and the implication for their control in this key commodity used as an important ingredient in animal feed.

The multi-biomarker approach was used to validate urinary biomarkers in piglets administered boluses contaminated with mixtures of deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone (ZEA) and ochratoxin A (OTA) at different concentrations. Boluses contaminated with mycotoxins were prepared by slurrying and freezedrying feed material fortified with culture extracts of selected toxigenic fungi. Piglets were individually placed in metabolic cages to collect urine before gavage and 24 h post dose. Urine samples were hydrolysed with β-glucuronidase and analysed by a multi-biomarker LC-MS/MS method developed and validated to identify and measure biomarkers of FB1, OTA, DON, ZEA and AFB1. Urinary levels of FB1, OTA, DON + de-epoxy-deoxynivalenol, ZEA + alphazearalenol and aflatoxin M1 were selected as biomarkers of FB1, OTA, DON, ZEA and AFB1, respectively. Mean percentages of dietary mycotoxins excreted as biomarkers in 24 h post dose urine were 36.8% for ZEA, 28.5% for DON, 2.6% FB1, 2.6% for OTA and 2.5% for AFB1. A good correlation was observed between the amount of mycotoxins ingested and the amount of relevant biomarkers excreted in 24 h post dose urine. Linear dose-response correlation coefficients ranged between 0.68 and 0.78 for the tested couples of mycotoxin/biomarker. The good sensitivity of the LC-MS/MS method and the good dose-response correlations observed in this study permitted to validate the selected mycotoxin biomarkers in piglets at dietary levels close to the maximum permitted levels reported in Commission Directive 2003/100/EC for AFB1 and the guidance values reported in Commission Recommendation 2006/576/EC for DON, ZEA, OTA and FB1.

This study was designed to develop a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples with particular focus on the optimization of the sample preparation protocol and method validation. All 25 mycotoxins were extracted in a single step with a mixture of methanol/ethyl acetate/water (70:20:10, v/v/v). The method limits of quantification (LOQ) varied from 0.3 μg/kg to 106 μg/kg. Good precision and linearity were observed for most of the mycotoxins. The method was applied for the analysis of naturally contaminated peanut cake, cassava flour and maize samples from the Republic of Benin. All samples analyzed (fifteen peanut cakes, four maize flour and four cassava flour samples) tested positive for one or more mycotoxins. Aflatoxins (total aflatoxins; 10-346 μg/kg) and ochratoxin A (<LOQ-2 μg/kg) were detected in peanut cake samples while fumonisin B(1) (4-21 μg/kg), aflatoxin B(2) (<LOQ-8 μg/kg), aflatoxin B(1) (<LOQ-9 μg/kg), diacetoxyscirpenol (<LOQ-6 μg/kg) and zearalenone (<LOQ-12 μg/kg) were detected and quantified in cassava flour samples. Fumonisin B(1) (13-836 μg/kg), fumonisin B(2) (5-221 μg/kg), fumonisin B(3) (<LOQ-375 μg/kg) and beauvericin (<LOQ-25 μg/kg) were detected in the maize samples.

Children's exposure to mycotoxins and risk characterization using urinary biomarkers in São Paulo, Brazil.

Grape pomace (pulp and skins) was investigated as a new biosorbent for removing mycotoxins from liquid media. In vitro adsorption experiments showed that the pomace obtained from Primitivo grapes is able to sequester rapidly and simultaneously different mycotoxins. Aflatoxin B1 (AFB1) was the most adsorbed mycotoxin followed by zearalenone (ZEA), ochratoxin A (OTA), and fumonisin B1 (FB1), whereas the adsorption of deoxynivalenol (DON) was negligible. AFB1 and ZEA adsorptions were not affected by changing pH values in the pH 3-8 range, whereas OTA and FB1 adsorptions were significantly affected by pH. Equilibrium adsorption isotherms obtained at different temperatures (5-70 °C) and pH values (3 and 7) were modeled and evaluated using the Freundlich, Langmuir, Sips, and Hill models. The goodness of the fits and the parameters involved in the adsorption mechanism were calculated by the nonlinear regression analysis method. The best-fitting models to describe AFB1, ZEA, and OTA adsorption by grape pomace were the Sips, Langmuir, and Freundlich models, respectively. The Langmuir and Sips models were the best models for FB1 adsorption at pH 7 and 3, respectively. The theoretical maximum adsorption capacities (mmol/kg dried pomace) calculated at pH 7 and 3 decreased in the following order: AFB1 (15.0 and 15.1) > ZEA (8.6 and 8.3) > OTA (6.3-6.9) > FB1 (2.2 and 0.4). Single- and multi-mycotoxin adsorption isotherms showed that toxin adsorption is not affected by the simultaneous presence of different mycotoxins in the liquid medium. The profiles of adsorption isotherms obtained at different temperatures and pH and the thermodynamic parameters (ΔG°, ΔH°, ΔS°) suggest that mycotoxin adsorption is an exothermic and spontaneous process, which involves physisorption weak associations. Hydrophobic interactions may be associated with AFB1 and ZEA adsorption, whereas polar noncovalent interactions may be associated with OTA and FB1 adsorption. In conclusion, this study suggests that biosorption of mycotoxins onto grape pomace may be a reasonably low-cost decontamination method.

Data on the natural occurrence of Alternaria mycotoxins in apple by-products is lacking in Argentina and the risk of exposure to these mycotoxins has not been characterized before. The levels of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tenuazonic acid (TeA), tentoxin (TEN), altertoxin-I (ATX-I), altertoxin-II (ATX-II), alternariol 3-sulfate (AOH-3-S), alternariol 3-glucoside (AOH-3-G), alternariol monomethyl ether 3-sulfate (AME-3-S), and alternariol monomethyl ether 3-glucoside (AME-3-G) were determined in clarified and cloudy apple juices, marmalades, and apple-based infant food from the Argentinean market, and the risk of exposure was characterized. Detectable levels of AME, TEN, TeA, AME-3-S and AOH-3-G were found in clarified juices, while the same mycotoxins plus AOH were found in cloudy apple juices in higher concentrations. AME, TEN, TeA and AOH-3G were detected in marmalades, and AOH, AME, TEN and TeA in apple infant food. Probabilistic exposure assessment and risk characterization were carried out for children between 6 months and 5 years old in Argentina. The highest risk of exposure affected children between 6 and 23 months from the consumption of apple infant food and mainly associated with the alternariols. Better control strategies to prevent the incorporation of Alternaria mouldy core into the process line and the establishment of legislation for Alternaria mycotoxins are needed in Argentina.

ABSTRACTThere is an increasing awareness of the deleterious effects attributed to mycotoxins during their fate within the gut, particularly for deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), and patulin (PAT). Evidence indicates that disruption of the epithelial barrier is well established. However, intestinal barrier function on its luminal side involves two other partners, mucus and microbiota, which have rarely been considered in the context of mycotoxin exposure. The current review aimed at providing a summary of DON, ZEN, OTA, FB1, AFB1, and PAT effects on intestinal barrier function, with special focus on mucus and microbiota. DON, ZEN, OTA, FB1, AFB1, and PAT are known to markedly affect epithelial cell integrity and functions. Regarding mucus, DON is the most documentated mycotoxin. In vivo, toxicological impact of DON generally has only been assessed through goblet cell number. Evaluation of the mycotoxins/mucus interplay considering other indicators such as composition, thickness, and penetrability of mucus, mucin O-glycosylation thus warrants further attention. With respect to microbiota, few short-term studies to date have been reported indicating deleterious effects. However, long-term exposure to mycotoxins may also produce significant changes in microbiota composition and metabolic activity, which requires further experimentation. In conclusion, mucus and microbiota are key targets for dietary mycotoxins although assessment of induced effects is preliminary. A significant research effort is now underway to determine the adverse consequences of mycotoxins on mucus and microbiota considered as individual but also as tightly connected gut players.

Liver samples from finisher pigs were collected at the slaughterhouses for the analysis of zearalenone (ZEA), alfa-/beta-zearalenone (α-ZE, β-ZE), zearalanone (ZA), alfa-/beta-ZA (α-ZA, β-ZA), aflatoxin B1 (AFB1) and aflatoxin M1, fumonisin B1 (FB1), ochratoxin A (OTA) and ochratoxin B, deoxynivalenol and deepoxi-deoxynivalenol (DOM-1). For the analysis liquid chromatography-triple quadrupole coupled with mass spectrometry was applied. Liver samples with detected FB1 were further histopathologically evaluated after hematoxylin and eosin staining. Various levels of liver mycotoxins were detected in all farms. Pig livers with 2.91-8.30 μg/kg of FB1 were detected in three farms, estimate of 850-2400 μg/kg of FB1 intake, whereas 0.54 μg/kg of OTA was detected in one farm, estimate of 75 μg/kg of OTA intake. Moreover, pig livers with 0.30 μg/kg of ZEA, 1.87 μg/kg of α-ZE, and 0.63 μg/kg of β-ZE were detected in one farm, estimate with of 300 μg/kg of ZEA intake. The histopathological analysis revealed that the lesions' grading and necrosis grading were analogously increased when FB1 concentration increased from 2.91 to 4.36-8.30 μg/kg. The severity of megalocytosis was analogously increased with FB1 detection levels and particularly in levels of 4.36-8.3 μg/kg. However, the increased FB1 detection levels did not show analogous behavior with the severity of hepatic cell vacuolization. Results showed that FB1 remained the most critical risk factor in the Greek pig industry, whereas ZEA and AFB1 were also prevalent. The OTA contamination in pig farms raised a high risk for animal and human health.

High levels of mycotoxin contamination have been reported in various food commodities in Pakistan, however, there has been no exposure assessment study using multiple mycotoxins’ biomarkers. This study aimed to simultaneously assess the exposure to the five major mycotoxins: aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin B1 (FB1), ochratoxin A (OTA) and zearalenone (ZEN) in a Pakistani population using an integrated approach of human biomonitoring. Human urine samples (n = 292) were analyzed by a super-sensitive liquid-chromatography tandem mass spectrometry (LC-MS/MS) method. Rice and wheat were also collected and analyzed for mycotoxins by the LC-MS/MS method. Food consumption data were collected using a 24 h recall method. A high prevalence of urinary AFM1 (66%, mean ± SD 20.8 ± 41.3 pg/mL) and OTA (99%, 134.7 ± 312.0 pg/mL) were found, whilst urinary DON, FB1 and ZEN levels were low. The probable daily intake (PDI) derived from the urinary biomarkers revealed that 89% of the participants had exposure to OTA exceeding the established tolerable daily intake (TDI = 17 ng/kg bw/day). The average PDI of AFB1 for the studied population was 43 ng/kg bw/day, with rice as the main source of AFB1 exposure. In summary, exposure to AFB1 and OTA are of health concern and require further management.

Analysis of mycotoxins in coffee and risk assessment in Spanish adolescents and adults