Determination of Urinary Mycotoxin Biomarkers Using a Sensitive Online Solid Phase Extraction-UHPLC-MS/MS Method

Using micro high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) a simple and fast method for the quantitative determination of 26 mycotoxins was developed. Sample preparation consists of a single extraction step and a dilute-and-shoot approach without further cleanup. With a total run time of 9 min and solvent consumption below 0.3 mL per chromatographic run, the presented method is cost-effective. All toxins regulated by the European Commission with maximum or guidance levels in grain products (fumonisins B1 and B2 (FB1 and FB2)); deoxynivalenol (DON); aflatoxins B1, G1, B2, and G2 (AFB1, AFG1, AFB2, and AFG2); ochratoxin A (OTA); T-2 and HT-2 toxins; and zearalenone (ZEN) can be quantified with this method. Furthermore, the enniatins B, B1, A, and A1 (EnB, EnB1, EnA, and EnA1); beauvericin (BEA); 3-acetyl-deoxynivalenol (3-AcDON); fusarin C (FusC); sterigmatocystin (STC); gliotoxin (GT); and the Alternaria toxins alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tentoxin (TEN), and altertoxin I (ATX I) can also be quantified. For all regulated compounds, recoveries ranged between 76 and 120%. For all other toxins, the recovery was at least 51%. The method was applied for the analysis of 42 maize samples from field trials in South Africa.

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.

Human exposure assessment to deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone (ZEA) and ochratoxin A (OTA) can be performed by measuring their urinary biomarkers. Suitable biomarkers of exposure for these mycotoxins are DON + de-epoxydeoxynivalenol (DOM-1), aflatoxin M1 (AFM1), FB1, ZEA + α-zearalenol (α-ZOL) + β-zearalenol (β-ZOL) and OTA, respectively. An UPLC-MS/MS multi-biomarker method was used to detect and measure incidence and levels of these biomarkers in urine samples of 52 volunteers resident in Apulia region in Southern Italy. The presence of ZEA + ZOLs, OTA, DON, FB1 and AFM1 were detected in 100%, 100%, 96%, 56% and 6%, of samples, respectively. All samples contained biomarkers of two or more mycotoxins. The mean concentrations of biomarkers ranged from 0.055 ng/mL (FB1) to 11.89 ng/mL (DON). Urinary biomarker concentrations were used to estimate human exposure to multiple mycotoxin. For OTA and DON, 94% and 40% of volunteers, respectively exceeded the tolerable daily intake (TDI) for these mycotoxins. The estimated human exposure to FB1 and ZEA was largely below the TDI for these mycotoxins for all volunteers.

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

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.

Development and application of an efficient, accurate, and environmentally friendly liquid chromatography-tandem mass spectrometry method for the determination of five Alternaria toxins in wheat.

The determination of mycotoxin and metabolite concentrations in human and animal urine is currently used for risk assessment and mycotoxin intake measurement. In this study, pig urine (n = 195) was collected at slaughterhouses in 2012 by the Swedish National Food Agency in three counties representing East, South and West regions of Sweden. Urinary concentrations of four mycotoxins, (deoxynivalenol (DON), zearalenone (ZEA), fumonisin B1 (FB1), and ochratoxin A (OTA)), and four key metabolites, (deepoxy-deoxynivalenol (DOM-1), aflatoxin M1 (AFM1, biomarker of AFB1), α-zearalenol (α-ZOL), and β-zearalenol (β-ZOL)) were identified and measured by UPLC-MS/MS. Statistically significant regional differences were detected for both total DON (DON + DOM-1) and total ZEA (ZEA + α-ZOL + β-ZOL) concentrations in pig urine from the three regions. These regional differences were in good agreement with the occurrence of Fusarium graminearum mycotoxins (DON + ZEA) in cereal grains harvested in 2011 in Sweden. There were no statistically significant differences in FB1, AFM1 and OTA urinary concentrations in pigs from the three regions. The overall incidence of positive samples was high for total ZEA (99–100%), total DON (96–100%) and OTA (85–95%), medium for FB1 (30–61%) and low for AFM1 (0–13%) in the three regions. Urinary mycotoxin biomarker concentrations were used to estimate mycotoxin intake and the level of mycotoxins in feeds consumed by the monitored pigs. The back-calculated levels of mycotoxins in feeds were low with the exception of seven samples that were higher the European limits.

A magnetic beads (MBs)-assisted fluorescence aptasensing approach based on dual DNA tweezers and magnetic separation was established for the detection of ochratoxin A (OTA) and fumonisin B1 (FB1). A dual DNA tweezers structure with four ends linked with fluorophores (FAM, ROX) and quenchers (BHQ1, BHQ2) was designed, and produced the high initial fluorescence signals because of the long spatial distance between FAM and BHQ1, ROX, and BHQ2. Bio-aptamer/anti-aptamer of OTA and bio-aptamer/anti-aptamer of FB1 were respectively annealed to form dsDNA, and immobilized to MBs coated with streptavidin (SA). With the existence of OTA and FB1, OTA and FB1 preferentially bound with their respective bio-aptamers, which made anti-aptamers dissociate from dsDNA coupled on MBs. After magnetic separation, the dissociated anti-aptamers reacted with dual DNA tweezers, respectively, which made DNA tweezers close and the fluorescence was quenched. The linear ranges of approach for OTA and FB1 detection were 0.05-20ng/mL and 0.1-40ng/mL, respectively. The limit of detection for OTA and FB1 was 0.029ng/mL and 0.061ng/mL. The prepared MBs-assisted fluorescence aptasensing approach was applied to detect OTA and FB1 in spiked red wine and corn samples, which showed good recoveries between 92 and 106%.

Aflatoxin M1 (AFM1) is the only toxin with the maximum residue limit in milk, and ochratoxin A (OTA) represents a common toxin in cereals foods. It is common to find the co-occurrence of these two toxins in the environment. However, the interactive effect of these toxins on hepatoxicity and underlying mechanisms is still unclear. The liver and serum metabolomics in mice exposed to individual AFM1 at 3.5 mg/kg b.w., OTA at 3.5 mg/kg b.w., and their combination for 35 days were conducted based on the UPLC-MS method in the present study. Subsequent metabolome on human hepatocellular liver carcinoma (Hep G2) cells was conducted to narrow down the key metabolites. The phenotypic results on liver weight and serum indicators, such as total bilirubin and glutamyltransferase, showed that the combined toxins had more serious adverse effects than an individual one, indicating that the combined AFM1 and OTA displayed synergistic effects on liver damage. Through the metabolic analysis in liver and serum, we found that (i) a synergistic effect was exerted in the combined toxins, because the number of differentially expressed metabolites on combination treatment was higher than the individual toxins, (ii) OTA played a dominant role in the hepatoxicity induced by the combination of AFM1, and OTA and (iii) lysophosphatidylcholines (LysoPCs), more especially, LysoPC (16:1), were identified as the metabolites most affected by AFM1 and OTA. These findings provided a new insight for identifying the potential biomarkers for the hepatoxicity of AFM1 and OTA.

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.

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.

In order to understand the current situation of mycotoxin contamination and risk assessment of dietary exposure of coix seed (Coix lacryma-jobi) sold in Gansu Province, China, a total of 67 coix seed samples were collected from the local markets and supermarkets in 14 prefectures and cities in Gansu Province. Sixteen mycotoxins were determined by isotope dilution liquid chromatography-tandem mass spectrometry. The results showed that 37.3% of coix seed was contaminated by mycotoxins. Among them, fumonisin B1 (FB1), fumonisin B2(FB2), fumonisin B3 (FB3) and zearalenone (ZEN) had the highest prevalence in coix seed, 10.5%, 11.9%, 17.9% and 10.5%, respectively. Coix seed was susceptible to mixed contamination of various mycotoxins, which was often detected in the forms of an FB1, FB2, and FB3 combination (35.7%) and an ZEN-FBs combination (21.5%). In this study, the exposure assessment was evaluated separately according to the use of coix seed for food or clinical use. It was found that the EDI values of ZEN, FB1, FB2, FB3, AME (alternariol monomethyl ether), TeA (tenuazonic acid), and TEN (tentoxin) calculated from the intake of coix seed for both medicinal and edible purposes were less than their TDIs, indicating that the health risk of exposure to the above toxins caused by the consumption of coix seed was within an acceptable range.

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

Alternaria alternata is an important mycotoxin-producing species that occurs on cereals, sunflower seeds, oilseed rape, tomato, carrot, lentils, olives, various fruits, etc. A. alternata produces a number of mycotoxins, including alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), L-tenuazonic acid (TeA), and other less-toxic metabolites. The objective of this study was a toxicological characterization of A. alternata strains isolated from wheat (34 strains) and grapes (11 strains). All the A. alternata strains were cultured on rice to evaluate their toxigenic potential (incubated in the dark at 25°C for 21 days). An instrumental high-performance thinlayer chromatography (HPTLC) for the quantification of ALT, AOH, AME, and TeAin rice cultures has been developed. The mycotoxins were extracted with methanol and ammonium sulfate in water and the extracted solutions were further cleaned up by liquid-liquid extraction. The silica gel layers pre-coated with oxalic acid in methanol were used ...

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.