Mycotoxins are important food contaminants responsible for health effects such as cancer, nephrotoxicity, hepatotoxicity or immunosuppression. The assessment of mycotoxin exposure is often based on calculations combining mycotoxin occurrence data in food with population data on food consumption. Because of limitations inherent to that approach, the direct measurement of biomarkers of exposure in biological fluids has been proposed as a suitable alternative to perform an accurate mycotoxin exposure assessment at individual level. For this reason, the BIOMYCO study was designed to assess mycotoxin exposure in Belgian adults and children using urinary biomarkers of exposure. Morning urine was gathered in a representative part of the Belgian population according to a standardised study protocol, whereby 155 children (3-12 years old) and 239 adults (19-65 years old) were selected based on random cluster sampling. These urine samples were analysed for the presence of 33 potential biomarkers with focus on aflatoxins, citrinin (CIT), fumonisins, trichothecenes, ochratoxin A (OTA), zearalenone and their metabolites using two validated LC-MS/MS methods. Nine out of the 33 analysed mycotoxins were detected whereby deoxynivalenol (DON), OTA, CIT and their metabolites were the most frequently detected. Deoxynivalenol-15-glucuronide was the main urinary DON biomarker and was found in all urine samples in the ng/mL range. Furthermore deoxynivalenol-3-glucuronide was quantified in 91% of the urine samples collected from children and in 77% of the samples collected from adults. Deoxynivalenol was detected in 70% and 37% of the samples of children and adults respectively. For the first time deepoxy-deoxynivalenol-glucuronide was detected in children's urine (17%). In the samples collected by adults, the prevalence was 22%. Whereas all these mycotoxins contaminated the urine samples in the ng/mL range, CIT and OTA were present in much lower concentrations (pg/mL). OTA contaminated 51% and 35% of the samples collected by children and adults respectively. CIT and its metabolite were present in 72% and 6% of children's urine, whereas they contaminated 59% and 12% of adult's urine. Finally, α-zearalenol and β-zearalenol-14-glucuronide were found in respectively one and two samples from adults. The exposure to DON, OTA and CIT was compared between subgroups and urinary mycotoxin concentrations differed significantly among age and gender. Based on the urinary levels, the daily intake of DON and OTA was estimated and evaluated whereby, depending on the used method, 16-69% of the population possibly exceeded the tolerable daily intake for DON and 1% for OTA. The BIOMYCO study is the first study whereby a multi-toxin approach was applied for mycotoxin exposure assessment in adults and children on a large-scale. Moreover, it is the first study that described the exposure to an elaborated set of mycotoxins in the Belgian population. Biomarker analysis showed a clear exposure of a broad segment of the Belgian population to DON, OTA and CIT. Th...

An LC-MS/MS method was developed and validated for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin, HT-2-toxin and metabolites, including 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol-3-glucoside, α-zearalenol, β-zearalenol, zearalenone-4-glucoside, α-zearalenol-4-glucoside, β-zearalenol-4-glucoside and zearalenone-4-sulfate in maize, wheat, oats, cornflakes and bread. Extraction was performed with acetonitrile/water/acetic acid (79/20/1, v/v/v) followed by a hexane defatting step. After filtration, the extract was evaporated and the residue was redissolved in mobile phase for injection. The mobile phase, which consisted of a mixture of methanol and water with 10 mM ammonium acetate, was adjusted to pH 3 with glacial acetic acid. A sample clean-up procedure was not included because of the low recoveries of free and masked mycotoxins and their differences in polarity. The method allowed the simultaneous determination of 13 Fusarium mycotoxins in a one-step chromatographic run using a Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer. The method was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, expanded measurement uncertainty and specificity. The limits of detection varied from 5 to 13 ng g⁻¹; those for the limit of quantification from 10 to 26 ng g⁻¹. The results of the performance characteristics of the developed LC-MS/MS method were in good agreement with the criteria mentioned in Commission Regulation (EC) No. 401/2006. Thirty samples of a variety of food and feed matrices were sampled and analysed between July 2010 and January 2011.

The aim of this study was to determine the toxicokinetic characteristics of ZEN and its modified forms, α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalenone-14-glucoside (ZEN14G), and zearalenone-14-sulfate (ZEN14S), including presystemic and systemic hydrolysis in pigs. Crossover pig trials were performed by means of intravenous and oral administration of ZEN and its modified forms. Systemic plasma concentrations of the administered toxins and their metabolites were quantified and further processed via tailor-made compartmental toxicokinetic models. Furthermore, portal plasma was analyzed to unravel the site of hydrolysis, and urine samples were analyzed to determine urinary excretion. Results demonstrate complete presystemic hydrolysis of ZEN14G and ZEN14S to ZEN and high oral bioavailability for all administered compounds, with further extensive first-pass glucuronidation. Conclusively, the modified-ZEN forms α-ZEL, β-ZEL, ZEN14G, and ZEN14S contribute to overall ZEN systemic toxicity in pigs and should be taken into account for risk assessment.

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A direct, fast and sensitive LC-MS/MS method was developed to measure biomarkers for mycotoxin exposure in human urine. In total, 32 biomarkers were quantitatively or semi-quantitatively measured in 32 urine samples of Belgian volunteers using two injections. All urine samples contained deoxynivalenol-15-glucuronide, the major detoxification metabolite of deoxynivalenol, in the ng/mL range. Also deoxynivalenol-3-glucuronide and de-epoxy-deoxynivalenol-glucuronide were present in, respectively, 90 and 25% of the samples, while deoxynivalenol was detected in 60% of the samples, in lower concentrations. Deoxynivalenol glucuronides were the major biomarkers for deoxynivalenol exposure. Ochratoxin A was detected in 70% of the samples in pg/mL. Citrinin and/or dihydrocitrinone were detected in 90% of the samples, also in concentrations of pg/mL. The presence of ochratoxin A and citrinin was confirmed by a second method using sample cleanup by immunoaffinity columns, followed by LC-MS/MS. Our data show that humans are much more exposed to citrinin than realized before and suggest further work on citrinin exposure in relation with ochratoxin A exposure, as both mycotoxins are nephrotoxic.