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.
Validation study on urinary biomarkers of exposure for aflatoxin B1, ochratoxin A, fumonisin B1, deoxynivalenol and zearalenone in piglets