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

The multi-biomarker approach was used to validate urinary biomarkers in piglets administered with boluses contaminated with different doses of a mixture of deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone (ZEA) and ochratoxin A (OTA). Boluses contaminated with target levels of mycotoxins were prepared by slurrying with water and freeze-drying feed material fortified with extracts of culture materials of selected toxigenic fungi. Piglets were individually placed in metabolic cages to collect urine before gavage and in 24 h urine post dose. Urine samples were hydrolysed with ?-glucuronidase enzyme 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 (DOM-1), ZEA + alpha-zearalenol (?-ZOL) and aflatoxin M1 (AFM1), were selected as biomarkers of FB1, OTA, DON, ZEA and AFB1, respectively. Mean percentages of dietary mycotoxins excreted as biomarkers in the 24 h post-dose urines 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 the relevant biomarkers excreted in 24 h post-dose urines. Linear dose-response correlation coefficients ranged between 0.68 to 0.78 for the tested couples 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 piglet at dietary levels close to the maximum permitted levels reported in the directive 2003/100/EC for AFB1 and the guidance values reported in the recommendation 2006/576/EC for DON, ZEA, OTA and FB1.