Special focus was given to the estimation of urinary excretion rates and the direct determination of major conjugation products. Methanol (LC gradient grade) and glacial acetic acid (p.a.) were purchased from find more Merck (Darmstadt, Germany), acetonitrile (ACN, LC gradient grade) from VWR (Leuven, Belgium), creatinine from Sigma

(Schnelldorf, Germany). Deoxynivalenol-3-O-glucuronide and zearalenone-14-O-glucuronide were synthesized by optimized procedures as described elsewhere ( Fruhmann et al., 2012 and Mikula et al., 2012). Other standards were purchased from Sigma (ZEN, α- and β-ZEL) and Romer Labs Diagnostic GmbH Tulln, Austria (DON, 13C15-DON, deepoxy-DON, nivalenol, T-2 toxin, HT-2 toxin, ochratoxin A, aflatoxin M1, fumonisins B1 and B2). Solid standard substances were dissolved in pure methanol (DON-3-GlcA, nivalenol) or ACN (DON, ZEN-14-GlcA, ZEN, α- and β-ZEL). All other standards were delivered in ACN or ACN/H2O (fumonisins B1 and B2) and stored at −20 °C. A combined multi standard working solution for preparation of calibrants and spiking experiments was prepared in ACN containing 10.0 mg/L DON, IDH inhibitor DON-3-GlcA, deepoxy-DON, nivalenol and HT-2, 5.0 mg/L fumonisin B1 and B2, 2.5 mg/L ZEN-14-GlcA, α-ZEL, β-ZEL and T-2, 1.0 mg/L ZEN and 13C15-DON and 0.125 mg/L aflatoxin M1 and ochratoxin A. DON-15-GlcA was separated

and subsequently fractionated from a highly contaminated human urine

sample which contained both, DON-3-GlcA and DON-15-GlcA (Warth et al., 2012a). Sulfate conjugates were not included in the study FER due to a lack of reference standard (DON-sulfate) and poor chromatographic behavior on the used chromatographic column (ZEN-sulfate). Enzymatic hydrolysis of the samples was done using β-glucuronidase from Escherichia coli (Type IX-A, Sigma). 500 μL urine were mixed with 500 μL PBS buffer (75 mM, pH 7.4) containing 3000 units of β-glucuronidase and incubated at 37 °C for 18 h. Digested samples were centrifuged and 200 μL of the supernatant was diluted with 800 μL dilution solvent to result in a total dilution factor of ten like the untreated samples. The study was conducted on a 27 year old, healthy male volunteer who consumed a special diet over a period of eight days as displayed in Fig. 1. On the first and the last two days the person ingested a mycotoxin reduced diet, which was based on rice, vegetables, fruits and milk products to reach Fusarium mycotoxin blank levels in excreted urine. During the four days in between, a diet naturally contaminated with high levels of DON was consumed, with exactly the same servings each day at the same times. This DON intervention diet consisted of cereals with wheat bran for breakfast, maize porridge (including maize flour) for lunch and bread, beer and pop-corn in the evening.