*2.3. Sample Preparation for LC-MS*/*MS*

Twenty-two mycotoxins were extracted from the samples according to the methodology described by Monbaliu et al. [75]. Five grams of dried maize sample was spiked with internal standards ZAN and DOM at a concentration of 200 and 250 μg/kg, resp. The spiked sample was kept in the dark for 15 min and extracted with 20 ml of extraction solvent (acetonitrile/water/acetic acid (79/20/1, *v*/*v*/*v*)), and then agitated on a vertical shaker for 1 h. After centrifuging for 15 min at 3300 g, the supernatant was passed through a preconditioned C18 solid phase extraction (SPE) column (Alltech, Lokeren, Belgium). The eluate was diluted to 25 ml with extraction solvent and defatted with 10 ml n-hexane. In order to recover all 22 mycotoxins, two different clean-up pathways were followed. In the first pathway, 10 ml of extract was diluted with 20 ml acetonitrile/acetic acid (99/1 *v*/*v*), passed through a Multisep®226, AflaZon+ multifunctional column from Romer Labs (Tulln, Austria) and washed with 5 ml acetonitrile/acetic acid (99/1 *v*/*v*). For the second pathway, 10 ml extract was filtered using a Whatman glass microfilter (VWR International, Zaventem, Belgium). Two milliliters of this filtered extract was combined with the MultiSep 226 eluate from the first pathway and evaporated to dryness. The residue was then redissolved into 150 μL of mobile phase (water/methanol/acetic acid (57.2/41.8/1, *v*/*v*/*v*)) and 5 mM ammonium acetate. Lastly, the solution was centrifuged for 5 min at 14,000 ×*g* using ultra free-MC centrifuge filters (Millipore, Bedford, MA, USA).
