*2.5. Mycotoxins Extraction and Analysis*

Before mycotoxin extraction, samples were dried at 60 ◦C for 24 h and then milled in a laboratory blender (Waring Commercial, Christian, UK). Mycotoxins accumulation of three replicate samples per combination of inoculation position and aw on alternate days (days 2, 4, 6, 8 and 10) were extracted by adding 500 μL of acetonitrile:water:formic acid (79:20.9:0.1, *v*:*v*:*v*) to 100 (± 10) mg of milled wheat and agitated for 90 min at 300 rpm at 25 ◦C on a rotary shaker (miniShaker VWR, Leighton Buzzard, UK). Then, samples were centrifuged for 10 min at 22,600 g (Centrifuge 5417S Eppendorf, Stevenage, UK), and 1 or 3 μL of the supernatant was injected into an Exion LC series HPLC linked to a 6500+ qTRAP-MS system in Electrospray Ionisation (ESI) mode (Sciex Technologies, Warrington, UK). An ACE 3-C18 column (2.1 × 100 mm, 3 μm particle size; Hichrom) with guard cartridge (4 × 3 mm, Gemini, Agilent) was conditioned at 40 ◦C. The elution gradient used water:acetic acid (*<sup>v</sup>*:*v*, 99:1, solvent A) and methanol:acetic acid (*<sup>v</sup>*:*v*, 99:1) (solvent B), both supplemented with 5 mM ammonium acetate. The 15 min-long gradient included: 0 min, 90% A; 0–2.0 min, 90–60% A; 2.0–10.0 min, 0% A and 10.0–11.50 min, 0% A; 11.50−12.0 min, 90% A; 12.00−15.0 min, 90% A at a 0.3 mL·min−<sup>1</sup> flow rate. 10 msec of dwell time per daughter ion (2 per metabolites) was used in an unscheduled Multiple Reaction Monitoring (MRM) in both positive and negative mode using the parameters listed in Table 1.



The source conditions used included: Curtain gas 40%, Collision Gas Medium, IonSpray voltage 4500 V/ 5500 V, Temperature 400 ◦C, both ion sources gas at 60 psi, with a 10 V Entrance Potential for all compounds. Data acquisition was conducted with Analyst® Data Acquisition version 1.6.3, and quantification through MultiQuant™ version 3.0.3.

The analyte recovery, the limit of detection (LOD) and the limit of quantification (LOQ) were calculated using recommendations stated elsewhere [22]. The calculated recoveries were used as correction factors to calculate the absolute concentration of the analytes. The LODs were 0.26 and 2.29 ng·g<sup>−</sup><sup>1</sup> and the LOQs 0.85 and 7.63 ng·g<sup>−</sup><sup>1</sup> for ZEN and DON, respectively.
