Microfuge r

The wheat was grinded in a Retsch Ultra-Centrifugal mill ZM200 (Haan, Germany) at a particle size of 0.5 mm. Homogenised dry wheat meal (2.5 g) was weighed in duplicates into a 50-ml Falcon tube and extracted using 5 ml H₂O and 5 ml ACN 0.1% FA. Following, the samples were shaken for 30 min, centrifuged (30 min; 5833×g; 10 °C) and 1 ml of supernatant was taken out to which 100 μl IS and 100 μl H₂O was added. Phase separation of organic and water phase was achieved by addition of 250 mg anhydrous MgSO₄, shaking for 30 s (VXR B, IKA) and centrifugation at 17,530×g for 10 min at 10 °C (Microfuge R, Beckmann). Supernatant (300 μl) was transferred into a HPLC crimp vial, mixed with 300 μl H₂O and either directly measured or stored at 3 °C for up to 6 days.

To improve the accuracy of the measurements, for the analytes for which U-[¹³C]-labelled homologues were available, standard isotope dilution assay (SIDA) was performed. In the case of the sulfates derivates, matrix-matched calibration was carried out. For that, several blank oat samples were extracted by weighting 5 g of oat flour and mixing it with 10 mL acetonitrile and 10 mL water, shaking for 30 min (Multi Reax, Heidolph Instruments GmbH and Co.KG, Schwabach), centrifuging at 3800 g for 30 min (Megafuge 16, Thermo Fisher Scientific, Braunschweig, Germany), collecting 1 mL of the supernatant and mixing it with 250 mg of anhydrous magnesium sulfate. Then, the samples were centrifuged at 17,000 g for 5 min (Microfuge R, Beckmann, Krefeld, Germany) and all the resulting extract collected in a vial for further use.
Finally, three eight-point series of calibration solutions were prepared: one containing ZEN (8.008–500.5 ng/mL), α-ZEL (1.1047–69.074 ng/mL), β-ZEL (2.776–173.5 ng/L) and their respective U-[¹³C]-labelled homologues, dissolved in acetonitrile:water (25:75, v/v); one containing a series of calibration solutions of ZEN-14-S (0.858–546.25 ng/mL) and another one containing α-ZEL-14-S (0.853–947.1 ng/mL) and β-ZEL-14-S (0.6092–676.35 ng/mL), both dissolved in blank matrix extract (25%).

Larval samples were grinded in a Planetary Micro Mill, Pulverisette 7 premium line (FRITSCH GmbH, Idar-Oberstein, Germany). A variation on the method for multi mycotoxin extraction in wheat was used for larval and residue extractions. Two hundred milligrams of either grinded larvae or residue was weighed in duplicates into a 7-ml glass vial and extracted using 1.5 ml H₂O and 1.5 ml ACN 0.1% FA. Following, the samples were mixed for 10 min in an ultrasonic bath, shaken for 30 min, centrifuged (30 min; 5833×g; 10 °C) and 1 ml of supernatant was taken out to which 100 μl IS and 100 μl H₂O was added. Phase separation of organic and water phase was achieved by addition of 250 mg anhydrous MgSO₄, shaking for 30 s (VXR B, IKA) and centrifugation at 17,530×g for 10 min at 10 °C (Microfuge R, Beckmann). Three hundred microliters of supernatant was transferred into a HPLC crimp vial, mixed with 300 μl H₂O and either directly measured or stored at 3 °C for up to 6 days. Due to varying dilutions used, the LOQs for this method were calculated to be DON: 251 μg/kg, ZEN: 40.9 μg/kg; α-ZEL is 42.0 μg/kg. Additionally, the LOD for DON is 76.1 μg/kg, for ZEN is 12.4 μg/kg and for α-ZEL is 12.8 μg/kg.