**2.2. Chemicals, standards and reference materials**

T-2 toxin (Art. No. 34071, 100 μg/mL in acetonitrile) and HT-2 toxin (Art. No. 34136, 100 μg/ mL in acetonitrile) standards were provided by Sigma-Aldrich Chemie GmbH (Steinheim, Germany). A RIDASCREEN® T-2/HT-2 toxin kit (Art. No. R3805) was provided by R-Biopharm (Darmstadt, Germany). PuriTox Total Myco-MS solid phase clean-up columns (Art. No. TC-MT3000) were produced by R-BiopharmRône LTD (Glasgow, Scotland). The Certified Reference oat flour Material (CRM) (Art. No. TET039RM) having the reference values of 85.3 ± 13.7 μg/kg for T-2 toxin and 86.9 ± 11.9 μg/kg for HT-2 toxin, was purchased from Fapas, Fera Science Ltd. (York, UK).

All chemicals used for ELISA and LC-MS/MS analyses were of an analytical grade (acetic acid, Art. No. 33209, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) or a HPLC grade (acetonitrile, Art. No. 34851, and methanol, Art. No. 34885 Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Ultrapure water was supplied by the Merck system Direct-Q 3 UV (New Jersey, USA).

#### **2.3. Analytical methods**

All samples were first analysed using the validated ELISA method. After grinding, to 5 g of a homogenised sample, 25 mL of methanol/distilled water solution (70/30; v/v) were added, except for oat samples, to which 25 mL of the appropriate extraction buffer provided with the ELISA kit were added. The extraction of T-2/HT-2 toxin was performed using a headover-head shaker for 10 min (100 rpm, room temperature) followed by an additional 10-min centrifugation (4000 rpm, room temperature). When it comes to oat samples, the obtained supernatant was diluted in methanol/distilled water (70/30; v/v) in 1:2 ratio, while the supernatant obtained with all other biological materials under study was diluted in distilled water in 1:2 ratio. The obtained solutions were then transferred into the wells of the ELISA microtitration plate. The ELISA tests were performed using a ChemWell autoanalyser (Awareness Technology Inc. 2910, Palm City, USA), observing thereby the instructions given by the kit provider. Once the stop solution had been injected, the absorbances were determined at 450 nm. In order to calculate the summary T-2/HT-2 concentration in an individual sample, the results provided by the calibration curve were multiplied by the corresponding sample dilution factor. The calculation of the summary toxin concentrations was guided by the average recovery values ascertained by method validation.

Samples, in which T-2/HT-2 concentrations higher than the ELISA's Limit of Detection were established, were further analysed using the LC-MS/MS method. To that effect, to 2.5 g of a test sample, 10 mL of 80%-acetonitrile were added and vortexed for 30 s; afterwards, the samples were put on a head-over-head shaker for 10 min (100 rpm, room temperature). The samples were then centrifuged (10 min, 4000 rpm, room temperature). Two millilitres of the obtained supernatant were acidified with 20 μL of (glacial) acetic acid; after that, 1.4 mL of the obtained solution was passed through the PuriTox Total Myco-MS columns (R-Biopharm, Glasgow, Scotland). Five hundred microliters were then evaporated under a nitrogen stream at 40°C and reconstituted in 250 μL of 1%-acetic acid in 20% acetonitrile. Fifty microliters were injected onto the LC-MS/MS system. The LC-MS/MS system consisted of a degasser, a binary pump, an auto-sampler and a column compartment (Infinity 1260, Agilent Technologies, Santa Clara, USA) coupled with a triple quadrupole mass detector (6410 QQQ, Agilent Technologies, Santa Clara, USA). The chromatography separation was performed on an XBridge BEH C18 column (particle size 2.5 μm, dimensions 4.6 × 150 mm) (Waters, Milford, Massachusetts, USA). The mobile phase consisted of 0.1%-acetic acid (constituent A) and acetonitrile (constituent B). The flow rate was 0.8 mL/min and the temperature was set at 40°C. A gradient elution program was employed: 0–3 min 90% A, 18 min 10% A, 18.1 min 90% A with a 3-minute post-run time. Mass spectrometry conditions were as follows: electrospray ionisation (ESI), positive polarity, source temperature 350°C, gas flow 9 L/min, nebulizer 45 psi, and capillary voltage 6 kV. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. **Table 1** shows the ions monitored within the frame of LC-MS/MS analyses targeted at T-2 and HT-2 toxin. The final concentrations of T-2 and HT-2 toxin were calculated based on the dilution factor and the average recovery values obtained during the validation process.
