## 2 Materials and Methods
### Validation of Screening and Confirmatory Methods  
The screening and confirmatory methods were validated in accordance with ISO/IEC 17025:2017 [18], taking the indications reported in Decision No. 2002/657/EC and Regulation EC No. 2017/625 as reference and some updated guidelines for analytical method validation [19; 20]. The full validation of two methods assured the compliance and conformity with the criteria established in Regulation EC No. 401/2006. Since 2016, these two methods have also been accredited by ACCREDIA, the Italian Organization for laboratory accreditation. The parameters considered during the validation of ELISA and UPLC-FLD analytical procedures were: selectivity, sensitivity, precision, recovery, and ruggedness. The measurement uncertainty was only estimated for UPLC-FLD, since the evaluation of this parameter is only compulsory for confirmatory methods. On the basis of the propagation of uncertainty law, the measurement uncertainty was calculated for each toxin by using the following equation:  
\[v=\sqrt{\left(v(C)\right)^{2}+v\left(V_{f}\right)^{2}+v(w)^{2}}\]  
where \(u\) indicates the relative uncertainty, \(C\) is the analyte concentration in the sample, \(V_{f}\) is the volume of the final extract, and \(w\) is the sample weight. The determination of \(v\) was performed by considering four sources of uncertainty: (a) standard preparation, (b) method reproducibility, (c) method recovery, and (d) calibration curve.  
For the selectivity and accuracy tests, the samples were spiked, prior to the extraction, with the proper amounts of the T-2/HT-2 toxin standard solutions for obtaining the required concentrations.  
Within the ELISA validation, the trust semi-interval was calculated by using the following equation:  
\[\pm\frac{t\cdot s_{0}}{\sqrt{n}}\]  
where \(s_{0}\) is the standard deviation of the signal obtained from the 20 sample blank tests, \(t\) is the Student's \(t\) value tabulated for 19 degrees of freedom (one-tail, \(p\) = 0.05), and \(n\) is the number of performed tests.  
Regarding UPLC/FLD, the limits of detection and quantification (LOD and LOQ, respectively) were calculated according to the following equations:  
\[LOD=3.9\times S_{b}/b\text{ and }LOQ=3.3\times X_{LOD}\]  
where \(S_{b}\) is the standard deviation of the pseudo-blank difference and \(b\) is the slope of the mean regression line. For selectivity evaluation, 20 blank samples (common wheat, durum wheat, oats, maize, and barley--4 each) were tested based on an immunoenzymatic assay to verify the absence of the T-2/HT-2 toxins.  
Method ruggedness was tested for both techniques by introducing seven minor changes in the analytical procedure (e.g., different centrifugation speeds and manual shaking time) and then assessing their influence on the final result. The effect of each factor was calculated by subtracting the mean result obtained using the variable at a high level (reference value) to the mean result at a low level (investigated factor).  
Validation results were statistically evaluated using of an ANOVA, [PERSON]'s exact test and Student's \(t\) test (\(p\) > 0.05). In 2019, the ELISA was submitted to a proficiency test round (Progetto Trieste, Mycotoxins 2019, round of October, test Veritas s.r.l., Trieste, Italy), and it obtained good results in quantifying the T-2 and HT-2 toxins in naturally contaminated common wheat (result: 185.5 \(\upmu\)g kg\({}^{-1}\); acceptability range: 89.5-220.9 \(\upmu\)g kg\({}^{-1}\)).