**3.1. Validation of analytical methods used**

The ELISA assay, used as a quantitative screening method for the determination of summary concentrations of T-2/HT-2 toxins, was first validated and then applied for the analyses of the sampled cereals. Its cross-reactivity declared by the kit manufacturer is approximately 85% for T-2 toxin and 100% for the HT-2 toxin. Validation of the ELISA method resulted in LODs ranging from 20.6 to 30.1 μg/kg and LOQs ranging from 26.7 to 37.4 μg/kg, depending on the type of cereal under consideration. The mean recovery value equalled to 90.1%, with the mean coefficient of variation (CV) of 7.8% (**Table 2**).

Validation of the LC-MS/MS method resulted in LODs spanning from 5.5 to 8.3 μg/kg, and LOQs ranging from 18.2 to 27.5 μg/kg for T-2 and HT-2 toxin, respectively. The mean recovery values were 89.6 and 77.0%, with the mean CV of 5.1% and 8.9%, respectively. The analyses of oat flour sample (CRM) used for the determination of trueness, resulted in concentrations of 77.9 μg/kg for T-2 (91.3% of the mean certified value) and 75.6 μg/kg for HT-2 (86.9% of the mean certified value). **Figure 1** presents a typical LC-MS/MS-MRM chromatogram of the analysed CRM. The determination of linearity resulted in correlation coefficients higher than 0.99 for both analytes (**Table 3**).

Given the obtained validation results, both analytical methods were recognised as suitable for analyses of different cereals, the ELISA assay thereby being employed as a screening method used for the determination of summary concentrations of T-2/HT-2 toxins, and the LC-MS/ MS thereby being used as a confirmatory method exploited to the effect of determination of individual concentrations of mycotoxins under study.
