*2.4. Method Validation*

The method was successfully validated for all tested mycotoxins in feedstuffs (Table 1). Good specificity of the methods was confirmed by analysis of 20 pseudo-blank samples. No interference peaks (S/N > 3) were detected in the retention time (±2.5%) for targeted analytes. The determined LOD and LOQ for all analytes were in the 1.78–15.0 and 5.87–49.5 µg/kg ranges, respectively. These low LOD and LOQ for DON and its modified mycotoxins were comparable with those disclosed in other publications [23,44], or even lower [45]. The calibration curves were linear over the calibration range for all compounds, resulting in R<sup>2</sup> values between 0.998 and 0.999. The REC was determined for each toxin based on a sample fortified at three VL (0.5 × VL, 1.0 × VL, and 1.5 × VL) and was above 90%, which fulfilled established criteria [46]. All values for repeatability and within-laboratory reproducibility were in the range of 4–24%, showing good precision for all toxins. Moreover, RSDr was between 4% and 22%, indicating that this method could be adopted for a wide range of feedstuffs. The expanded measurement uncertainty U (%) was satisfactory at below 35% for all tested toxins, showing acceptable method performance. Due to the large variability and complexity of the analysed samples, significant ME was expected [47]. In our study, ME values ranged from 61% to 120%. To compensate for this enhanced or suppressed signal, matrix-matched calibration curves were used as well as IS for DON and DON-3Glc. The MIX IS was added after extraction only to compensate for possible ME, to limit use of the expensive IS. This result shows that the developed procedure can be applied as a confirmatory method for determination of DON, its metabolites and others type B trichothecenes in feedstuffs.
