4.5. Method Validation

The linearity range, limit of detection (LOD, analyte concentration at which signal:noise = 3:1), limit of quantification (LOQ, analyte concentration at which signal:noise = 10:1), recovery rate, and method repeatability (expressed as relative standard deviation, RSD) were determined for each analyte to validate the analytical method developed in this study. To eliminate matrix effects that could suppress analyte ions, calibration curves were obtained using blank solutions of wheat and its green malt, with added internal ¹³C-labelled standards at nine concentrations spanning the following ranges: 83–2000 μg/kg for NIV, 33–792 μg/kg for NIV3G, 167–20,000 μg/kg for DON, and 83–6000 μg/kg for DON3G. The coefficient of determination (R²) exceeded 0.99 in all cases. If the concentration of an analyte in a sample exceeded the method’s upper limit of linearity for that analyte, the sample was diluted and re-analysed. Recovery rates (R) were determined on the basis of analyses of samples to which known amounts of analytes were added at four fortification levels (apart from the internal standards). These samples were processed in the same manner as the unknowns (Section 4.3). The analysed ions, retention times, LOD, and LOQ for individual analytes are shown in Table 3. Further, the recovery rate (R%) and method repeatability (RSD%) are shown in Table 4.

The analysed ions, retention times, LOD, and LOQ for individual analytes.

Recovery rate (R%) and method repeatability (expressed as relative standard deviation, RSD%) for individual analytes at four different fortification levels.

The European Commission has established some criteria for analytical methods for various mycotoxins, including DON. The regulation No. 401/2006 of 23 February 2006 [40] lays down methods of sampling and analysis for the official control of mycotoxin levels in foodstuffs. For concentrations of mycotoxins ranging from 100–500 µg/kg, the regulation requires DON recovery rates within 60–110%. For concentrations greater than 500 µg/kg, the DON recovery rates should be within 70–120%. RSD, which reflects the method’s precision, must not be higher than 20% for both concentration ranges. No criteria have been set in the regulation for the remaining analytes investigated in this study. However, they all are trichothecenes of a similar chemical structure, so we adopted the same criteria for them, as specified for DON. The method developed in this study meets these criteria (see Table 4).