An analytical method for the simultaneous determination of trichothecenes-namely, nivalenol (NIV), deoxynivalenol (DON) and its acetylated derivatives (3- and 15-acetyl-DON), T-2 and HT-2 toxins-and zearalenone (ZEN) in wheat, wheat flour, and wheat crackers was validated through a collaborative study involving 15 participants from 10 countries. The validation study, performed within the M/520 standardization mandate of the European Commission, was carried out according to the IUPAC (International Union of Pure and Applied Chemistry) International Harmonized Protocol. The method was based on mycotoxin extraction from the homogenized sample material with a mixture of acetonitrile-water followed by purification and concentration on a solid phase extraction column. High-performance liquid chromatography coupled with tandem mass spectrometry was used for mycotoxin detection, using isotopically labelled mycotoxins as internal standards. The tested contamination ranges were from 27.7 to 378 μg/kg for NIV, from 234 to 2420 μg/kg for DON, from 18.5 to 137 μg/kg for 3-acetyl-DON, from 11.4 to 142 μg/kg for 15-acetyl-DON, from 2.1 to 37.6 μg/kg for T-2 toxin, from 6.6 to 134 μg/kg for HT-2 toxin, and from 31.6 to 230 μg/kg for ZEN. Recoveries were in the range 71-97% with the lowest values for NIV, the most polar mycotoxin. The relative standard deviation for repeatability (RSDr) was in the range of 2.2-34%, while the relative standard deviation for reproducibility (RSDR) was between 6.4% and 45%. The HorRat values ranged from 0.4 to 2.0. The results of the collaborative study showed that the candidate method is fit for the purpose of enforcing the legislative limits of the major Fusarium toxins in wheat and wheat-based products.

A strong trend toward using highly selective mass spectrometry technologies for screening of multiple mycotoxins has been observed in recent years. In the present study, the process of validation of a multimycotoxin screening method based on liquid chromatography-high-resolution mass spectrometry method is presented. The method was intended for the simultaneous screening of the major Fusarium toxins (deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, and B1, and beauvericin) in wheat. The sample preparation protocol was based on a double extraction (methanol followed by acetonitrile/water mixture) and purification through solid-phase extraction C18 column. To provide insights for full exploitation of the potential of the double-stage high-resolution mass spectrometry detection, a full-scan acquisition event followed by a sequence of 5 fragmentation events (variable data-independent acquisition) was set for mycotoxin detection, the latter to be exploited for confirmatory purposes. Method analytical performances were evaluated through in-house validation and small-scale interlaboratory study, designed according to Commission Regulation 519/2014/EU, setting performance requirements for screening methods for mycotoxins. Screening target concentrations were close to European Union maximum permitted or indicative levels. The in-house validation provided the precision of the response under repeatability conditions and the intermediate precision (both resulting lower than 30%), the cutoff value, and the rate of false suspect results for negative (free of the mycotoxin of interest) samples, which resulted lower than 0.1% in all cases. The collaborative study provided reproducibility and laboratory independent cutoff values. Analysis of reference materials proved method trueness and suitability for screening of the major Fusarium mycotoxins in wheat. Finally, the applicability of the full-scan/variable data-independent acquisition detection approach was successfully tested on a set of naturally contaminated wheat samples, where 2 characteristic product ions could be detected for all identified mycotoxins even at levels in the low μg/kg range.