**Determination of Zearalenone and Trichothecenes, Including Deoxynivalenol and Its Acetylated Derivatives, Nivalenol, T-2 and HT-2 Toxins, in Wheat and Wheat Products by LC-MS**/**MS: A Collaborative Study**

**Annalisa De Girolamo, Biancamaria Ciasca, Michelangelo Pascale and Veronica M.T. Lattanzio \*** Institute of Sciences of Food Production, National Research Council of Italy, via Amendola 122/O, 70126 Bari, Italy; annalisa.degirolamo@ispa.cnr.it (A.D.G.); biancamaria.ciasca@ispa.cnr.it (B.C.);

michelangelo.pascale@ispa.cnr.it (M.P.)

**\*** Correspondence: veronica.lattanzio@ispa.cnr.it

Received: 12 November 2020; Accepted: 8 December 2020; Published: 10 December 2020

**Abstract:** 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.

**Keywords:** trichothecenes; zearalenone; *Fusarium* toxins; wheat; liquid chromatography–mass spectrometry; official control; collaborative study

**Key Contribution:** A method for the determination of trichothecenes and zearalenone by high-performance liquid chromatography coupled with tandem mass spectrometry after clean-up by solid phase extraction has been validated in a collaborative study involving 15 participant laboratories following the provisions given in the AOAC/IUPAC International Harmonized Protocol. Results show 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.
