*2.2. Chromatographic Separation*

Due to the different polarities of the analysed toxins, several chromatographic columns (Table S2) and mobile phases were tested. Based on the literature data, hydrophilic interaction liquid chromatography (HILIC) was tested as a first choice. Satisfactory separation on DON and DON-3Glc was achieved with Luna HILIC column (Phenomenex, Torrance, CA, USA), For other toxins no satisfactory retention times were obtained, and poor separation of all analytes was observed (Figure 1A). Kinetex C18 and Kinetex Biphenyl columns (Phenomenex) were also compared (Figure 1B,C). Although, these columns enable DON, DON-3Glc, NIV and FUS-X to be separated, 3Ac- and 15Ac-DON were not. In our study, the best results were achieved using a Luna Omega Polar C18 column (Phenomenex; 100 × 2.1; 1.6 µm) which is designed for polar compounds. Application of this column with MeOH as the organic mobile phase allowed all analytes to be separated except 3Ac and 15Ac-DON, their peaks still broadening (Figure 2A), and co-eluted.

**Figure 1.** LC–MS/MS chromatograms of analysed toxins, tested in the same mobile phase but at different chromatographic columns: (**A**) Phenomenex Luna HILIC (**B**) Phenomenex Kinetex Biphenyl 100 × 2.1mm, 1.7 µm; (**C**) Phenomenex Kinetex C18 100 × 2.6 mm, 2.1 µm.

To obtain full separation of peaks, ACN was used as an organic mobile phase with specific gradient mode (2–6 min with an almost isocratic gradient from 15–18% of ACN) (Figure 2B). Moreover, the choice of ACN shortened retention time for all analytes, as well as the time of analysis. To date a lot of studies have described the chromatographic separation of DON, its modified mycotoxins, and other type B Trichothecenes in different biological matrixes [13,14,16,18,27–34]. Nevertheless, most of the authors did not achieve baseline separation of acetylated forms of DON. Only a few studies described the full chromatographic separation of these compounds [17,35,36]. Yoshinari et al. (2013) obtained retention times for 3Ac-DON and 15Ac-DON of 5.48 and 5.60 min, respectively. In a different study Goncalves et al. [17] achieved partial separation of the isomeric form (both peaks co-eluted). Contrary results were demonstrated by Slododchikova et al. [37] who concluded that the best for separation of the isomeric forms of acetylated DON was a pentafluorophenyl column and MeOH as the mobile phase.

**Figure 2.** Chromatographic separation of tested compounds on the same column (Phenomenex Luna ® Omega C18 100 × 2.1, 1.6 µm) with different organic mobile phase: (**A**) MeOH; (**B**) ACN.

In conclusion, the usage of a Phenomenex Luna Omega Polar C18 column (100 × 2.1; 1.6 µm) column with the combination of 0.2% CH3COOH with ACN as the mobile phase in a specific gradient mode achieves full separation of all tested compounds with a total run time of 12 min (Figure 2B).
