*4.7. LC-ID-MS*/*MS Separation*

Toxins were separated on a Zorbax Extend C-18 (150 mm × 3 mm, 5 μm) HPLC column (Agilent; Waldbronn, Germany) using a binary gradient elution. Solvent A contained 5.0 mM ammonium acetate

in water (pH adjusted to 8.8 with ammonium hydroxide) and solvent B was methanol. The mobile phase consisted of 10% B at 0 min; 10% B at 1 min; 100% B at 10 min; 100% B at 14.0 min; 10% B at 14.1 min. Stop time was 21 min. The flow rate was 0.5 mL/min. The column thermostat was maintained at 30 ◦C. The injection volume was 10.0 μL. Compounds were detected in APCI negative ionization mode and using multiple reaction monitoring (MRM) scan type in the triple quadrupole MS/MS instrument. The ion transitions are given in Table 1. The LC-ID-MS/MS analysis was carried out using all corresponding isotopically labeled ISTDs. The ISTDs were employed to compensate the signal suppression/enhancement in the ion source (matrix effect) caused by the co-eluting matrix constituents.

During the method development, the positive ionization mode (Table 1) with both APCI and ESI sources and the ESI negative ionization mode were also tested. When the positive ionization was employed in the ion source, the separation was performed with 0.3% (*v*/*v*) acetic acid in water (mobile phase A) and 0.3% (*v*/*v*) acetic acid in methanol (mobile phase B) eluent composition using the same gradient elution as written above.

The ion source settings were as follows: nebulizer current (only with APCI ion source): −4 (negative ion mode) or 4 (positive ion mode); drying gas temperature: 600 ◦C; nebuliser pressure: 30 unit; drying gas flow: 30 unit; curtain gas: 20 unit; capillary voltage: −4200 V (negative ion mode) or + 5000 V (positive ion mode); collision gas (N2): medium unit; interface heater: on.

The optimal ion transitions are given in Table 1, and the optimal ionization mode was APCI negative. These detection parameters were used during validation and real sample analysis.

**Supplementary Materials:** The following are available online. Table S1: Existing LC-MS/MS methods for *Alternaria* toxins; Table S2: The validation results for Alternaria toxins in sunflower oil samples and the results of analyzing sunflower seed QC samples by the modified method.

**Author Contributions:** Methodology, Á.T. and L.K.; validation, L.K. and Á.T.; formal analysis, L.K. and Á.T.; investigation, L.K. and Á.T.; writing—Original draft preparation, Á.T., V.K.S. and L.K.; writing—Review and editing, Á.T., V.K.S. and L.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** Authors like to thank Carlos Gonçalves for providing the QC samples. We thank Mingbao Feng for his comments on the revised manuscript. We thank the anonymous reviewers for their comments, which improve the manuscript greatly.

**Conflicts of Interest:** The authors declare no conflict of interest.
