*4.1. Standards, Reagents, Equipment, Samples*

Tenuazonic acid (TEA), altenuene (ALT), alternariol (AOH), tentoxin (TEN), and alternariol monomethyl ether (AME) analytical standards were obtained from Romer Labs (Tulln, Austria) and individual 100 <sup>μ</sup>g/mL stock solutions in methanol were prepared and then kept at <sup>−</sup>18 ◦ C for a year. The isotopically labeled analogues (ISTDs) were purchased from ASCA GmbH (Berlin, Germany). An ISTD mixture containing TEA-13C2 (2.5 μg/mL), ALT-d6 (1 μg/mL), AOH-d3 (0.5 μg/mL), TEN-d3 (0.5 μg/mL), and AME-d3 (0.5 μg/mL) in methanol was prepared and stored at −18 ◦C for a half year. Methanol, acetonitrile, *n*-hexane, ethyl acetate, ammonia (25%), acetic acid, and ammonium acetate were either of LC-MS or HPLC grade, purchased from the Merck-Sigma group (Schnelldorf, Germany). The PTFE syringe filters (13 mm, 0.45 μm), Strata-XL-C mixed-mode polymeric strong cation exchange SPE cartridges (3 mL, 200 mg), and Strata-Si-1 silica SPE cartridges (6 mL, 500 mg) were acquired from Gen-lab Ltd. (Budapest, Hungary). The LC-MS/MS analysis was carried out by an Agilent 1100 HPLC pump (Agilent; Waldbronn, Germany), which was coupled to an AB Sciex 3200 QTRAP triple quad MS detector, equipped with a Turbo Ion Spray APCI or ESI sources (Sciex; Warrington, Cheshire, UK). Data acquisition and evaluation were performed using the Analyst software version 1.5.2. (Sciex; Warrington, Cheshire, UK). Sample shaking and centrifugation were done using horizontal shaker SM 30 B (Edmund Bühler, Bodelshausen, Germany) and Jouan B4i centrifuge (Thermo Fisher Scientific, Budapest, Hungary), respectively. Sunflower oil samples of different brands and lots were purchased at local shops and originated from the EU. Three sunflower quality check (QC) samples (i.e., W54 NC, C08 SP, and Q25 SP) were leftovers from MVS organized by JRC in 2018. The stability of toxins in both food samples and sample extracts was studied by JRC during the MVS in 2016 and 2018 [6,7]. *Alternaria*

toxins are stable at least up to 4 months in samples and they do not degrade in the autosampler in the aqueous injection solution during validation.

#### *4.2. Sample Extraction*

Sunflower oil samples (2.00 g) were weighed in polypropylene (PP) centrifuge tubes, followed by the addition of 2 mL *n*-hexane. The oils were completely dissolved in the tubes by vortex-mixing for 5 s. The sample-to-hexane ratio was 1.0. Then, 8 mL methanol–water (80/20, *v*/*v*) mixture was added to the samples and the tubes were capped, followed by vortex-mixing for 5 s. The sample-to-extraction solvent ratio was 4.0. Afterwards, the samples were shaken for 45 min at 180 rpm at ambient temperature. Then, the extracts were centrifuged at 4000× *g* for 2 min at ambient temperature and the hexane layer was discarded.
