Academic Editor: Ping-Chung Kuo Received: 27 February 2020; Accepted: 3 April 2020; Published: 7 April 2020

**Abstract:** *Alternaria* toxins have gained attention as a potential health risk and can be classified as emerging mycotoxins. As a result, they are candidates to be regulated by the European Commission. This paper describes a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for analyzing five *Alternaria* toxins in sunflower oil, which is a rather different type of sample to those matrices investigated in earlier published papers. An optimal sample preparation condition was achieved when samples were dissolved in *n*-hexane and extracted with methanol/water mixture, followed by sample pre-concentration with solvent evaporation. This study is the first focusing only on this lipophilic matrix and in using all corresponding isotopically labeled internal standards (ISTD) to compensate the matrix effect that strongly influences the LC-MS/MS analysis of toxins. Target compounds were separated on Zorbax Extend C-18 column enabling the analysis at alkaline pH of 8.8 that was necessary to obtain appropriate peak shape of tenuazonic acid and to separate the analytes at baseline. The method was validated according to the EU 2002/657/EC Decision and all the analytical performance characteristics met the requirements. The recovery was between 74% and 122% in fortified sunflower oil samples and the precision varied from 9% to 22%. The method was successfully demonstrated for sunflower seed quality check (QC) samples. Finally, 16 different sunflower oil samples were measured; and tenuazonic acid and tentoxin toxins were detected at levels close to LOQ concentrations.

**Keywords:** *Alternaria* toxins; LC-MS/MS; isotope dilution; sunflower oil; validation; real sample analysis
