| Summary: | A comprehensive strategy combining a quantitative method for 28 mycotoxins and a post-target screening for other 245 fungal and bacterial metabolites in dry pet food samples were developed using an acetonitrile-based extraction and an ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method. The proposed method showed satisfactory validation results according to Commission Decision 2002/657/EC. Average recoveries from 72 to 108% were obtained for all studied mycotoxins, and the intra-/inter-day precision were below 9 and 14%, respectively. Results showed mycotoxin contamination in 99% of pet food samples (<i>n</i> = 89) at concentrations of up to hundreds µg/kg, with emerging <i>Fusarium</i> mycotoxins being the most commonly detected mycotoxins. All positive samples showed co-occurrence of mycotoxins with the simultaneous presence of up to 16 analytes per sample. In the retrospective screening, up to 54 fungal metabolites were tentatively identified being cyclopiazonic acid, paspalitrem A, fusaric acid, and macrosporin, the most commonly detected analytes.
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