A validated multi-residue ultra-high-performance liquid chromatography-high-resolution mass spectrometry method for emerging mycotoxins in pork, liver and pig feed

Emerging mycotoxins produced by Fusarium and Alternaria species, such as enniatins (ENN), beauvericin (BEA), alternariol (AOH), and alternariol monomethyl ether (AME), are increasingly detected along the feed-food chain and raise growing concerns for animal health and food safety. Despite their widespread occurrence, regulatory limits are currently lacking, mainly due to insufficient occurrence and exposure data, highlighting the need for robust analytical methods applicable to both feed and edible animal tissues. In this study, a multi-residue ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method was developed and validated for the simultaneous determination of seven emerging mycotoxins (AOH, AME, BEA, ENA, ENA1, ENB, and ENB1) in pork, liver, and pig feed. Sample preparation was based on solvent extraction followed by a defatting step, while chromatographic separation was achieved using a reversed-phase column coupled to Orbitrap high-resolution mass spectrometry operating in full-scan and product ion scan modes. Method validation was performed according to Commission Implementing Regulation (EU) 2023/2782, evaluating selectivity, linearity, precision, recovery, and sensitivity. Excellent linearity was obtained for all analytes in all matrices, with coefficients of determination >0.99. Recoveries ranged from 70 to 91% in liver, from 72 to 95% in muscle and from 73 to 120% in feed, while intra- and inter-day precision values were consistently below 15%. The limit of quantification was established at 1 ng g⁻¹ for all compounds. Overall, the validated UHPLC-HRMS method proved to be sensitive, selective, and reproducible, providing a reliable analytical tool for monitoring emerging mycotoxins in swine production and associated feedstuffs and supporting future occurrence studies, exposure assessments, and risk evaluation activities along the agri-food chain.