Mycotoxins are compounds produced by several fungi that contaminate agricultural fields and, either directly or by carry-over, final food products. Animal exposure to these compounds through contaminated feed can lead to their excretion into milk, posing threats to public health. Currently, aflatoxin M1 is the sole mycotoxin with a maximum level set in milk by the European Union, as well as the most studied. Nonetheless, animal feed is known to be contaminated by several groups of mycotoxins with relevance from the food safety point of view that can be carried over into milk. To evaluate the multi-mycotoxin occurrence in this highly consumed food product it is crucial to develop precise and robust analytical methodologies towards their determination. In this sense, an analytical method for the simultaneous identification of 23 regulated, non-regulated, and emerging mycotoxins in raw bovine milk using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) was validated. A modified QuEChERS protocol for extraction purposes was used, and further validation was performed by assessing the selectivity and specificity, limits of detection and quantification (LOD and LOQ), linearity, repeatability, reproducibility, and recovery. The performance criteria were compliant with mycotoxin-specific and general European regulations for regulated, non-regulated, and emerging mycotoxins. The LOD and LOQ ranged between 0.001 and 9.88 ng mL⁻¹ and 0.005 and 13.54 ng mL⁻¹, respectively. Recovery values were between 67.5 and 119.8%. The repeatability and reproducibility parameters were below 15 and 25%, respectively. The validated methodology was successfully applied to determine regulated, non-regulated, and emerging mycotoxins in raw bulk milk from Portuguese dairy farms, proving the importance of widening the monitoring scope of mycotoxins in dairy products. Additionality, this method presents itself as a new strategic and integrated biosafety control tool for dairy farms for the analysis of these natural and relevant human risks.

Mycotoxins are abiotic hazards whose contamination occurs at the pre- and post-harvest stages of the maize value chain, with animal exposure through contaminated feed leading to their excretion into milk. Currently, only aflatoxin M1 is regulated in milk products. Since feed materials and complete feed present a multi-mycotoxin composition and are the main mycotoxin source into milk, it is important to recognize the occurrence of multiple toxins and their co-occurrence in this highly consumed food product. The aim of this study was to determine the content of regulated and emerging mycotoxins in milk samples, which allowed for evaluating the occurrence and co-occurrence patterns of different mycotoxins known to contaminate feed materials and complete animal feed. Human exposure considering the occurrence patterns obtained was also estimated. Aflatoxins, fumonisins, zearalenone, and emerging mycotoxins were among the mycotoxins found to be present in the 100 samples analyzed. Concentrations ranged from 0.006 to 16.3 μg L⁻¹, with no sample exceeding the AFM1 maximum level. Though several mycotoxins were detected, no exceeding values were observed considering the TDI or PMTDI. It can be concluded that the observed exposure does not pose a health risk to milk consumers, though it is important to recognize vulnerable age groups.

Milk is one of the most widely consumed foods in the world, despite the increasing consumption of plant-based alternatives. Although rich in nutrients and believed by consumers to be free of undesirable contaminants, milk, whether of animal or plant origin, is not always free from residues of chemical substances, including veterinary medicines. For instance, in intensive livestock production, antibiotics are often used to treat animals or, illicitly, to improve their growth performance, which can lead to their presence in the final food. Additionally, the continuous use of veterinary drugs in intensive animal production can lead to their occurrence in agricultural soils and therefore are absorbed by plants as another source of entering the food chain. An effective and accurate multi-detection quantitative screening method to analyze 89 antibiotics in milk was optimized by ultra-high-performance liquid chromatography coupled with a time-of-flight detector (UHPLC-ToF-MS) and further validated in accordance with the Commission Implementing Regulation (CIR) 808/2021 and the International Council for Harmonization (ICH) guidelines on the validation of analytical procedures. Apart from the specific parameters required by CIR 808/2021, the aim was to access the lower limits of the method, limits of detection (LoD) and quantification (LoQ), regardless of the maximum residue limits (MRLs) defined in the legislation. The method was then applied in the analysis of 32 supermarket samples, resulting in four positive findings, including one plant-based sample. The antibiotics found were from the macrolides and sulphonamides families. Nevertheless, the concentrations detected were below the established maximum residue level (MRL).