Mycotoxin occurrence and risk assessment in plant-based meat, cheese, and fish alternatives based on an adapted UHPLC-MS/MS multi-method

Plant-based alternatives to animal-derived products have gained significant prominence in recent years, with the market for these products experiencing continuous growth. In response to this trend, we developed an ultra-high-performance liquid chromatography method, coupled with tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous quantification of 15 mycotoxins from Fusarium (deoxynivalenol (DON), DON-3-glucoside (D3G), 3-acetyl-DON, 15-acetyl-DON, HT-2 toxin, T-2 toxin), Alternaria (alternariol, alternariolmonomethylether (AME), tenuazonic acid (TeA)), and Aspergillus species (aflatoxin (AF) B1, B2, G1, G2, sterigmatocystin (STC), ochratoxin A) in plant-based meat, cheese, and fish substitutes. Sample extraction was performed using a QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach, followed by dispersive solid phase extraction (dSPE) for clean-up. Quantification was achieved through stable isotope dilution analysis (SIDA). The method demonstrated limits of detection (LOD) ranging from 0.01 µg/kg (AFB2 and STC) to 14.1 µg/kg (D3G) and limits of quantification (LOQ) between 0.02 µg/kg (STC) and 41.9 µg/kg (D3G). This method was successfully applied to quantify mycotoxins in 32 plant-based alternative products, revealing contamination predominantly with Alternaria toxins such as AME and TeA. Seitan-based products were identified as having the highest levels of contamination. In total, 27 out of 32 analyzed products contained at least one of the target mycotoxins. With the obtained data, we performed a risk assessment and revealed a potential risk, especially for toddlers, but also for individuals of all ages, due to the presence of Alternaria toxins. However, none of the detected concentrations exceeded the established regulatory limits or recommended values, as plant-based alternatives are not specifically included in the relevant food safety regulations.