Quantification of aflatoxins and sterigmatocystin in walnuts, cashews, pistachios, peanuts, and hazelnuts by using UHPLC-MS/MS following Aspergillus flavus inoculation.

Nuts are widely consumed worldwide and valued for their high nutritional quality. Tree nuts are, however, prone to colonization by various fungal genera that can cause spoilage and lead to the formation of toxic secondary metabolites, among which mycotoxigenic Aspergillus (A.) species are of particular concern because they produce hepatotoxic aflatoxins (AFs). In this study, untreated, shelled walnuts, hazelnuts, cashews, pistachios, and peanuts were surface-sterilized, inoculated with different A. flavus strains and incubated under controlled conditions. The concentrations of AFs and sterigmatocystin (STC) in the edible kernels were quantified by an ultra-high-performance liquid chromatography method, coupled with tandem mass spectrometry (UHPLC-MS/MS). In walnuts inoculated with five A. flavus strains, toxin production was highly strain dependent: four strains produced AFB1 in a wide range from 0.18 µg/kg to > 11,000 µg/kg, three strains formed AFB2 (3.58-1,411 µg/kg), and three strains synthesized STC (0.26-262 µg/kg), whereas one strain did not generate any AFs or STC, and none of the strains produced AFG1 or AFG2. In pistachios, inoculation with strains AF70 and CBS119.62 did not result in detectable AF formation, in contrast to hazelnuts, cashews, and peanuts, on which these strains yielded measurable toxin levels (AFB1: hazelnuts 0.11-3.56 µg/kg, cashews 0.15-0.16 µg/kg, peanuts 0.06-17.9 µg/kg; AFB2: hazelnuts 0.13-0.18 µg/kg, cashews < LOQ, peanuts 0.26-7.59 µg/kg; STC: hazelnuts 0.08 µg/kg, peanuts 5.28 µg/kg). Overall, markedly higher concentrations of A. flavus toxins were detected in walnuts than in the other nut types, identifying walnuts as a particularly susceptible matrix and indicating that AF and STC contamination of walnuts may pose an increasing food-safety challenge under future climate-change scenarios.