LC-MS/MS Determination of Cereulide in Food and Infant Formula: Method Validation and Six-Year Survey in Italy.

Among pathogenic microbes that can produce toxins, Bacillus cereus plays an important role in foodborne disorders. It is responsible for the production of cereulide, a cyclic dodecadepsipeptide toxin, very stable to heat and acidic pH, that can cause emetic syndromes. The study describes the secondary validation of the ISO 18465 method for food matrices in the concentration range 1-100 μg/kg and the in-house validation of a more sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for infant formula at the lower concentration levels required by the EFSA since 2026. To improve the applicability of cereulide determination in infant formula, the solid matrix was reconstituted before extraction and the analysis was performed on the SCIEX QTRAP 7500 system. The method showed good linearity (0.010-1.000 μg/L, R2 = 1000), a limit of quantification equal to the half-maximum level (LM = 0.054 μg/L, LOQ = 0.025 μg/L), good recovery (100-109%) and acceptable within-laboratory reproducibility (RSD% < 15%). This method provides a reliable analytical tool for cereulide monitoring in a broad variety of food commodities not considered before by ISO 18465 and makes it possible to reach the new lower limits established for infant formula. Furthermore, data on 332 samples involved in suspected food poisoning in Italy between 2019 and 2024 are presented in this retrospective study.