Casein determination in açaí: method validation and estimation of measurement uncertainty

Milk allergy is one of the most prevalent adverse food reactions, representing a relevant risk to public health. Accidental ingestion of traces of milk can trigger severe reactions in sensitized individuals, requiring sensitive, specific, and reliable analytical methods for their detection. The validation of these methods is essential, especially in complex food matrices, with a high risk of cross-contamination and of commercial relevance, such as açaí. According to RDC 512/2021/ANVISA, methods used in the analysis of products subject to health surveillance must be verified or validated under the conditions of use, demonstrating their suitability for the purpose. This study aimed to validate an enzyme-linked immunosorbent method (ELISA, R-Biopharm^® R4612) for the quantification of casein in frozen açaí, a food naturally free of dairy ingredients, aiming to expand the analytical scope of the Central Laboratory of Public Health of Minas Gerais (LACEN-MG) to evaluate the conformity of allergen labeling, according to RDC 727/2022/ANVISA. A commercial sample of açaí without milk or dairy products declared on the label was used, whose absence of dairy proteins was previously confirmed. The samples were then fortified with standard casein solution (NIST 1549A), at levels of 5.0; 25.0 and 50.0 mg/kg, in addition to white, being analyzed in 16 independent replicates by concentration, in two different analytical batteries