Quantitative Analysis and Health Risk Assessment of Heterocyclic Aromatic Amines in Plant-Based Milk Beverages

Heterocyclic aromatic amines (HAAs) are mutagenic and potentially carcinogenic compounds formed during the thermal processing of protein- and sugar-rich foods, yet their occurrence in plant-based milk alternatives remains largely unexplored. To the best of our knowledge, this is the first study to report on the presence of HAAs in plant-based milk beverages. The aim of this study was to develop a robust and environmentally friendly µSPE-UHPLC-MS/MS method for the quantification of ten HAAs in plant-based milk beverages and to assess the potential health risks associated with their formation under varying thermal treatment conditions. A novel analytical method was applied to both commercially available and homemade beverages prepared from almonds, soy, cashews, and peanuts, including pasteurized and unpasteurized variants with and without added sugar. Chemometric tools were used to optimize retention and enrichment strategies. Detection limits ranged from 0.01 to 0.04 µg L^-1, while quantification limits ranged from 0.01 to 0.05 µg L^-1. Recovery rates ranged between 84% and 100%, with enrichment factors spanning 43 to 50. HAA concentrations varied from 0.09 to 13.66 µg L^-1, with significantly higher levels observed in beverages subjected to thermal treatment, particularly those with added sugar and higher protein content. The health risk assessment indicated that the cumulative incremental lifetime cancer risk (ILCR) values were below the unacceptable threshold (10^-4), though some scenarios approached 10^-5, suggesting a moderate risk for frequent consumers of plant-based milk alternatives.