Quantification of nine N-nitrosamines in cocoa and cocoa-based products: development and validation of a matrix-adapted LC–MS/MS method

In 2023, the European Food Safety Authority (EFSA) published a comprehensive risk assessment on N-nitrosamines in food. These substances occur ubiquitously and may be formed unintentionally during food processing. Due to their potentially carcinogenic and mutagenic properties, they are of considerable toxicological concern. Consequently, the European Commission issued a draft recommendation for monitoring these compounds across selected food categories, including cocoa and chocolate products. A quick and selective analytical method was developed via LC-MS/MS for the quantitation of nine out of ten N-nitrosamine compounds identified by EFSA as relevant in food (N-nitrosodibutylamine, N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitrosodipropylamine, N- ni­tro­someth­ylani­line, N-nitrosomethylethylamine, N-nitrosomorpholine, N-nitrosopiperidine and N-nitrosopyrrolidine) in cocoa and chocolate matrices. The experimentally determined limits of quantification ranged from 0.20 to 0.90 µg kg−1 and thus met the analytical performance criterion of <1 µg kg−1 specified in the draft European Commission monitoring recommendation for the ten N-nitrosamines considered carcinogenic and genotoxic by EFSA. The successfully developed method was applied to investigate the occurrence and potential formation of N-nitrosamines in semi-finished (N = 77) and finished (N = 21), cocoa and cocoa-based products. In all cocoa and chocolate samples analysed, N-nitrosamine concentrations were below the respective limits of quantification.