In-house validation of an LC–MS method for the multiplexed quantitative determination of total allergenic food in chocolate

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow’s milk, hen’s egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µgTAFP/gfood), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µgTAFP/gfood, for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µgTAFP/gfood, respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide.