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 (µg_TAFP/g_food), 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 µg_TAFP/g_food, for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µg_TAFP/g_food, 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.

质谱法已被广泛接受为一种验证工具，用于灵敏检测未声明的过敏成分的存在。迄今为止，已经开发了多种方法，实现了不同水平的灵敏度和稳健性，但仍然缺乏分析验证的协调性，并损害了结果的可比性。在这项研究中，我们内部验证了一种定量方法，用于测定巧克力基质中的六种过敏成分（牛奶、鸡蛋、花生、大豆、榛子和杏仁）。后者是在食品中试工厂生产的，为根据官方指南正确评估方法性能特征提供真实且特征良好的矩阵。特别是，欧洲标准化委员会最近发布的考虑因素已被遵循，以指导严格的单一实验室验证并体现主要方法性能，例如选择性、线性和灵敏度。肽标记物的合成替代品以天然形式和标记形式分别用于基质匹配校准曲线中作为外部校准物和内部标准物。研究了两个数量级的范围，重点关注低浓度范围，以便通过严格的校准方法正确评估检测和定量限（LOD 和 LOQ）。首次确定了所有六种致敏成分的换算系数，以将最终定量信息报告为每质量食品中总致敏食品蛋白 (TAFP) 的分数（μg _TAFP /g_食品），因为这样的报告单位可用于过敏风险评估计划。 该方法对除鸡蛋和大豆之外的所有成分均实现了良好的灵敏度，LOD 值范围为 0.08 至 0.2 µg _TAFP /g_食品，其定量标记报告的限值略高（分别为 1.1 和 1.2 µg _TAFP /g_食品）。对在接近当前可用阈值剂量的四个定义浓度水平下产生的巧克力棒的不同样品进行了分析，以测试分析方法的定量性能，并适当估计来自不同变异源的测量不确定度。所达到的灵敏度符合全球发布或推荐的各种阈值剂量。