Development of innovative methods for multi allergen quantification in processed foods

The growing global incidence of allergy to nuts and peanuts has constantly increased the need of new strategies to protect sensitive consumers. Nowadays, the best choice to avoid unawares immunological reactions still remains the total removal of the culprit products from the diet. However, traces of nuts and peanuts could contaminate other foods, especially processed products, as consequence of cross-contamination events occurring during production. In the light of this, the development of a multi-target method based on mass spectrometry for the detection of traces of allergens could represent a useful tool to protect allergenic individuals also preventing the removal of safe food from their diet. In this work, an ultra-high-performance liquid chromatography coupled with high resolution Orbitrap™-based mass spectrometry (UHPLC-HRMS/MS), for the simultaneous detection of five nuts species (almonds, hazelnuts, walnuts, cashews and pistachios) and peanuts in an incurred bakery product (cookie) is described. Experiments were accomplished on an incurred cookie model food produced in-house by adding roasted and powdered nuts/peanut upon dough preparation and before baking. The following pipeline was used for the LC-MS/MS method development: i) LC-HRMS/MS untargeted analysis of both raw and roasted ingredients for the selection of the most appropriate candidate marker peptides for detecting nuts and peanut allergens, ii) constraining the resulting peptides according to the specific criteria previously reported in literature [1], iii) assessing of the impact of processing on the detection of candidate peptides in cookie extract, iv) selection of the most appropriate marker peptides for tracing nuts/peanut in cookie food, v) evaluation of method performance by building up specific curve calibration for each investigated peptide and assessment of the most relevant method parameters (linearity, limit of detection (LOD), limit of quantification (LOQ), repeatability inter-day and inter-day), vi) comparison of the method performance with the VITAL 3.0 thresholds. Suitable and thermostable peptide markers for each allergenic ingredient were selected and the LOD and LOQ values obtained for each allergen calculated in incurred cookies (referred to the protein content) allowed to detect levels of contamination complying with the reference thresholds set for each allergen and recommended (action level 1) by the VITAL program v 3.0. Additionally, method precision provided good results for all the allergenic ingredients analyzed in this matrix [2]. This method could represent an important tool to support the risk assessment for allergen management also preventing the overuse of PAL (Precautionary Allergen Labelling). References 1. Pilolli, R. et al. Food Research International, 128, (2020) pp 108747. 2. Luparelli, A. et al. Foods, 12, (2023), pp 1–23