Development of an innovative quantitative method for allergen detection in food using isotopic dilution mass spectrometry

PhD thesis defended by Maxime GAVAGE (Prof. Thierry ARNOULD) - 02/09/2020

Prof. Thierry ARNOULD, UNamur, Laboratory of Cellular and Molecular Biology (URBC)

  • Prof. Martine RAES (UNamur), President
  • Prof. Thierry ARNOULD (UNamur), Promotor and Secretary
  • Prof. Patsy RENARD (UNamur), Co-promotor
  • Prof. Fabrice BUREAU (ULiège)
  • Prof. Bruno DE MEULENAER (UGent)
  • DVM. Philippe DELAHAUT (CER Groupe)
  • Prof. Gauthier EPPE (ULiège)
  • PhD. Nathalie GILLARD (CER Groupe)
  • Prof. Georges LOGNAY (ULiège)

PhD thesis in the framework of a collaboration between UNamur and CER Groupe

Food allergen analysis is an essential tool in the development of a risk-based approach for allergen management. Robust, specific and sensitive detection methods are still needed to protect allergic patients and guarantee correct food labelling. During the last decade, mass spectrometry became the method of choice for allergen analysis. This approach is predominantly performed by specific analysis of peptides obtained by an enzymatic digestion of the proteins of the sample, including the proteins of the allergenic ingredients.

Here, a strategy to develop a food allergen quantitative analysis method was proposed. This strategy was based on isotope dilution considering an original approach using a stable isotope-labelled concatemer as internal standard and was applied to elaborate a UHPLC-MS/MS method for the simultaneous detection and quantification of four allergenic ingredients (egg, milk, peanut and hazelnut) in processed food products.

A list of potential peptide biomarkers was first identified with an experimental approach based on high resolution mass spectrometry and the analysis of the four considered allergenic ingredients submitted to different representative food processing techniques. Among the hundreds of identified peptides, potential peptide biomarkers were selected using a set of selection criteria to ensure the specificity, sensitivity and robustness of the quantitative method. This list was further reduced during the development of the UHPLC-MS/MS method, based on sensitivity and selectivity criteria.

The 19 selected peptides biomarkers were combined in a 15N stable isotope-labelled concatemer. Design and labelling strategy of the concatemer was optimized to reach high production yield with a sufficient isotopic enrichment. These conditions were necessary to set up a cost-effective method when compared to the synthetic peptides internal standard and avoid any introduction of false positive results, respectively.

The developed UHPLC-MS/MS was validated using different food matrices incurred or spiked with the four allergenic ingredients. Performance parameters including selectivity, LOD, LOQ, linearity, trueness and precision were evaluated. The use of concatemer as internal standard was finally compared to synthetic peptides and labelled protein approaches.

In conclusion, the combination of mass spectrometry and stable isotope dilution using concatemer allowed the development of a method for the simultaneous quantification of egg, milk, peanut and hazelnut, four major allergenic ingredients, in processed food products.