Absolute quantification of Bet v 1 in birch polymerized allergenic extracts via mass spectrometry-targeted analysis

Background Quantifying major allergens is essential for evaluating the quality and efficacy of allergenic extracts. They are usually measured in non-polymerized extracts using immunoassays. However, the direct measurement of allergens in allergoids is currently not supported. This study set out to develop a method for quantifying Bet v 1 in polymerized birch extracts using mass spectrometry-based targeted analysis. Methods Three isotopically labelled peptide sequences of Bet v 1 were synthetized and used as internal standards for the development of a mass spectrometry-based targeted analysis. The calibration curves of the three peptides to assess the linearity and limit of detection, as well as reverse calibration curves with a constant amount of sample, were constructed. The Bet v 1 content was determined and measured in 18 batches of depigmented (native extracts purified by a mild acid treatment) and depigmented-polymerized extracts. Results Bet v 1 isoforms were identified in both type of extracts by mass spectrometry. According to mass spectrometry-targeted analysis depigmented and depigmented-polymerized extracts exhibited mean values of 70.5 and 73.5 mu g Bet v 1/mg of lyophilized extract, respectively. A statistically significant correlation between the allergen content of both extracts was identified. Statistically significant differences were observed when the Bet v 1 content in non-polymerized extracts was measured via mass spectrometry (70.5 +/- 11.6 mu g/mg) or immunoassay (83.7 +/- 19.8 mu g/mg). Conclusions These results represent the first direct quantification of Bet v 1 in allergoids using mass spectrometry-based targeted analysis. The proposed method demonstrates robustness and reliability and constitutes a promising alternative for detailed characterization of polymerized allergenic extracts.