Development of a green LC-MS/MS method for the detection of peanuts allergens in oil samples

Cross-contamination of foods by allergens represents a safety risk for allergic consumers [1]. Currently available and routinely used methods are mainly based on immuno assay kits for the detection of allergenic ingredients in food products. On the other hand, application to selected and highly processed foods can raise issues making the whole analysis complex and challenging; this is the case of oil rich products where there is a lack of reliable and sensitive methods available [2,3]. In addition, processing modifies protein structure, and this complicates the identification and quantification of allergens especially in ultra-processed foods. Methods capable of detecting low amounts of allergens in oily matrices, would be needed to answer the question about the amount of residual allergenic proteins in oil, in order to properly estimate the risk for allergic consumers. In this note we present the optimization of reliable and robust reference LC-MS/MS based methods that can be widely used for the quantification of peanut allergens in oily matrices. We herein describe the optimization of a whole analytical protocol for peanut identification and quantification in oil samples by a stepwise approach. First, sample preparation for the extraction of allergenic proteins from complex lipid matrices was optimized, with a specific focus on edible oils. Two extraction strategies were compared: conventional saline buffers and Deep Eutectic Solvents. Further, MSMS method was duly tuned in order to develop a sensitive targeted MSMS method on a Triple Quadrupole Mass Spectrometer. Method optimization was carried out on sunflower oil spiked with peanut proteins at 1000 ppm. Five saline extraction buffers were compared, and the best extractive conditions were further investigated by MSMS analysis. The overall optimized LC MS method used a EVOQ® DART TQ+ system to enable the simultaneous detection of 27 peanut derived marker peptides. Based on precursor ion acquisition results, a targeted MRM method was built up for the simultaneous quantification of 13 peanut reliable markers, where both the quantifier and the qualifier fragment ions were monitored. The MRM method allowed a sensitive and robust preliminary assessment of the extraction efficiency of the tested protocols.