Background: Food allergen cross-contact during food preparation and production is one of the causes of unintentional allergen presence in packaged foods. However, very little is known about allergen cross-contact in shared frying or roasting oil, which prevents the establishment of effective allergen controls and may put allergic individuals at risk. To better understand the quantity of allergen transferred to frying oil and subsequent products, an analytical method that is capable of quantifying protein in oil is of utmost importance. Objective: The goal of this study was to develop a parallel reaction monitoring LC-MS/MS method to quantify the amount of cashew protein in shared roasting oil. Methods: The sample preparation method was evaluated to improve protein extractability and peptide performance. Four quantitative peptides representing cashew 2S and 11S proteins were selected as targets based on their sensitivity, heat stability, and specificity. A calibration strategy was developed to quantify the amount of total cashew protein in oil. Method performance was evaluated using a heated cashew-in-oil model system. Results: The method showed high recovery in oil samples spiked with 100 or 10 ppm total cashew protein heated at 138 or 166 °C for 2-30 minutes. Samples (100 ppm total cashew protein) heated for 30 minutes had more than 90% recovery when treated at 138 °C and more than 50% when heated at 166 °C. Conclusion: The method is fit-for-purpose for the analysis of cashew allergen cross-contact in oil. Highlights: A novel MS-based method was developed that can accurately quantify the amount of cashew protein present in heated oil.

Figure 1: 20190904_Organic_Aqueous_Extraction_Oil_Spike_24pep; Figure 2: 20191007_HeatedOilSpike_Extraction_method_24pep; Figure 3: 20191112_Diff-TempConc_Oil-Spiked_24pep; Figure 4: 20200622_PeptideSpecificityTest; 20200715_PeptideSpecificity_SignalRatio; Figure 5: 20210212 Low range exploration 140k-fragmod_Pub; Figure 6: 20210607 Calibration Curve_DilutionDigest; 20210301 Calibration Dev_DilutionOil; Figure 7 and 8: HeatedOilSpike-LowTemp_HighTemp_Combined_Final

Soybean oil spiked with known amounts of total cashew protein (in the form of cashew butter); Unheated or heated at low (138C) or high (166C) temperatures for up to 30 minutes