Abstract: In this study, we established a novel method for the simultaneous detection of allergenic foods based on multiplex polymerase chain reaction (PCR) coupled with liquid chromatography-high resolution mass spectrometry (LC-HRMS). Multiplex PCR primers were designed based on the specific sequences of five common allergenic foods: peanut (Mat K), soybean (Atp A), sesame (Ses i 1), walnut (Jug r 3), and cashew (Ana o 3). The resulting amplicons contained high-frequency restriction enzyme recognition sites with product lengths of 409, 93, 99, 104, and 88 bp, respectively. After restriction enzyme digestion, characteristic oligonucleotide fragments were generated, followed by separation and detection by LC-HRMS under optimized conditions. Based on the retention times (4–7 min) and precise mass-to-charge ratios (e.g., peanut m/z 843.704, soybean m/z 854.142) of the oligonucleotide fragments from the digestion products, different food allergen sources could be effectively distinguished and identified. Two characteristic fragments were selected from each allergenic food for qualitative identification. The proposed method exhibited a sensitivity of 1 mg/kg. This study successfully achieved the multiplex simultaneous detection of the five allergenic foods, providing a new strategy for rapid food safety analysis and further expanding the application of nucleic acid mass spectrometry in the field of food testing.

Key words: food allergy; allergen; oligonucleotide; nucleic acid mass spectrometry; qualitative detection