[Identification of a Chemically Modified Nutrient for Direct Evidence of Ethylene Oxide Fumigated Foods]

Ethylene oxide (ETO) is a highly reactive sterilizing agent classified as a carcinogen, and its use in foods is prohibited in many countries. Regulatory monitoring currently relies primarily on the detection of ETO and/or 2-chloroethanol (2CE), a chloride-derived reaction product. However, ETO is volatile and difficult to detect directly in commercial foods, and 2CE may occur naturally or arise from non-food-related sources, raising scientific concerns regarding its specificity as a fumigation marker. In this study, we applied a food-processing metabolomics approach to comprehensively investigate the chemical alterations induced by ETO fumigation of cumin (Cuminum cyminum L.) seeds using gas chromatography-mass spectrometry. Fumigated and non-fumigated samples were clearly discriminated by principal component analysis of the data containing fumigation-specific metabolites. Here, we report that high-resolution gas chromatography quadrupole time-of-flight mass spectrometric analysis identified a characteristic compound with a molecular formula consistent with a trimethylsilylated derivative of C₆H₁₀O₆ containing a single ETO incorporation. Fragmentation patterns and accurate mass measurements suggested that this compound was an ETO adduct of malic acid. In vitro fumigation experiments using authentic malic acid reproduced the same chromatographic retention time and mass spectrum pattern, confirming its formation. A targeted gas chromatography-tandem mass spectrometric analysis further demonstrated the presence of the malic acid-ETO adduct (m/z 233 → 73) in fumigated sesame and soybean, indicating its potential broader presence across ETO fumigated plant-derived matrices. Collectively, these findings provide the first direct evidence that ETO fumigation induces the formation of a nutrient-derived adduct in foods. The identified ETO-modified nutrient represents a promising chemical marker for verifying ETO fumigation beyond conventional 2CE-based approaches, thereby contributing to improved analytical specificity and regulatory reliability in food safety control.