An ultra-performance liquid chromatography–high-resolution mass spectrometry method for comprehensive profiling and quantification of free fatty acids in edible oils

A novel strategy for analyzing free fatty acids (FFAs) in edible oils was developed using isotope-coded derivatization reagents—4-(dimethylamino)benzoylhydrazine (DABA) and d 6 -4-(dimethylamino) benzoylhydrazine ( d 6 -DABA)—synthesized via a two-step process. These reagents selectively derivatized FFAs at 35°C in 30 min, considerably enhancing mass spectrometry sensitivity. Ultra-performance liquid chromatography–high-resolution mass spectrometry (UPLC–HRMS) enabled multidimensional identification via isotope ratio analysis, retention time prediction, isotope peak matching, and carbon count–based retention behavior, excluding 47.5% of initial proposals and identifying 42 FFAs. A relative quantification method using d 6 -DABA-derivatized pentadecanoic acid as the internal standard showed good linearity ( R = 0.9914–0.9993), precision, repeatability, and stability (relative standard deviation (RSD) ≤ 13.0%). The primary FFAs in five edible oils included palmitic, stearic, linoleic, arachidonic, and linolenic acids. Specifically, linoleic acid was predominant in rapeseed oil (117525.5 ng/mL) and peanut oil (525880.0 ng/mL), stearic acid was notable in soy oil (21255.2 ng/mL) and corn oil (29349.7 ng/mL), and palmitic acid was predominant in olive oil (97834.5 ng/mL). This method offers a sensitive analytical platform with excellent tolerance to substrate interference, facilitating the study of the composition and content of FFAs in edible oils. • Two-step synthesized DABA/ d 6 -DABA enabled rapid FFAs derivatization (35°C, 30 min). • DABA derivatization boosts sensitivity by 528–3677-fold, with LODs at 0.04-10 ng/mL. • UPLC-HRMS profiled 42 FFAs with 47.5% exclusion, no cis/trans/DB isomers resolution. • Key FFAs in 5 edible oils: palmitic, stearic, linoleic, arachidonic, linolenic acid. • FFAs analysis aids edible oil composition/distribution studies.