Intramolecular Ring–Chain Equilibrium Elimination Strategy for Pinpointing CC Positional and Geometric Isomers of N‑Alkylpyridinium Unsaturated Fatty Acid Derivatives via Ion Mobility-Mass Spectrometry

Free unsaturated fatty acids (UFA) are key intermediates of lipid metabolism and participate in many metabolic pathways with specific biological functions. Although various fragmentation-based methods for pinpointing CC locations in UFA were developed, the current mass spectrometry methods are difficult to simultaneously differentiate geometric isomers and positional isomers in trace samples due to low ionization efficiency, low conversion, and low resolution. Herein, an intramolecular ring–chain equilibrium elimination strategy via 4-plex stable isotope labeling dual derivatization-assisted ion mobility-mass spectrometry was developed, thereby one-pot specifically labeling CC and carboxyl groups among the trace and unstable UFA with high sensitivity, high efficiency, and good substrate generality. It achieved fast separation of both CC positional and geometric isomers with high resolution, which benefited from eliminating the intramolecular ring–chain equilibrium by suppressing the formation of salt bridges between free carboxyl groups and pyridine cations. 4-plex stable isotope labeling reagents showed similar reactivity, enabling high-throughput quantitative analysis of omics. This method was successfully applied for accurate and rapid identification of the UFA composition in olive oil extract. These results suggest that the developed method provides new insight for rapid characterization of UFA CC positional and geometric isomers in complex samples to explore disease biomarkers and food quality control indicators.