Cyclic Ion Mobility Separations of Free Oxylipins

Abstract Oxylipins are oxygenated lipid metabolites derived from polyunsaturated fatty acids (PUFAs). They play interconnected roles in physiological processes such as inflammation and vascular function, and their dysregulation has been linked to diseases, including cardiovascular, metabolic, and immunological disorders. Liquid chromatography (LC) has a limited ability to resolve the large number of isomers and isobars within this diverse lipid class. Ion mobility spectrometry (IMS) offers an orthogonal gas-phase separation technique and has been shown to improve peak capacity for isomers that differ by more than 1–2% in collision cross section (CCS). High-resolution IMS platforms have progressively enabled the separation of isomers with increasingly similar gas-phase structures and offer the potential to separate complex mixtures in experiments such as mass spectrometry imaging where LC is not available. Here, we demonstrate the application of high-resolution cyclic ion mobility spectrometry–mass spectrometry (cIMS–MS) to separate and characterize six complex mixtures of authentic oxylipin standards. Results show that deprotonated oxylipin species often exhibit multiple mobility features that emerge only after extended separations, whereas metal-adducted species, especially sodium adducts, yield clearer single peaks. Optimized traveling wave parameters enabled the separation of mixtures containing three to five oxylipins within 3–10 m of effective path length before ion wrap-around occurred, resolving features with ΔDTCCS% of approximately 0.5% without front-end chromatographic separation. Multipass cIMSCCSN2 measurements under optimized conditions were highly reproducible and closely matched the DTCCS reference values from Moran-Garrido et al. and da Silva et al., with average deviations of 0.33–0.76%. These findings support the use of multipass cIMS experiments and cIMSCCS measurements as valuable tools for oxylipin separation and annotation, particularly in the absence of chromatographic retention time data.