Cyclic ion mobility-mass spectrometry to enhance the separation of pyrrolizidine alkaloid epimers

Background: Cyclic ion mobility spectrometry (cIMS) has emerged as a powerful tool for enhancing the resolution of isomeric and epimeric species that remain unresolved by traditional LC-MS or single-pass ion mobility techniques. In this study, we integrated multipass cIMS into a UPLC-HRMS workflow to address the long-standing challenge of separating epimeric pyrrolizidine alkaloids (PAs), a class of plant-derived toxins of regulatory concern. Four representative PA epimer pairs—lycopsamine/indicine, rinderine/echinatine, rinderine-N-oxide/echinatine-N-oxide, and integerrimine-N-oxide/senecivernine-N-oxide—were subjected to offline high resolution cyclic ion mobility separation. Results: Baseline or near-baseline separations were achieved for all PA epimer pairs, with multi pass mode, either in their protonated or sodium adduct forms, and no significant loss of signal intensity was observed at the number of passes applied. Single-pass CCS values were measured and compared with previously reported values obtained using linear IMS, revealing limited resolution for several epimers. In contrast, multipass CCS values were then calculated for each epimer, enabling differentiation in cases where single-pass values were identical. Finally, the optimized cyclic sequences were integrated into a LC–cIMS–HRMS method, targeting specific retention time windows and m/z to introduce an additional dimension of separation. This approach was successfully applied to the analysis of a green tea extract, demonstrating the method's suitability for real food matrices. Significance: This work highlights, for the first time, the integration of multiple targeted multipass cIMS separations within an untargeted LC–HRMS workflow, underscoring its potential to expand separation power and analytical confidence for structurally related small molecules. The proposed workflow is especially valuable for the analysis of complex mixtures where epimeric compounds co-occur, as commonly found in naturally contaminated foods.