Spatial Mapping of Stereoisomeric and Isobaric Alkaloids in <i>Mitragyna speciosa</i> Tissues by High-Resolution DESI-cIM-MS

Conventional mass spectrometry imaging (MSI), even when combined with low-resolution ion mobility, lacks the resolving power to distinguish stereoisomers. To address this limitation, we developed a high-resolution desorption electrospray ionization cyclic ion mobility mass spectrometry (DESI-cIM-MS) method for in situ separation and spatial mapping of stereoisomeric compounds, using <i>Mitragyna speciosa</i> (kratom) as a model system. We characterized and validated the separation of four mitragynine-type stereoisomers─mitragynine (MG), speciogynine (SG), mitraciliatine (MC), and speciociliatine (SC)─using chemical standards. Notably, SC exhibited two gas-phase conformers, fast (SC-F) and slow (SC-S), supported by quantum chemical calculations. Using multipass separation and targeted ion slicing, we resolved and mapped SG, MC, and SC-S in surface-spotted standards. To address coelution between MG and SC-F, we developed a pixel-wise subtraction strategy based on the SC-F/SC-S intensity ratio to mitigate SC-F interference in the MG ion image. Direct analysis of kratom twig tissue revealed distinct spatial distributions for each stereoisomer. MG was found broadly throughout the twig except in the xylem. MC was concentrated in the pith, with some presence in the bark. SC and SG were predominantly localized in the bark, especially the epidermis. Furthermore, we resolved two additional important alkaloids, paynantheine and 7-OH-mitragynine, from their isobaric compounds; both were distributed throughout the twig except the xylem. These findings demonstrate the importance of high-resolution ion mobility in MSI for accurately resolving structurally similar compounds and improving spatial analysis in metabolomics and natural product research.