Glycosphingosines are neuroactive lipids whose stereochemical variations are increasingly implicated in neuronal dysfunction and neurodegenerative disease processes. However, their close structural similarity renders stereoisomer-resolved analysis inaccessible to conventional LC–MS approaches, limiting mechanistic interpretation of lipid-associated neuropathology. In previous work, we demonstrated that complexation of glycosphingosine isomers with crown ethers enabled cyclic ion mobility spectrometry to resolve most of the species. Here, we investigate the energetics and structural effects of crown ether binding to interrogate the mechanism of separation enhancement. Systematic evaluation of crown ether derivatives shows that separation efficiency does not scale with macrocycle size or structural complexity, but is maximized by 18-crown-6, indicating a geometry-dependent interaction. Molecular modeling supports a mechanism in which the protonated amine preferentially associates with the crown ether cavity and further induces conformational reorganization rather than simple collision cross-section expansion. Saccharide-driven reactivity in boronic acid–diols interaction provided complementary modification of glycosphingosine sugar headgroups to further enhance isomer discrimination. The combination of noncovalent sphingosine backbone modification via crown ether complexation and covalent sugar headgroup modification provides a tunable analytical platform for stereochemistry-resolved glycosphingolipid analysis and may facilitate more precise characterization of lipid species implicated in neurodegenerative disease, such as alpha-synuclein aggregation and lysosomal dysfunction.

Glycosphingolipid, Glycosphingosine, Stereoisomer, Crown ether, Ion mobility, Mass spectrometry