Abstract
Many biologically important molecules are structural isomers that share the same molecular mass yet differ in shape, bonding and function. This creates an ambiguity for mass spectrometry imaging (MSI), where chemically distinct species can be indistinguishable on the mass-to-charge ( m/z) scale and therefore cannot be reliably mapped in heterogeneous materials. Here we demonstrate that Orbitrap secondary ion mass spectrometry (OrbiSIMS) can provide an orthogonal contrast that enables both identification and spatial separation of isomeric molecules directly on surfaces. By exploiting structure-dependent secondary-ion dissociation and gas-phase collisional effects during ion transmission, we discriminate isomeric trisaccharides raffinose, maltotriose and melezitose and spatially resolve raffinose and maltotriose in mixtures. We benchmark these measurements against ion mobility spectrometry and observe consistent trends between OrbiSIMS transmission behaviour and ion mobility separation. These results establish OrbiSIMS as an approach for isomer-resolved MSI at surfaces and interfaces, opening opportunities to map isomeric biomolecules in complex systems where spatial distribution is central to function.