Advantages of Alkali Metal Adduct Formation in Cyclic Ion Mobility‐Mass Spectrometry of O‐Methylated Cyclodextrin Isomers—Separation and Crossring Fragmentation

The expanding use of O‐methylated cyclodextrins (MeCDs) demands analytical strategies capable of rapidly and reliably distinguishing regioisomeric species, which remain challenging to characterize using conventional mass spectrometry. Here, we demonstrate that cyclic ion mobility–tandem mass spectrometry (cIM‐MS/MS) provides a powerful and structurally selective approach for detailed analysis of isomeric MeCD mixtures. Using heptakis(2,3‐di‐O‐methyl)‐β‐cyclodextrin (2,3‐DIMEB) and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin (2,6‐DIMEB), we comparatively evaluate the suitability of different adduct ions and show that cIM enables clear separation of these regioisomers. We reveal that they can be unambiguously identified by postseparation tandem mass spectrometry (MS/MS) through characteristic crossring fragmentation of their di‐O‐methyl‐glucoside subunits by selecting their [M + Li/Na] + ions in positive ionization mode and the [M – H] − ions in negative ionization mode. These diagnostic fragments allow confident assignment of methyl substitution positions. Moreover, analysis of crystalline methylated‐β‐cyclodextrin (CRYSMEB) and random‐O‐methyl‐β‐cyclodextrin (RAMEB) demonstrates that both substitution degree and pattern markedly influence fragmentation behavior. Overall, this study establishes cIM‐MS/MS as an effective and highly discriminative platform for regioisomeric MeCD characterization, offering new mechanistic insight and practical guidance for future analytical applications.