SLIM-Based High-Resolution Ion Mobility Reveals New Structural Insights into Isomeric Vitamin D Metabolites and their Isotopologues.

Testing for vitamin D deficiency remains a high-volume clinical assay, much of which is done using mass spectrometry-based methods to alleviate challenges in selectivity associated with immunoassays. Ion mobility-mass spectrometry (IM-MS) has been proposed as a rapid alternative to traditional LC-MS/MS methods, but understanding the structural ensemble that contributes to the ion mobility behavior of this molecular class is critical. Herein we demonstrate the first application of high-resolution Structures for Lossless Ion Manipulations (SLIM) IM separations of several groups of isomeric vitamin D metabolites. Despite previous IM studies of these molecules, the high resolving power of SLIM (Rp ∼ 200) has revealed additional conformations for several of the compounds. The highly similar collision cross sections (CCS), some differing by as little as 0.7%, precluded adequate characterization with low-resolution IM techniques where, in some cases, wider than expected peak widths and/or subtle shoulders may have hinted at their presence. Importantly, these newly resolved peaks often provided a unique mobility that could be used to separate isomers and provides potential for their use in quantification. Lastly, the contribution of isotopic labeling to arrival time distribution for commonly used 13C- and deuterium-labeled internal standards was explored. Minor shifts of ∼0.2-0.3% were observed, and in some instances these shifts were specific to the conformer being measured (i.e., "closed" vs "open"). Accounting for these shifts is important during raw data extraction to ensure reproducible peak area integration, which will be a critical consideration in future quantitative applications.