Investigating Differences in Gas-Phase Conformations of 25-Hydroxyvitamin D3 Sodiated Epimers using Ion Mobility-Mass Spectrometry and Theoretical Modeling

Christopher D. Chouinard, Vinícius Wilian D. Cruzeiro, Christopher R. Beekman, Adrian E. Roitberg, Richard A. Yost

Drift tube ion mobility coupled with mass spectrometry was used to investigate the gas-phase structure of 25-hydroxyvitamin D3 (25OHD3) and D2 (25OHD2) epimers, and to evaluate its potential in rapid separation of these compounds. Experimental results revealed two distinct drift species for the 25OHD3 sodiated monomer, whereas only one of these conformations was observed for its epimer (epi25OHD3). The unique species allowed 25OHD3 to be readily distinguished, and the same pattern was observed for 25OHD2 epimers. Theoretical modeling of 25OHD3 epimers identified energetically stable gas-phase structures, indicating that both compounds may adopt a compact "closed" conformation, but that 25OHD3 may also adopt a slightly less energetically favorable "open" conformation that is not accessible to its epimer. Calculated theoretical collision cross-sections for these structures agreed with experimental results to <2%. Experimentation indicated that additional energy in the ESI source (i.e., increased temperature, spray voltage) affected the ratio of 25OHD3 conformations, with the less energetically favorable "open" conformation increasing in relative intensity. Finally, LC-IM-MS results yielded linear quantitation of 25OHD3, in the presence of the epimer interference, at biologically relevant concentrations. This study demonstrates that ion mobility can be used in tandem with theoretical modeling to determine structural differences that contribute to drift separation. These separation capabilities provide potential for rapid (<60 ms) identification of 25OHD3 and 25OHD2 in mixtures with their epimers. Graphical Abstract ᅟ.