Ozone-Induced Cleavage of Endocyclic C═C Double Bonds within Steroid Epimers Produces Unique Gas-Phase Conformations

Abstract

Herein we demonstrate the first application of ozone-induced cleavage of endocyclic CC double bonds for improved steroid isomer separation using ion mobility-mass spectrometry. Steroids represent a challenging biomolecular class for ion mobility (IM) separations due to their structural rigidity and subtle stereochemical differences. In this work, we compare the effects of ozonolysis on the relative mobilities of a model stereoisomer pair, testosterone and epitestosterone. A solution-phase ozonolysis approach is used due to its simplicity, relatively low cost, and potential for rapid, online analysis. Despite the presence of solvent-based addition products, we observe that these steroids undergo an ozone-based cleavage resulting in unique, stable gas-phase conformations. The resulting resolution between testosterone and epitestosterone, with collision cross section values of 176.6 and 193.3 Å2, respectively, demonstrates a significant improvement in comparison with previous IM-based approaches. The significantly smaller conformation observed for epitestosterone is stabilized by a three-point interaction between the oxygen-containing functional groups and a sodium ion; this same conformation cannot be sterically achieved by testosterone. Identification of this specific structural difference is strengthened by experimental results showing the disappearance of this conformation following in-source water loss, which eliminates the potential for that three-point interaction. Computational modeling of the lowest energy gas-phase structures for these ozone products corroborates the experimental results. In conclusion, this approach provides tremendous potential as a rapid IM separation method for steroid isomers and other endocyclic CC double bond containing molecules.

Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC–Ion Mobility–HRMS

“…Ion mobility spectrometry (IMS) is a gas-phase separation technique that distinguishes ions based on their size, shape, and charge state. − The IMS size and shape measurement takes the form of an ion collision cross section (CCS), which is a coarse-grained surface area measurement (reported in square angstroms, Å 2 ) encompassing the ion size as well as its interaction with the neutral gas. IMS separates ions based on differences in gas-phase electrophoretic mobility.…”

“…Gas-phase IMS analyses are rapid, typically occurring on a time scale of 10–100 ms per spectrum, whereas condensed-phase LC–MS is on the time scale of minutes. Therefore, IMS can be included in existing LC–MS workflows without compromising analytical throughput, providing an additional separation dimension and an associated molecular descriptor (CCS) to support analyte detection and identification. , Previously, IMS has been utilized to characterize intact AASs along with their phase I and phase II metabolites. ,,− Various IMS techniques, such as field asymmetric IMS (FAIMS), traveling wave IMS (TWIMS), and drift tube IMS (DTIMS), have been utilized for AAS analyses. ,− The Yost research group investigated the effect of Group 1 cation adducts on resolving AAS isomers using FAIMS, providing direct separation of metal-coordinated AAS isomers with no increase in analysis time or modifications to the instrumentation, which is useful for high-throughput screening . Hernández-Mesa et al have successfully cross-validated the first TWIMS database for steroids in calves urine and obtained CCS values within 2% of literature databases entries .…”

Improved Identification of Isomeric Steroids Using the Paternò-Büchi Reaction with Ion Mobility-Mass Spectrometry

“…The ion funnel trap was filled for 2000 μs with release time of 150 μs. Collision cross sections (CCS) were measured using the stepped-field method (five drift fields) similar to previous work by our group …”

“…Our group has recently demonstrated the first application of UV-catalyzed solution-phase ozonolysis for application to isomeric steroids . This study involved formation of ozone in a sample solution, which reacted with the CC bond in testosterone and epitestosterone and resulted in cleavage of the steroid A-ring.…”

Targeted glucocorticoid analysis using ion mobility-mass spectrometry (IM-MS)

“…Most notably, these have included changes to ionic structure/conformation and drift gas environment (e.g., temperature, gas composition, volatile solvent modifiers, etc.). First, simple chemical modifications have been demonstrated to improve resolution of various isomeric steroids; these have included derivatization with p-toluenesulfonyl isocyanate, [23] ozonolysis, [24] and the Paternò-Büchi reaction. [25] Investigation of ion adducts as alternatives to the most common protonated/deprotonated/sodiated has led to interesting separation results, with examples of alkali, alkaline earth, and transition metal complexes as well as negatively charged species, such as chlorides.…”