Improved Ion Mobility Separation and Structural Characterization of Steroids using Derivatization Methods

Steroids are an important class of biomolecules studied for their role in metabolism, development, nutrition, and disease. Although highly sensitive GC- and LC-MS/MS-based methods have been developed for targeted quantitation of known steroid metabolites, emerging techniques including ion mobility (IM) have shown promise in improved analysis and capacity to better identify unknowns in complex biological samples. Herein, we couple LC-IM-MS/MS with structurally selective reactions targeting hydroxyl and carbonyl functional groups to improve IM resolution and structural elucidation. We demonstrate that 1,1-carbonyldiimidazole derivatization of hydroxyl stereoisomer pairs such as testosterone/epitestosterone and androsterone/epiandrosterone results in increased IM resolution with ΔCCS > 15%. Additionally, performing this in parallel with derivatization of the carbonyl group by Girard's Reagent P resulted in unique products based on relative differences in number of each functional group and C17 alkylation. These changes could be easily deciphered using the combination of retention time, collision cross section, accurate mass, and MS/MS fragmentation pattern. Derivatization by Girard's Reagent P, which contains a fixed charge quaternary amine, also increased the ionization efficiency and could be explored for its potential benefit to sensitivity. Overall, the combination of these simple and easy derivatization reactions with LC-IM-MS/MS analysis provides a method for improved analysis of known target analytes while also yielding critical structural information that can be used for identification of potential unknowns.