Recent advances in multidimensional separations (i.e., LC-IM-MS) have resulted in significant increases in the size of data sets. While incorporating separations techniques like chromatography and ion mobility provides some level of chemical/structural characterization (retention time indicates polarity, collision cross section indicates size, etc.), the ability to more comprehensively characterize and identify unknown compounds in complex mixtures remains a significant challenge in the molecular -omics.<br/><br/>We have recently developed a set of structurally selective reactions targeting common functional groups within steroids. The suite of reactions holds promise for anabolic steroids, endocrine hormones, bile acids, and even Vitamin D. From a practical standpoint, reactions are selected based on simplicity and speed; in most cases, incubation for ≤30 minutes results in nearly 100% yield. From a molecular standpoint, the reactions offer substantial analytical benefits, both qualitative and quantitative.<br/><br/>Recently we have completed a study targeting carbonyl and hydroxyl groups, common to nearly all steroids. Benefits to this approach are as follows: (1) predictable chromatographic shifts; (2) increased ionization efficiency; (3) improved IM resolution; (4) functional group(s) elucidation; and (5) confident identification based on MS/MS fragmentation. First, derivatization with known reagents (or combinations thereof) results in predictable changes to RT. For example, introduction of fixed-charge reagents (e.g., Girard’s reagents) results in ~20% reduction in RT, whereas more nonpolar imidazole derivatives result in ~20% increase. Second, fixed-charge reagents result in significant improvements to quantitation, allowing nearly an order of magnitude improvement with limits of detection below 20 pg/mL. Third, reactions especially targeting stereochemical differences such as hydroxyl epimers have promoted baseline IM separation for even the most challenging isomer groups. Fourth, such reactions occur selectively based on the number of functional groups and adjacent chemistry (such as methylation), resulting in straightforward elucidation. Finally, MS/MS provides predictable fragmentation pattern for a given reaction substrate, such that these can be easily filtered in a complex data set. Overall, it our expectation that these reaction suites will pave the future path for untargeted identification of steroids, and potentially other biomolecular classes.

Leveraging structurally selective reactions and ion mobility-mass spectrometry for improved biomolecular identification

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.