Date of Award

5-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering and Sciences

First Advisor

Rudolf Wehmschulte

Second Advisor

Christopher Chouinard

Third Advisor

Pavithra Pathirathna

Fourth Advisor

Andrew Palmer

Abstract

This dissertation presents a comprehensive study on the analysis of steroid hormone isomers using liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS). The dissertation is divided into three main parts: the first part focuses on research using different drift gases and ion adducts to modify the mobility separations allowing for resolution between the isomers. The second part focuses on research exploring derivatization reactions that target structural differences between the steroid hormone isomers to amplify the differences and resolve the isomers in the ion mobility region. The final part covers a proposal on future study directions to include simultaneous derivatization reactions, mass spectrometry fragmentation, and high-resolution ion mobility instrumentation to resolve isomers in human urine and serum with the goal of collaborating with medical teams to test these workflows on real-world samples. The first study demonstrated the use of IM-MS to detect and separate glucocorticoids. By utilizing various experimental conditions, such as different cation adducts and drift gases, improved separations for several isomeric pairs were achieved. Using IM-MS allowed for improved sensitivity and selectivity in the detection of glucocorticoids compared to traditional LC-MS methods. This suggests that IM-MS could be useful for clinical applications, particularly in analyzing complex biological samples where isomer separation is crucial. The second study demonstrated the effectiveness of using derivatization reactions to improve IM-MS analysis of steroid hormones. We applied this approach to challenging separations, such as between flunisolide and triamcinolone acetonide. The structural information obtained from patterns seen for derivatized isomers can help identify the location of specific functional groups within the molecules. Overall, this strategy is encouraging for improving the quality of difficult IM analyses in the field of steroid hormone isomer analysis. The final section of this dissertation delves into the possibility of conducting simultaneous site-specific reactions to attack the steroids’ carbonyl and hydroxyl groups during a single sample preparation. This research could provide valuable structural information and aid in isomer resolution by targeting differences in the number and location of functional groups between isomers. Expanding on this idea, conducting MS/MS fragmentation studies in conjunction with using new high-resolution ion mobility instrumentation may be beneficial. These studies could allow for a more detailed examination of the different structural features of the various isomers, further expanding our understanding of their unique properties and characteristics. The final section of this dissertation also delves into using high-resolution ion mobility instrumentation to resolve steroid hormone isomers. The proposed method involves the use of Structures for Lossless Ion Manipulation (SLIM), which allows the isomers to spend an extended amount of time in the ion mobility drift region. This would allow for improved resolution, enabling researchers to distinguish between isomers that may have previously been indistinguishable. In conclusion, the research presented in this dissertation has provided valuable insights into the analysis of steroid hormone isomers using LC-IM-MS. The studies conducted have demonstrated the usefulness of IM-MS for detecting and separating glucocorticoid isomers, offering improved sensitivity and selectivity compared to traditional LC-MS methods. Different experimental conditions and derivatization reactions have also shown promise in enhancing the quality of the analysis, particularly in difficult separations. Furthermore, the proposed future study directions offer exciting possibilities for further improving the resolution of isomers in human urine and serum, with the goal of collaborating with medical teams to test these workflows on real-world samples. Using simultaneous derivatization reactions, MS/MS fragmentation studies, and high-resolution ion mobility instrumentation holds great potential for expanding our understanding of the unique properties and characteristics of steroid hormone isomers, ultimately aiding in clinical applications where accurate isomer separation is crucial.

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