MSACL 2026 Abstract

Metabolites, Microbes, and Mobility: Oh My!

Heidi M. Sabatini, Jude Sulieman, Sabrina Fernandez, Elijah Hood, Angela Acateca, Bradley B. Garrison, Christopher D. Chouinard
Clemson University, Clemson, SC

Christopher Chouinard, PhD (Presenter) Clemson University

Presenter Bio: I received my PhD from University of Florida in 2016, where I developed ion mobility-mass spectrometry (IM-MS) methods for steroids and vitamin D metabolites. I then worked as a post-doctoral research at Pacific Northwest National Laboratory, building Structures for Loss Ion Manipulations (SLIM) ion mobility instrumentation for application in metabolomics and proteomics. In 2018, I began my independent career as an Assistant Professor at Florida Institute of Technology. I have since moved to Clemson University in August 2022. Work in my research group focuses on ion mobility-mass spectrometry (IM-MS)-based methods and technology, including structurally selective reactions for improved characterization of steroids and other controlled substances.

Relevant Financial Disclosures (within past 24 months, reported on Apr 22, 2026) Grant/Research Support MOBILion Systems Discussing Products? YES, presenter will present educational content related to products/services of one or more ineligible company(ies) listed above. Using Generic Terms? YES Conflict mitigation IN PROCESS.

INTRODUCTION:
The metabolome is an inherently complex and dynamic system that yields significant insights into human health, development, and disease. Despite modern technological advances, some current challenges include isomeric heterogeneity and wide-ranging concentrations that push the qualitative and quantitative performance of mass spectrometry-based assays. The coupling of ion mobility spectrometry with high-resolution and/or tandem mass spectrometry (i.e., IM-HRMS/MS) offers a unique avenue toward overcoming these challenges. This presentation will discuss various high-resolution ion mobility (HRIM) strategies, including multiplexed acquisition and long-path structures for lossless ion manipulations (SLIM), applied to interesting metabolomics samples. Specific examples will include low-mass enabled SLIM methods for endocrine hormones and anabolic steroids, chemical methods for 11-oxosteroids, and high-resolution demultiplexing data interpretation for global metabolomics in microbial systems.

METHODS:
All analyses were performed using either an Agilent 6560 IM-QTOF or MOBILion Systems MOBIE SLIM/Agilent 6546 QTOF, both coupled to Agilent 1290 UHPLC via Agilent JetStream and/or MultiMode Ionization (MMI) sources. Agilent 6560: all data were obtained using 4-bit multiplexed acquisition (i.e., 8 injections per frame) with the maximum 3900 usec ion accumulation time, and a drift tube maintained at ~18.5 V/cm with 3.95 Torr nitrogen at ambient temperature. All-ion fragmentation (AIF) was performed with alternating low/high collision energy on successive injections. Data processing was performed using a combination of Agilent IM-MS Browser (visualization), PNNL PreProcessor (demultiplexing/smoothing), Agilent Mass Profiler (feature finding), and Agilent HRdm 2.0 (high-resolution demultiplexing). MOBIE SLIM: all data were obtained using optimized traveling wave conditions following 100 ms accumulation period. The SLIM module was maintained at 2.5 Torr nitrogen or 3.0 Torr helium. All-ion fragmentation (AIF) was performed with alternating low/high collision energy on successive injections. Data processing was performed using a combination of Mobilion EyeOn (data conversion), Agilent IM-MS Browser (visualization), PNNL PreProcessor (demultiplexing/smoothing), and Agilent Mass Profiler (feature finding). All samples were prepared according to previous protocols.

RESULTS:
In the first example, we demonstrate a low-mass enabled SLIM platform for analysis of several steroid subclasses including anabolic androgens, endocrine hormones, and bile acids. The modified SLIM board geometry includes rounded turns which, coupled with helium buffer gas and higher RF frequency (1.2 MHz), improved sensitivity across all compounds surveyed. The resolving power of Rp ~100 (in helium) still provided adequate sensitivity to resolve structural and stereochemical isomers, such as testosterone/1-testosterone, corticosterone/21-deoxycortisol, and glycohyochenodeoxycholic acid/glycochenodeoxycholic acid. Studies are underway to investigate the quantitative performance (limits of detection, sensitivity, etc.) of these assays applied to relevant blood plasma and/or urine extracts. In the second example, we demonstrate that exploiting hydrazine chemistry through targeted derivatization (i.e., Girard’s reagents) can simultaneously improve sensitivity and selectivity via IM-MS/MS. For this demonstration, we focused on 11-oxosteroids relevant to several adrenal disorders, and showed that carbonyl derivatization yielded improved mobility separation and limits of detection. Furthermore, MS/MS also yielded structural information about functional group position and molecular stereochemistry, and location of derivatization(s). In the final example, we apply multiplexed acquisition IM-MS to a microbial system for metabolomic identifications. The multiplexed acquisition improved signal abundance by nearly an order of magnitude while the high-resolution demultiplexing software improved mobility resolution to ~200, which allowed us to identify numerous co-eluting isobaric/isomeric metabolites. This could be coupled with all-ion fragmentation to achieve 4-dimensional datasets: retention time, mobility, accurate mass, and fragmentation pattern for each feature identified.

CONCLUSION:
Combining traditional LC-HRMS/MS with several high-resolution IM strategies and chemical approaches provided added selectivity and sensitivity across wide range of steroids and other metabolites. This workflow shows tremendous promise both for untargeted/discovery-based metabolomics as well as high-throughput/targeted analyses where simultaneous speed and isobar/isomer separation is required.