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Abstract INTRODUCTION:
With speed of analysis in mind, ion mobility (IM) has presented intriguing potential for clinical applications due to its ability to separate isomers on a millisecond timeframe. As an alternative and/or complement to traditional chromatographic separations, some of the remaining challenges to its routine implementation in the clinical lab include sensitivity due to low ion utilization and resolution to differentiate stereoisomers. While significant advances in high-resolution ion mobility (HRIM), including the recent introduction of structures for lossless ion manipulations (SLIM), have allowed improvement upon the latter, sensitivity (especially for low mass analytes) remains a barrier. This presentation will focus on three major approaches, applied in tandem, to overcome this challenge using a high-throughput integrated LC-IM-MS/MS workflow: use of helium buffer gas, rounded SLIM turns, and targeted chemical derivatizations.
METHODS:
All experiments were performed using a recently developed commercially available structures for lossless ion manipulations (SLIM) platform (MOBILion Systems, Inc.) tailored for low mass applications by introducing rounded turns in contrast to the typical 90° geometry.[1] The SLIM is coupled to an Agilent 1290 UHPLC for chromatographic separations and an Agilent 6546 QTOF to provide both high-resolution and tandem mass spectrometry. Pooled human urine and plasma samples were prepared by spiking isotopically labeled standards of target analytes, concentrated using solid phase extraction (SPE), dried down under nitrogen, and reconstituted in mobile phase. Chemical derivatizations (when performed) were done using established methods following SPE. Short (2-5 min) modified LC gradients were developed to increase throughput. Ion mobility separations were performed in helium buffer gas at 2.5 Torr and ambient temperature, and traveling wave (TW) conditions were optimized for each molecular class. Following acquisition and processing using software packages from MOBILion Systems (EyeON), Pacific Northwest National Laboratory (PNNL PreProcessor), and Agilent (IM-MS Browser), all data was interpreted for quantification using Skyline.
RESULTS:
The goal of this project was to develop a high-throughput LC-IM-MS/MS method demonstrating quantification of isomers at clinically relevant concentrations in representative biological matrixes. First, a simple LC method (with gradient as short as 2 mins) was developed to remove interferences outside of the target polarity range. This approach has been applied previously[2] and provided only minimal separation of isomeric analytes. However, coupling this with SLIM ion mobility yielded the opportunity to rapidly differentiate even stereoisomers. Analyte classes surveyed included endocrine hormones, anabolic steroids, and drug metabolites. Additionally, we were able to precisely measure collision cross sections (CCS) for standards of all analytes and use these values to improve confidence of identification by further filtering data extraction using this descriptor. This included challenging isomer pairs such as methyclostebol and fluoxymesterone (CCS 179.0 and 177.3 Å2, respectively), 7α-hydroxydehydroepiandrosterone and methandriol (CCS 178.1 and 179.2 Å2, respectively), and boldenone and androstenedione (CCS 170.9 and 171.8 Å2, respectively), demonstrating the ability to differentiate species with CCS difference as little as 0.53%. These compounds were quantified by constructing calibration curves and in most cases provided limits of detection in the pg/mL range. Where sensitivity was insufficient, targeted chemical derivatizations including using carbonyl-diimidazole and various Girard’s reagents were demonstrated to both improve ionization efficiency and in some cases improve mobility resolution, which has been previously shown for several classes of steroids.[3,4]
CONCLUSION:
Endocrine hormones, anabolic steroids, and drug metabolites in biological matrix were quantified using a newly developed high-throughput LC-IM-MS/MS. In most cases, sensitivity and limits of detection (~pg/mL) were adequate to meet clinical testing requirements. However, targeted chemical derivatizations were also implemented for some challenging compounds, providing both improved sensitivity and IM separations. This approach continues to push ion mobility-mass spectrometry towards implementation in the clinical field, now offering requisite sensitivity to couple with its superior separation speeds.
REFERENCES:
1. Deng, L., et al., Rounded Turn SLIM Design for High-Resolution Ion Mobility Mass Spectrometry Analysis of Small Molecules. Anal Chem, 2024. 96(51): p. 20179-20188.
2. Wedge, A., et al., Development of a rapid, targeted LC-IM-MS method for anabolic steroids. J Am Soc Mass Spectrom, 2023. 34(8): p. 1708-1714.
3. Neal, S.P., et al. Improved analysis of derivatized steroid hormone isomers using ion mobility-mass spectrometry (IM-MS). Anal Bioanal Chem, 2023. 415(27): p. 6757-6769.
4. Velosa, D.C., et al., Improved ion mobility separation and structural characterization of steroids using derivatization methods. J Am Soc Mass Spectrom, 2022. 33(9): p. 1761-1771.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
