Integrating Ion Mobility into Routine Drug Metabolite Identification Studies Using the Vion IMS QTof Mass Spectrometer

Significance of the Topic

Ion mobility spectrometry (IMS) coupled with high-resolution mass spectrometry (HRMS) addresses key challenges in drug metabolite identification by improving spectral clarity, resolving isobaric and co-eluting species, and providing collision cross section (CCS) values as robust molecular descriptors. Incorporation of IMS into routine workflows enhances confidence in structural assignments and enables detection of low-abundance metabolites in complex biological matrices.

Objectives and Study Overview

This application study demonstrates the integration of IMS into routine drug metabolism analyses using the Waters ACQUITY UPLC I-Class system, Vion IMS QTof mass spectrometer, and UNIFI software. The primary goals are to show how data independent acquisition with HDMSE improves metabolite tracking, to illustrate CCS measurement stability across varying conditions, and to highlight enhanced identification of co-eluting and isomeric metabolites.

Methodology and Instrumentation Used

Samples of nefazodone and buspirone were incubated with rat hepatocytes over a time course, quenched with acetonitrile, and analyzed by UPLC-IMS-QTof in HDMSE mode. Key conditions included:

• LC: ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 µm), 45 °C, gradient run times of 5, 10, and 15 min
• MS: Vion IMS QTof with ESI+, source 120 °C, desolvation 450 °C, drift gas enabled, HDMSE acquisition at 0.1 s scan time
• Data Processing: UNIFI software for automatic CCS calibration and drift-time alignment of precursor and fragment ions

Main Results and Discussion

IMS-resolved spectra showed substantial background reduction and revealed diagnostic fragments for dihydroxylated glucuronides of buspirone and nefazodone. Drift-time filtered extracted ion chromatograms exhibited up to threefold improvement in signal-to-noise ratios. CCS values for nefazodone remained constant (0.2 % RSD) over three orders of magnitude concentration change and across different chromatographic gradients, enabling reliable metabolite tracking. Co-eluting metabolites, including hydroxylated and desaturated species, were deconvolved based on distinct CCS values, ensuring accurate structural elucidation without lost identifications.

Benefits and Practical Applications

IMS integration into routine workflows reduces data processing time, increases confidence in identifications, and extends detection capabilities for trace metabolites. CCS measurements serve as orthogonal identifiers that are matrix- and concentration-independent. HDMSE acquisition ensures comprehensive fragment ion collection for all ions, eliminating reliance on data dependent MS/MS.

Future Trends and Potential Applications

Expansion of CCS libraries and standardized CCS databases will facilitate cross-laboratory metabolite annotation. Real-time IMS-HRMS screening, machine learning-based CCS prediction, and coupling IMS with other separation techniques promise further improvements in throughput and structural insight. Applications may extend to large-scale metabolomics, environmental monitoring, and quality control in drug development.

Conclusion

The integration of ion mobility into routine drug metabolite identification using the Vion IMS QTof and UNIFI software offers a streamlined, high-confidence approach for resolving complex metabolite profiles. Automatic CCS generation and HDMSE acquisition deliver cleaner spectra, enhanced sensitivity, and reliable tracking of isomeric species, driving efficiency in DMPK studies.

References

1. Wrona M, Naughton S, Alelyunas Y, Mortishire-Smith R, Kirk J. Metabolite Identification Solution: Working with Large Preclinical Multispecies Data Sets. Waters Application Note 720006106EN, October 2017.
2. Holdsworth C, Clayton R, Robinson H, Lord-Mears C, Kendrick J. Utilization of Ion Mobility Enabled Collision Cross Section Measurements for a Comparison of Metabolites Across Differing Chromatographic Methods. Poster DMDG, 2016.
3. Clayton R, Holdsworth C, Tomczyk N, Plamer M, Hewitt D, Weston D. Resolution and Characterization of Co-eluting Metabolites by Collision Cross Section Measurements Using a Novel Geometry Traveling Wave IMS QTof Mass Spectrometer. Poster ASMS, 2016.