Evaluating Dynamic Range of High Resolution Demultiplexing of Drift Tube Ion Mobility - Mass Spectrometry Analysis

Significance of the Topic

Ion mobility mass spectrometry combines gas phase separation based on collision cross section with accurate mass analysis to provide complementary information on molecular size and structure. Improved resolving power and dynamic range are crucial for distinguishing isomeric species in complex mixtures. Such enhancements advance targeted and untargeted analyses in fields ranging from lipidomics to pharmaceutical quality control.

Objectives and Study Overview

This work evaluates dynamic range gains achieved by applying high resolution demultiplexing to drift tube ion mobility data. Three isomeric pairs of hydroxyeicosatetraenoic and epoxyeicosatrienoic acids with CCS separations of 1.7, 2.7 and 3.2 percent were analyzed across relative concentrations from equimolar to 1:200 dilution. The performance of standard resolution mode (resolving power 55) was compared to high resolution demultiplexing (HRdm, resolving power over 200).

Methodology and Instrumentation

• Sample Preparation: Equimolar mixtures of isomer pairs were serially diluted in LC MS grade methanol to establish dynamic range limits.
• Liquid Chromatography: Agilent 1290 Infinity II UHPLC performed flow injection at 0.2 mL per minute using a mobile phase of 70 percent methanol in water.
• Ion Mobility MS: Agilent 6560B IM QTOF operated in positive ion mode over a 60 millisecond drift time window, summing 20 IM MS frames per 1.2 second scan.
• Data Processing: Raw data were interpolated with PNNL PreProcessor drift bin interpolation, demultiplexed with HRdm 2.1.4, feature detected in IM MS Browser and CCS calibrated using single field tune mix infusion.

Major Results and Discussion

• Resolution Modeling: Simulations demonstrated that HRdm resolving power near 200 achieves complete valley separation for all isomer spacings, even at low relative abundance, whereas standard resolution mode showed limited separation.
• Analytical Measurements: Standard mode separated 2.7 percent spaced isomers to 1:8 dilution and 3.2 percent spaced pairs to 1:25. HRdm extended detection limits to 1:16 for 1.7 percent spacing, 1:100 for 2.7 percent and 1:200 for 3.2 percent.
• Resolving Power Dependence: HRdm resolving power decreased from approximately 200 to 100 at lowest signal levels, indicating dependence on available ion signal, while standard mode remained constant near 55.
• CCS Precision: Maximum CCS shifts remained below one percent across the full dilution series, confirming high precision CCS assignments with HRdm even at low analyte abundance.

Benefits and Practical Applications

• Extended Dynamic Range: Enables quantitative detection of low abundance isomeric species in complex matrices.
• Compatibility with Untargeted Workflows: Maintains full mobility range and acquisition rate for high throughput analyses without sacrificing resolution.
• Single Run Multiplexing: Provides both standard and high resolution data from the same acquisition, facilitating flexible data mining.

Future Trends and Opportunities

Ongoing algorithmic refinements in demultiplexing and regularization are expected to further boost resolving power and dynamic range. Integration with ultra high performance chromatography and capillary electrophoresis could expand coverage in metabolomics and lipidomics. Implementation of real time processing and machine learning based acquisition may enable adaptive resolution optimization for targeted analytes.

Conclusion

High resolution demultiplexing significantly enhances isomer separation, dynamic range and CCS precision in drift tube ion mobility mass spectrometry without compromising throughput. These advances support more comprehensive and quantitative analyses across biomolecular research environmental monitoring and industrial quality control.

References

1. J C May et al Anal Chem 2020 92(14) 9482 9492
2. Bilbao et al Journal of Proteome Research 2021