HRdm 2.0: Maximize Your IM Resolution Without Sacrificing Drift Range, Mass Range, or Data Acquisition Time

Significance of Topic

The combination of ion mobility separation with mass spectrometry has become essential for probing complex mixtures in biological and chemical research. Accurate collision cross section (CCS) measurements aid in structural characterization of ions. Conventional drift tube IM-MS offers reliable CCS values but is constrained by limited resolving power and duty cycle when applied in untargeted, chromatography-linked workflows.

Study Objectives and Overview

This whitepaper introduces HRdm 2.0, a software enhancement for the Agilent 6560 Ion Mobility LC/Q-TOF platform. The goal is to elevate drift resolution up to 250 while preserving broad mass and drift range, short acquisition times, and precise CCS determination. The work builds on earlier HRdm versions by integrating high-resolution demultiplexing with peak deconvolution for improved sensitivity and resolution.

Methodology and Used Instrumentation

HRdm 2.0 employs multiplexed gating of ion packets using pseudo-random binary sequences (Hadamard transforms) to boost duty cycle to 50%. Through extensive mathematical modeling of the 6560 instrument, expected Gaussian drift peak shapes are predicted. Advanced maximum likelihood deconvolution is then applied to overlay theoretical peak profiles onto raw multiplexed data, sharpening peaks and enhancing signal-to-noise.

Used Instrumentation:

• Agilent 6560 Ion Mobility LC/Q-TOF system
• Agilent MassHunter Acquisition Software 11.0
• HRdm 2.0 high-resolution demultiplexing software
• PNNL PreProcessor for TOF transient interpolation
• Supporting tools: MassHunter IMMS Browser, Mass Profiler, Skyline

Main Results and Discussion

Multiplexing experiments demonstrated that 4-bit gating sequences increase instrument duty cycle and sensitivity compared to single-pulse mode. Interpolation of TOF transients (from 8,000 to 24,000 per second) yields smooth Gaussian drift peaks. When four leucine isomers were infused together, HRdm 2.0 achieved baseline resolution with resolving power near 200, a significant improvement over stair-step limited peaks from prior methods. Across tests, drift resolution improved from a baseline of 50 to as high as 250 without extending analysis time.

Benefits and Practical Applications

HRdm 2.0 enables fast, untargeted LC-IM-MS workflows with enhanced peak capacity, increased dynamic range, and accurate CCS determinations for complex samples. Short trapping times reduce space charge effects and detection saturation. The software processes thousands of LC-IM peaks on standard workstations, supporting discovery and QA/QC applications in proteomics, metabolomics, lipidomics, and industrial analytics.

Future Trends and Opportunities

• Extending multiplexing bit depth and refining deconvolution algorithms for even higher resolution
• Integration with real-time data acquisition for adaptive experimental control
• Broader adoption in hybrid workflows combining trapped ion mobility and drift tube separations
• Development of CCS databases and machine-learning models trained on high-resolution IM-MS data

Conclusion

HRdm 2.0 represents a breakthrough in drift tube IM-MS, delivering resolutions up to 250 while retaining full drift and mass range and rapid LC-compatible acquisition. This advance empowers researchers to resolve isomeric and complex mixtures in untargeted analyses and to obtain precise CCS values in high-throughput settings.

References

• Stow S et al Anal Chem 2017 89 9048-9055
• May J et al Anal Chem 2020 92(14) 9482-9492
• Causon T et al Anal Bioanal Chem 2019 411 6265-6274
• Clowers B et al Anal Chem 2008 80 2464-2473
• Bilbao AJ Proteome Res 2021 doi 10.1021/acs jproteome 1c00425