Targeted High Resolution Quantification with Tof-MRM and HD-MRM

Significance of the Topic

High‐resolution mass spectrometry (HR‐MS) combined with advanced ion mobility separations delivers rich qualitative data across fields such as proteomics, metabolomics, and biopharmaceutical analysis. Incorporating targeted quantification on the same HR‐MS platform enhances workflow efficiency by avoiding method migration between instruments and offers superior selectivity based on accurate mass, isotope patterns, and drift time separation.

Objectives and Study Overview

This study introduces two targeted quantification workflows—Tof‐MRM and HD‐MRM—implemented on the Waters SYNAPT G2‐Si system. The goals are to demonstrate routine quantification at sub‐picogram and femtogram levels, to compare sensitivity gains against conventional full‐scan HR‐MS, and to highlight the benefits of combining multiple selectivity dimensions in quantitative assays.

Methodology and Workflow

Tof‐MRM and HD‐MRM synchronize the TOF pusher timing with selected m/z ranges to achieve a 100% detection duty cycle. Two enhancement modes are used:

• Target Enhancement: Narrow m/z windows with optimized collision energy for each transition.
• Wideband Enhancement: Full m/z range monitoring leveraging ion mobility separation for uniform collision energy.

Both methods integrate RADAR (m/z, RT, intensity) and HD‐RADAR (m/z, RT, drift time, intensity) to capture full‐scan accurate mass data at regular intervals, facilitating rapid method development and matrix effect evaluation.

Used Instrumentation

• Waters SYNAPT G2‐Si High‐Resolution QTOF Mass Spectrometer
• TriWave Ion Mobility Device
• ACQUITY UPLC System with 2.1 × 100 mm Column
• TargetLynx Software for Automated MRM Data Processing

Main Results and Discussion

Comparative experiments with testosterone in solvent demonstrated:

• At 1.25 pg on column, Tof‐MRM achieved a >4× improvement in signal‐to‐noise versus full‐scan MS.
• At 250 fg, the analyte peak was detectable by MRM yet undetectable by standard MS, establishing the LOD for Tof‐MRM.

HD‐MRM further leveraged Wideband Enhancement to enrich all fragment ions of a 527.75+ peptide at 500 amol, enabling post‐acquisition transition selection without sensitivity loss. Ion mobility separation in HD‐MRM also resolved isobaric interferences and permitted fragmentation after drift time gating.

Benefits and Practical Applications

• Up to 10× sensitivity increase for targeted analytes versus conventional HR‐MS full scan.
• Enhanced selectivity by combining m/z, collision fragment transitions, accurate mass, isotope ratios, and ion mobility drift times.
• Streamlined workflows: compound‐driven method editor and RADAR scanning support rapid method optimization.
• Potential to quantify trace analytes in complex matrices without instrument switching.

Future Trends and Opportunities

Emerging applications may include multi‐omics quantitation, real‐time quality control in biomanufacturing, and environmental trace analysis. Further integration of data‐independent acquisition with targeted duty cycle enhancement and machine‐learning–driven transition selection could push detection limits lower and automate method refinement across diverse sample types.

Conclusion

Tof‐MRM and HD‐MRM on the SYNAPT G2‐Si platform deliver sub‐picogram to femtogram quantification with unmatched selectivity by unifying traditional MRM, accurate mass, and ion mobility. This approach simplifies workflows, reduces method transfer challenges, and extends the quantitative capabilities of high‐resolution instruments.

References

• Tomczyk N, Wallace A, Richardson K, Grzyb A, Wildgoose J. Targeted High Resolution Quantification with Tof‐MRM and HD‐MRM. Waters Corporation; 2013.