Multiple C-Trap or HCD Fills as a Tool for Massive Parallelization of Orbitrap Mass Spectrometry – A New Concept for Targeted Mass Analysis

Importance of Spectrum Multiplexing

Spectrum multiplexing is a transformative approach that greatly increases throughput and dynamic range in Orbitrap mass spectrometry.
By utilizing idle times between scans to sequentially inject multiple precursor ions, this technique enhances analytical speed without sacrificing resolution or sensitivity.

Objectives and Study Overview

This work implements and evaluates spectrum multiplexing on a Thermo Scientific Q Exactive benchtop Orbitrap LC-MS/MS.
The primary goals are to demonstrate stepwise increases in scan speed, assess dynamic range improvements, and validate stepped collision energy experiments.
A targeted analysis of a 48-compound pesticide mixture serves as a real-world application.

Methodology and Instrumentation

• Instrument setup: Q Exactive hybrid quadrupole-Orbitrap with S-lens electrospray source, quadrupole mass filter, C-Trap, and HCD collision cell.
• Spectrum multiplexing principle: Multiple selected precursors are co-injected into the C-Trap (or HCD cell) during idle periods, followed by a single high-resolution Orbitrap detection.
• Multiplexing parameters: Up to 10 sequential fills per scan; stepped collision energy achieved by repeated injections at different energies.

Key Results and Discussion

• Scan speed enhancement: More than 48 precursors per second at standard resolutions; rates exceeding 100 precursors per second in 8-plex mode at 17,500 resolving power.
• Dynamic range boost: Up to 320,000 analytical dynamic range achieved in a single Orbitrap scan via spectrum multiplexing.
• Stepped collision energy: Ability to combine multiple HCD energy spectra into one composite spectrum without loss of acquisition speed.
• Targeted pesticide analysis: Four-fold multiplexing of 48 pesticides yielded complete coverage with cycle times under one second and high signal-to-noise ratios.

Practical Benefits and Applications

• High-throughput workflows: Ideal for (ultra)fast chromatography, significantly increasing sample throughput.
• Analytical flexibility: Facilitates exploration of optimal fragmentation energies for unknown compounds in a single experiment.
• Trace compound detection: Extended dynamic range supports sensitive analysis of low-abundance analytes in complex matrices.

Future Trends and Opportunities

• Quantitative performance: Systematic evaluation of linearity and limits of quantitation across different multiplexing levels.
• Real-time acquisition strategies: Integration with adaptive control and machine-learning algorithms to optimize precursor selection on the fly.
• Expanded applications: Potential for large-scale screening in pharmaceutical, environmental, and food safety laboratories.

Conclusion

Spectrum multiplexing on benchtop Orbitrap instruments offers a substantial advancement in MS/MS capability, delivering unprecedented throughput, dynamic range, and fragmentation flexibility.
This method stands to accelerate both targeted quantitation and discovery-driven analyses in complex sample matrices.