Real-Time Collisional Energy Optimization on the Orbitrap Fusion Platform for Confident Unknown Identification

Significance of Real-Time Collision Energy Optimization

Accurate fragmentation of small molecules is critical in complex sample analysis, as each compound class often requires distinct collision energies for optimal fragmentation. Traditional fixed or stepped collision energy approaches may miss key fragment ions or require multiple sample reinjections. Real-time collision energy optimization addresses this by dynamically selecting the ideal energy for each precursor ion during acquisition, enhancing identification confidence and throughput.

Objectives and Study Overview

This work introduces Assisted Collision Energy (CE) on the Orbitrap Fusion Tribrid platform, aiming to determine optimal collision energies in real time. The study evaluates:

• The implementation of hidden ion trap scans to build pseudo-breakdown curves.
• Impact on duty cycle and acquisition speed.
• Performance in a complex herbal extract application.

Methodology and Instrumentation

The approach embeds five rapid ion trap scans around each precursor (±2 Th) at incrementally increasing CE values. The unreacted precursor intensity is monitored against a threshold (e.g., 20%). Once crossed, the corresponding CE is applied to the subsequent high-resolution FTMS2 scan.

Instrumentation:

• Thermo Scientific Orbitrap ID-X Tribrid mass spectrometer, software version 3.1.
• Thermo Scientific Vanquish UHPLC with Accucore C18 column (2.1 × 100 mm).
• Source: HESI for standard compounds and herbal extract.

Main Results and Discussion

• Assisted CE produced pseudo-breakdown curves within the parallel FT transient, minimally affecting duty cycle. Comparative tests on top-speed methods (3 s cycle at 120K/60K resolution) showed less than 5% increase in cycle time.
• A database of 5,575 compounds revealed a broad distribution of optimal HCD energies (mean 43 ± 27 NCE), underlining the need for individualized optimization.
• In a blended ginseng extract, Assisted HCD improved FTMS3 signal-to-noise by ~30% versus stepped HCD, yielding richer fragment spectra for confident compound characterization.

Benefits and Practical Applications

• Enhanced spectral quality and identification confidence without sample reinjection.
• Increased throughput by reducing repeat analyses and optimizing each compound in a single run.
• Available as a feature in standard method editor for all Tribrid instruments (v3.1).

Future Trends and Possibilities

• Integration with data-dependent acquisition workflows to further prioritize targets based on real-time spectral quality metrics.
• Expansion of assisted CE strategies to alternative dissociation modes (e.g., ETD, UVPD).
• Machine learning-driven prediction of breakdown curves to reduce the number of required ion trap scans.

Conclusion

Assisted Collision Energy on the Orbitrap Fusion platform represents a robust solution for real-time optimization of fragmentation conditions. By leveraging rapid ion trap scans during the parallel FTMS transient, this method delivers higher-quality MS/MS spectra and improved confidence in unknown identification, all with minimal impact on instrument duty cycle.

References

• Bailey D.J., McAlister G.C., Sharma S., Remes P.M., Tautenhahn R., Ntai I. Real-Time Collisional Energy Optimization on the Orbitrap Fusion Platform. Thermo Fisher Scientific, 2018.
• Thermo Fisher Scientific. Orbitrap ID-X Tribrid Mass Spectrometer Technical Note, 2018.