High quality curated HRAM MSn spectral libraries and real time library search for the confident annotation of metabolites

Importance of the Topic

Confident annotation of metabolites in untargeted metabolomics is critical for converting raw mass spectrometry data into meaningful biological insights. Flavonoids represent a large and functionally important class of plant-derived compounds with significant roles in nutrition, pharmacology, and quality control. An analytical workflow that selectively deep-scans and accurately annotates flavonoids improves coverage, confidence, and relevance of metabolomic studies.

Goals and Study Overview

This work aimed to develop a compound-class–specific deep scan workflow by combining a high-quality curated High Resolution Accurate Mass (HRAM) MSn spectral library with Real Time Library Search (RTLS) on a Thermo Scientific™ Orbitrap IQ-X™ Tribrid™ mass spectrometer. The study focused on:

• Building a flavonoid spectral library from authentic standards.
• Implementing RTLS to trigger higher-order MSn acquisition for library-matched flavonoid candidates in real time.
• Comparing the number of putative flavonoid annotations in tea extracts with and without RTLS.

Materials, Methods and Instrumentation

Sample Preparation:
Green, black, and herbal tea infusions were prepared in replicates, pooled for quality control, and analyzed alongside blanks.

UHPLC Conditions:

• System: Thermo Scientific™ Vanquish™ Horizon.
• Column: Accucore™ Vanquish™ C18+ (2.1 × 150 mm, 1.5 μm) at 55 °C.
• Mobile Phases: 0.1% formic acid in water (A) and methanol (B).
• Gradient: 100% A to 98% B over 9 min, returning to initial conditions by 15 min.
• Flow Rate: 200 μL/min; Injection Volume: 2 μL.

MS Acquisition and Library Building:
MS1 full scans and MS2 were acquired in a 1.2 s cycle using AcquireX deep-scan mode to generate inclusion/exclusion lists. Authentic flavonoid standards (40 compounds) underwent multiple fragmentation strategies (HCD, CID, UVPD) to build an HRAM MSn spectral library in mzVault™ 2.3 via Compound Discoverer™ 3.3 and Mass Frontier™ 8.0.

Real Time Library Search (RTLS):
The Met-IQ template on the IQ-X™ system compared incoming MS2 spectra against the mzVault library. Matching spectra above similarity thresholds automatically triggered MS3 acquisition for enhanced structural elucidation.

Results and Discussion

Integration of the curated spectral library with RTLS led to the annotation of nearly 100 additional putative flavonoids in tea extracts compared to workflows without RTLS. RTLS-guided MS3 acquisition enriched data quality by focusing higher-order fragmentation on structurally relevant candidates. Differential analysis between green and black tea by Compound Discoverer™ 3.3 demonstrated improved coverage and fold-change insights for flavonoid metabolites.

Benefits and Practical Applications

• Enhanced depth and confidence in flavonoid annotation for natural product research.
• Strong applicability in quality control of botanicals, functional food profiling, and nutraceutical development.
• Streamlined workflow reduces data complexity and manual filtering efforts.

Future Trends and Applications

Advances may include integration with machine learning for spectral prediction, expansion of library content to broader compound classes, real-time cloud-based library sharing, and coupling intelligent acquisition strategies with emerging fragmentation techniques to further improve metabolome coverage.

Conclusion

The combined use of a curated HRAM MSn flavonoid library and RTLS on the Orbitrap IQ-X™ platform delivers a powerful, targeted deep-scan workflow. This approach significantly increases the number and confidence of flavonoid annotations in complex biological matrices while maintaining high-throughput capability.

References

1. Deshpande R, Amer B, Hermanson D, et al. High quality curated HRAM MSn spectral libraries and real time library search for the confident annotation of metabolites. Thermo Fisher Scientific; 2021.
2. Thermo Fisher Scientific. Orbitrap IQ-X Tribrid Mass Spectrometer User Guide.
3. Thermo Fisher Scientific. Compound Discoverer 3.3 Software Manual.
4. MetaSci Inc. Flavonoid Library Catalog.