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

Importance of the Topic

Flavonoids are a key group of plant metabolites with diverse biological activities, making them critical targets in tea metabolomics. Confident identification of these compounds supports quality control, authentication, and nutritional studies of tea products. However, untargeted metabolomics workflows often struggle to extract deeply informative structural data from complex plant matrices, limiting the reliability of flavonoid annotation.

Study Objectives and Overview

This work aimed to develop a compound-class-specific workflow for the reliable annotation of flavonoids in tea extracts. The approach combined a high-quality curated spectral library of authentic flavonoid standards with a real-time library search (RTLS) strategy on an advanced Orbitrap Tribrid mass spectrometer. By guiding on-the-fly MSn acquisition toward relevant analytes, the method sought to expand the coverage of putative flavonoids in green, black and herbal teas.

Methodology

Sample Preparation

• Tea extracts (green, black, herbal) were prepared by hot‐water extraction and pooled for quality control.
• Blank and pooled samples generated inclusion/exclusion lists for intelligent data acquisition.

Chromatography

• UHPLC separation on a C18+ column (2.1 × 150 mm, 1.5 µm) at 55 °C, 200 µL/min flow rate.
• Gradient elution from 100 % aqueous to 98 % methanol with 0.1 % formic acid.
• 2 µL injection volume per run.

Data Acquisition

• AcquireX Deep Scan workflow: iterative inclusion/exclusion list generation for full‐scan MS1 (200–1000 m/z) and targeted MS2.
• Real-Time Library Search (RTLS) using an offline mzVault library to trigger higher-order MSn (up to MS5) on matching flavonoid spectra.
• Spectral library creation: MSn data from 40 flavonoid standards collected with HCD, CID and UVPD fragmentation, processed via Mass Frontier 8.0 and Compound Discoverer 3.3.

Used Instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific Orbitrap IQ-X Tribrid Mass Spectrometer
• Thermo Scientific Compound Discoverer 3.3 software
• Thermo Scientific mzVault 2.3 and Mass Frontier 8.0

Main Results and Discussion

Implementation of RTLS driven by a high-resolution MSn spectral library led to the annotation of nearly 100 additional putative flavonoids in tea extracts versus analysis without real-time guidance. Compound Discoverer identified over 590 flavonoid-like features with triggered MS3 spectra under RTLS conditions, compared to approximately 416 features without RTLS. Comparative volcano‐plot analysis highlighted differential flavonoid profiles between green and black teas, demonstrating improved depth and confidence in metabolite coverage.

Benefits and Practical Applications

• Significantly expanded flavonoid annotation in complex plant samples
• Improved structural confidence through guided MSn fragmentation
• Enhanced throughput by automating library‐driven acquisition
• Applicability to quality control, authentication and nutritional profiling in tea and other botanicals

Future Trends and Potential Applications

Advances in spectral library curation, coupled with artificial intelligence–driven spectral matching, will further streamline compound‐class-focused metabolomics. Integration of larger flavonoid and polyphenol libraries, addition of ion mobility separation, and dynamic RTLS thresholds may enhance specificity and depth. The approach holds promise for broader application to glycosides, alkaloids and other phytochemical classes in food, nutraceutical and environmental studies.

Conclusion

The combination of a curated HRAM MSn spectral library and real‐time library search on a Tribrid Orbitrap platform delivers a powerful workflow for flavonoid annotation in tea. Intelligent acquisition strategies like AcquireX Deep Scan and RTLS significantly increase the number and confidence of putative identifications, addressing key challenges in untargeted metabolomics of complex matrices.

References

No external literature list was provided in the source document.