Differential analysis of natural products using fast polarity switching TOFMS acquisition with high mass accuracy and metabolomics-based approach

Importance of the Topic

Metabolomics-driven analysis of natural products enables comprehensive profiling of complex sample matrices influenced by factors such as origin, cultivation, harvest time and extraction method. High-mass-accuracy, fast-polarity-switching mass spectrometry streamlines the capture of both positive and negative ion species, facilitating biomarker discovery, quality control and comparative studies across diverse plant-derived substances.

Study Objectives and Overview

This study aimed to integrate ultra-high-performance liquid chromatography (UHPLC) with fast polarity-switching quadrupole ion trap time-of-flight mass spectrometry (QIT-TOF MS) and multivariate metabolomics to:

• Profile water extracts of kakkonto from four commercial sources.
• Demonstrate metabolite differentiation by origin and degradation status.
• Identify marker compounds with high mass accuracy and MSn data.

Methodology and Instrumentation

Sample Preparation:
Water extracts of kakkonto (500 mg in 5 mL water) were ultrasonicated, centrifuged, filtered (0.45 μm) and diluted 20×. Quality control pools were generated from sample aliquots.

Použitá instrumentace:

• UHPLC systems: Nexera and Prominence UFLC.
• Column: Shim-pack XR-ODS II (100 × 2.0 mm, 2.2 μm).
• Mass Spectrometer: LCMS-IT-TOF with electrospray ionization and fast polarity switching (100 ms).
• Mobile Phase: Water/ACN with 0.1% formic acid; flow 0.45 mL/min; column at 45 °C.
• Scan Range: m/z 120–1500; probe ±4.5 kV; temperatures 250 °C (CDL) and 200 °C (desolvation).
• Software: Profiling Solution for data alignment; SIMCA-P+ v12 for PCA and OPLS-DA.

Key Results and Discussion

Detection & Identification:
Approximately 6400 positive and 4500 negative ion features were captured within a 15-min UHPLC run. Known constituents such as puerarin, paeoniflorin, glycyrrhizin and flavonoids were confirmed with <4 ppm mass accuracy and MSn fragmentation. Formula prediction matched ions (e.g. m/z 419.0979 as C20H20O10).

Statistical Analysis:
Principal component analysis of pooled QC and sample data showed tight clustering of QC injections and clear separation of samples by origin. OPLS-DA on heated vs non-heated extracts highlighted specific degradation markers (m/z 315.072, 419.098, 469.056) and demonstrated the method's power for stability studies.

Benefits and Practical Applications

• Rapid, high-throughput profiling of complex herbal matrices.
• Simultaneous capture of positive and negative ions improves coverage.
• Accurate biomarker discovery for quality control and authenticity assessment.
• Applicability to food safety, industrial chemicals and TCM research.

Future Trends and Applications

Advancements may include integration with larger metabolite databases, automated MSn data mining, higher-throughput chromatography, and application to broader natural product libraries. Combining environmental metadata with metabolomic fingerprints will enhance provenance tracking and product standardization.

Conclusion

The fast polarity-switching UHPLC-ESI-QIT-TOF MS workflow, coupled with multivariate metabolomics, enables detailed differential analysis of natural product extracts. This approach affords high-confidence identification of markers, supports quality control, and can be extended to degradation studies and diverse application areas.

References

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