Quantitative Analysis of Flavonols in Tea Leaves

Significance of the Topic

Flavonols are important polyphenolic constituents in tea leaves associated with antioxidant and health-promoting properties. A rapid, accurate method to quantify both glycoside and aglycone forms without hydrolysis streamlines quality control, cultivar evaluation, and functional food research.

Objectives and Study Overview

The study aimed to establish a simple high-performance liquid chromatography method for simultaneous quantification of eight key flavonols (kaempferol, quercetin, isoquercitrin, hyperoside, rutin, myricetin, myricetin 3-glucoside, myricetin 3-galactoside) in two tea cultivars (Yabukita and Benifuuki) without prior hydrolysis.

Methodology and Instrumentation

• Sample preparation: 250 mg crushed tea leaves extracted with 80% methanol (sonication 30 min), centrifuged, diluted 10-fold with water, filtered.
• HPLC system: Shimadzu Nexera X3 equipped with Shim-pack GIST C18 column (150 × 4.6 mm, 3 μm).
• Mobile phases: A = 0.1% formic acid in water, B = acetonitrile; gradient from 15% to 95% B (0–24 min), re-equilibration to 15% B (24–30 min).
• Operating conditions: flow rate 1.0 mL/min, column temperature 40 °C, injection volume 10 μL, UV detection at 370 nm.

Main Results and Discussion

• Calibration: all analytes exhibited linearity across 0.1–20 µg/mL (quercetin 0.1–2 µg/mL) with R² ≥ 0.997.
• Repeatability: relative standard deviations for glycosides ranged below 3.4%, aglycones were below the lower limit of quantification.
• Tea leaf analysis: Isoquercitrin, hyperoside, rutin, and myricetin glycosides were detected at 0.13–1.01 mg/g; aglycone forms were generally below quantification limits.
• Benifuuki showed higher levels of rutin and myricetin glycosides compared to Yabukita.

Benefits and Practical Applications

• Eliminates time-consuming hydrolysis, preserving native glycoside profiles.
• Offers rapid, high-throughput analysis suitable for quality control and cultivar assessment.
• Supports research on functional components in agricultural and food products.

Future Trends and Potential Applications

• Integration with automated sample handling for large-scale screening.
• Extension to other polyphenol classes and diverse plant matrices.
• Coupling with mass spectrometry for enhanced structural characterization.
• Application in nutraceutical development and pharmacokinetic studies.

Conclusion

The developed HPLC-UV method on the Nexera X3 platform provides reliable, simultaneous quantification of major flavonols in tea leaves without hydrolysis, demonstrating strong linearity, repeatability, and suitability for cultivar comparison and functional analysis.

Reference

1. Monobe et al. Food Science and Technology Research. 2015;21(3):333-340.
2. Nobuya Shirai. Nippon Shokuhin Kagaku Kogaku Kaishi. 2018;65(7):357-362.