Quantitative Analysis of Anthocyanins in Tea Leaves and Barley

Significance of the Topic

Anthocyanins are plant pigments responsible for red, purple and blue coloration in fruits, vegetables, tea leaves and cereals. They have attracted attention for their antioxidant properties and potential health benefits. Reliable quantification of anthocyanin glycosides in agricultural and food matrices supports quality control, nutritional studies and breeding programs aimed at improving functional food content.

Objectives and Study Overview

This work describes the development and validation of a rapid, robust HPLC method for simultaneous quantitation of four major anthocyanin glycosides (delphinidin-3-galactoside, delphinidin-3-glucoside, cyanidin-3-galactoside and chrysanthemin-3-glucoside) in tea leaves and barley. The method avoids acid hydrolysis by measuring glycoside forms directly and can be implemented on conventional HPLC systems within 25 minutes per run.

Methodology

Sample pretreatment involved weighing 1.0 g of crushed sample into a centrifuge tube, adding 15 mL of 0.5 % trifluoroacetic acid/methanol/water (40/60 v/v) and shaking at 250 rpm for 10 min. After centrifugation (10,000×g, 5 min), the supernatant was transferred to a 25 mL volumetric flask. A second extraction with 10 mL of the same solvent mixture was performed, and the combined solution was diluted to volume and filtered through a membrane before HPLC analysis.

Chromatographic separation used a reversed-phase C18 column with a gradient of 2 % formic acid in water (mobile phase A) and 2 % formic acid in a 1:1 water/acetonitrile mixture (mobile phase B). The gradient ran from 15 % B to 30 % B over 15 min, then to 100 % B by 20 min, returning to 15 % B by 25 min. The flow rate was 1.0 mL/min, column temperature 40 °C and detection wavelength 520 nm.

Used Instrumentation

• HPLC system: Shimadzu Nexera X3
• Column: Shim-pack GIST-HP C18, 150 mm × 4.6 mm, 3 μm
• Detector: UV-VIS at 520 nm (W lamp)
• Centrifuge capable of 10,000×g

Main Results and Discussion

Calibration curves for all four anthocyanins were linear over 0.20–20 µg/mL with R² ≥ 0.9999. A representative chromatogram at 10 µg/mL showed baseline separation within 25 min. Repeatability assessed on seven replicates yielded %RSD values of 1.08–2.03 % for tea extracts and 3.34–4.72 % for barley extracts, demonstrating high precision. Quantitative analysis of two tea cultivars (Yabukita and Sunrouge) and one barley variety (Daishimochi) indicated that Sunrouge contained 0.1876 mg/100 g delphinidin-3-galactoside, 0.0746 mg/100 g delphinidin-3-glucoside, 0.0978 mg/100 g cyanidin-3-galactoside and 0.0307 mg/100 g chrysanthemin-3-glucoside, while other samples were below the lower limit of quantification or not detected.

Benefits and Practical Applications

• Simultaneous analysis of four anthocyanin glycosides without hydrolysis simplifies sample preparation.
• Quantification in glycoside form preserves native profiles and avoids complex derivatization.
• Use of standard HPLC equipment facilitates adoption in routine QA/QC laboratories.
• Short analysis time (25 min) supports medium-throughput screening of plant and food samples.

Future Trends and Opportunities

Extending this method to other anthocyanin-rich matrices, coupling with mass spectrometry for enhanced sensitivity and structural confirmation, and integrating high-throughput sample preparation techniques may broaden its application. Portable or benchtop HPLC systems could enable field analysis of crop samples during breeding or harvest. Further exploration of anthocyanin stability, bioavailability and functionality in processed foods represents an important research direction.

Conclusion

A rapid, accurate and precise HPLC method for direct quantitation of four anthocyanin glycosides in tea leaves and barley was developed and validated. The approach uses conventional instrumentation, obviates hydrolysis and delivers high linearity and repeatability. It offers a practical tool for quality assessment and research on functional components in agriculture and food products.

Reference

1. Oki, T., Analysis of anthocyanins in purple sweet potato, Food Functionality Evaluation Manual Vol. I (Revised 2nd Edition).
2. Oki, T., Analysis of anthocyanins in purple sweet potato, Food Functionality Evaluation Manual Vol. IV.
3. Masada, S., Uchiyama, N., Goda, Y., Hakamatsuka, T., An analysis of anthocyanins in “Foods with Functional Claims” containing bilberry extracts, Japanese Journal of Food Chemistry and Safety, Vol. 24(1), 32–38 (2017).