Quantitative Analysis of Carotenoids in Tea Leaves

Importance of the topic

Carotenoids are lipid-soluble pigments possessing strong antioxidant activity and play key roles in human health and plant physiology. Reliable quantification in tea leaves supports nutritional evaluation and quality control of agricultural products.

Objectives and study overview

This work aimed to establish a rapid, simple, and accurate HPLC method for simultaneous determination of seven carotenoids in tea leaves, based on Japanese Agricultural Standard procedures and Shimadzu Nexera X3 system.

Methodology and Pretreatment

Samples (100 mg) were saponified with ethanolic pyrogallol and KOH at 70 °C, followed by hexane/ethyl acetate extraction. To preserve astaxanthin, enzymatic hydrolysis is required as the standard saponification degrades this compound. Extracts were dried under nitrogen, redissolved with BHT in ethanol, filtered, and analyzed by HPLC.

Used Instrumentation

• Nexera X3 high performance liquid chromatograph
• YMC Carotenoid column (150 mm × 4.6 mm I.D., 3 µm)
• Mobile phases: 5 g/L ammonium acetate in methanol/acetonitrile (4/15, v/v) and ethanol
• Gradient: 5–50% B (0–20 min), 50–95% B (20.01–25 min), 95–5% B (25.01–30 min)
• Flow rate 1.0 mL/min, column temperature 40 °C, injection volume 10 µL
• PDA detection at 438 nm (neoxanthin), 445 nm (α-carotene, lutein), 450 nm (β-carotene, β-cryptoxanthin, zeaxanthin), and 470 nm (astaxanthin)

Results and discussion

Calibration curves for all seven standards exhibited excellent linearity (R2 ≥ 0.999) over defined ranges. Repeatability tests (n = 7) in Yabukita tea showed RSDs below 10% for all quantifiable carotenoids. Quantitative analysis of Yabukita and Benifuuki cultivars revealed cultivar-dependent variations, with Benifuuki displaying higher levels of α-carotene, β-carotene, neoxanthin, and lutein compared to Yabukita.

Benefits and practical applications

The developed HPLC method enables rapid profiling of key carotenoids in tea leaves, facilitating nutritional assessment, breeding programs, and quality control in agricultural and food industries.

Future trends and potential applications

Future improvements may include coupling with mass spectrometry for enhanced sensitivity, expanding the method to diverse plant matrices, and automating sample preparation for high-throughput screening.

Conclusion

A robust HPLC method using Shimadzu Nexera X3 allows simultaneous, accurate quantification of major carotenoids in tea leaves, demonstrating reliable performance and clear differentiation between tea cultivars.

References

• Japanese Agricultural Standard Determination of the lutein in spinach – High-performance liquid chromatographic method (JAS 0008)
• Standards for Labelling of Food Items (Consumer Affairs Agency Notification No. 139, 2015)
• Analytical Manual for Standard Tables of Food Composition in Japan 2015, Chapter 3 Vitamins
• Japanese Agricultural Standard Determination of the β-cryptoxanthin in Satsuma Mandarin – High-performance liquid chromatographic method (JAS 0003)