Automated Method Development in HPLC for the Quantitative Determination of Catechins in Tea

Importance of the topic

The analysis of catechins in tea is critical due to their health-promoting antioxidant properties and widespread consumption of tea varieties worldwide. Reliable quantification of these phenolic compounds supports quality control, nutritional studies and product authentication in both research and industrial laboratories.

Cíle a přehled studie

This work aimed to establish a rapid, robust and sensitive HPLC-UV method for simultaneous determination of seven major catechins in white, green and black tea. A software-driven automated method development strategy was applied to minimize analyst effort and accelerate gradient optimization across multiple stationary and mobile phase combinations.

Použitá metodika a instrumentace

The development workflow consisted of three key stages:

• Column and mobile phase scouting across four reversed-phase columns and combinations of four aqueous buffers with acetonitrile or methanol.
• Rapid optimization generating up to five experimental gradients per condition, totalling 69 method variants.
• Fine adjustment of the selected column-buffer combination using sample profiling to resolve matrix interferences.

Sample preparation involved brewing commercial tea bags in hot water, diluting extracts, and triplicate injections. Quantitative calibration of catechins ranged from 1 to 100 µg/mL. Peak purity was assessed by diode array detection to ensure co-elution was addressed during fine optimization.

Použitá instrumentace

The final method was implemented on a Thermo Scientific™ UltiMate™ 3000RS HPLC system featuring:

• Quaternary pump with 4-channel degasser
• Thermostatted column compartment
• Autosampler with plate sampler
• Photodiode array detector at 280 nm

ChromSwordAuto 5 software modules (Scout, Developer and ReportViewer) controlled automated scouting and optimization, while Chromeleon 7 CDS managed data acquisition and reporting.

Hlavní výsledky a diskuse

The automated workflow identified an Accucore Phenyl-X column with 20 mM ammonium acetate pH 3.8 and acetonitrile as optimal. The final gradient achieved baseline separation of all seven catechins within a 9.4 min run time. Calibration curves exhibited excellent linearity (R² = 0.9984–0.9995), and limits of detection were below 0.2 µg/mL for most analytes (LOQ < 0.7 µg/mL, except GCG and EGC). Application to tea infusions revealed catechin concentrations of approximately 107 mg per cup in green tea, 53 mg in white tea and 4 mg in black tea.

Přínosy a praktické využití metody

This automated approach significantly reduced method development time to under five days of instrument use and eight analyst hours, compared to manual scouting. The strategy enhances reproducibility and lowers the expertise barrier for complex gradient optimization, making it well suited for routine quality control and research laboratories.

Budoucí trendy a možnosti využití

Future developments may integrate mass spectrometric detection to further improve selectivity and sensitivity. Expansion of the software-driven workflow to additional compound classes and complex matrices can broaden its analytical applicability. Machine learning algorithms hold potential to accelerate method optimization even further by predicting optimal conditions based on historical data.

Závěr

An automated HPLC-UV method for catechin quantification in tea was successfully developed, offering rapid, sensitive and reproducible analysis. Software-guided optimization delivered a robust protocol amenable to routine application, highlighting the value of automation in modern chromatographic method development.

Reference

1. Neves M.F., Trombin V.G., Lopes F.F., Kalaki R., Milan P. World consumption of beverages. In: The orange juice business. Wageningen Academic Publishers; 2011.
2. Mohan M., Jeevanandan G., Raja S. M. The role of green tea in oral health – A review. Asian Journal of Pharmaceutical and Clinical Research. 2018;11(4):1.
3. Zuo Y., Chen H., Deng Y. Simultaneous determination of catechins, caffeine and gallic acids in green, Oolong, black and pu-erh teas using HPLC with a photodiode array detector. Talanta. 2002;57:307–316.