A RAPID HPLC-DAD METHOD FOR ANALYSIS OF THEAFLAVINS USING C₁₂ AS STATIONARY PHASE*

Abstract

Theaflavins are the major pharmacologically active antioxidant compounds present in the black tea. In this study we have developed a rapid, simple, and sensitive HPLC-DAD method for estimation and analysis of theaflavins using a RP-C₁₂ (4.6 × 250 mm, 4 micron) column. The present study aims at rapid estimation and analysis of theaflavins in black tea and black tea based products (Ready to Drink Teas and Instant Teas) for their qualitative analysis. The use of RP-C₁₂ as stationary phase with a simple gradient method enables the excellent separation of all the four major tea theaflavins: theaflavin (TF-1), theaflavin-3-gallate (TF-2), theaflavin-3, 3′-digallate (TF-3), and theaflavin-3′-gallate (TF-4) within 7 min. A linear calibration curve (r² = 0.999) was plotted against standard theaflavin. The purification of theaflavins from black tea (CTC) was done by reverse phase solid phase extraction (RP-SPE) technique and quantification of individual theaflavins were carried out as theaflavin equivalent per mg of total purified theaflavins (TFS) from black tea. The results of present study clearly shows that use of C₁₂ as a new stationary phase owes high sensitivity and faster analysis of theaflavins that otherwise are not easy to separate within 10 min.

Keywords:

• antioxidants
• black tea
• catechins
• C₁₂
• RP-HPLC
• theaflavins

ACKNOWLEDGMENT

The authors are very thankful to Council of Scientific and Industrial Research New Delhi, India for the financial support (Project SIP-0003) to carry out the work.

Notes

^a Percentage measured as theaflavin equivalent per mg of purified theaflavin mixture.

^b Percentage measured as theaflavin equivalent per mg of standard theaflavin mixture.

*IHBT Communication no. 2183.