DEVELOPMENT AND OPTIMIZATION OF AN HPLC METHOD FOR THE ROUTINE ANALYSIS OF CATECHINS, CAFFEINE, AND GALLIC ACID IN TEA (CAMELLIA SINENSIS)

Abstract

An efficient and rapid high performance liquid chromatographic (HPLC) assay was developed for the quantification of catechins [(+) – catechin, (−) – epigallocatechin, (−) – epigallocatechingallate, (−) – epicatechin, and (−) – epicatechingallate], caffeine, and gallic acid in tea (Camellia Sinesis var. sinesis). The assay was optimized by varying sample strength, column temperature, gradient type, and detection wavelength. A curved gradient using a Thermo Hypersil ODS column with 0.05% orthophosphoric acid and methanol as mobile phase A and B, respectively, and UV detection at 277.5 nm was employed. It was observed that a curved gradient along with an optimal temperature, dramatically improves the signal to noise ratio and separation profile. The limit of detection (LOD) and limit of quantification (LOQ) were found to be in the range of 1.04–22.81 µg mL⁻¹ and 3.47–76.05 µg mL⁻¹ respectively. The overall precision values obtained from the analysis of Kangra and Darjeeling orthodox tea samples were within the range of 0–0.34 (standard error).

Keywords:

• camellia sinensis
• catechins
• gradient
• HPLC
• method development
• temperature

ACKNOWLEDGMENTS

The authors are thankful to IHBT and the Palampur and Palampur Tea Cooperative Factory, Himachal Pradesh, India for providing the authentic tea samples.

Notes

^a Each value represents mean (%w/w) ± Standard deviation.

^a Each value represents mean ± standard deviation.

^a R.T., Mean Retention time.

^b RSD (%) are given in parentheses.

^a Each value represents mean (%w/w) ± Standard error where n = 5.