Analysis of Theanine in Tea Leaves by HPLC with Fluorescence Detection

Theanine, one of the main amino acid components in tea, is known as a precursor of the non‐peptide antigen ethylamine, which mediates a memory response leading to secretion of IFN‐γ. Tea which contains theanine is alleged to have various therapeutic benefits to man. Different types of tea contain various amounts of theanine. A method for reversed‐phase high performance liquid chromatographic separation of theanine with fluorescence detection and its application are described. The method was applied to determining theanine in different types and grades of tea samples, which were extracted in boiling water, followed by filtration through a 0.45 µm filter. Theanine was derivatized with o‐phthaldialdehyde (OPA) prior to analysis. Separation of theanine, using an isocratic elution with a mobile phase containing 15 mM sodium acetate, isobutanol, isopropanol, and acetonitrile (75∶3∶2.5∶8, pH 7.1) was achieved in less than ten minutes. The relative fluorescence of derivatized theanine remained steady during analysis. The limit of detection of theanine standard was 33.2 picograms, while the limit of quantitation of theanine in tea extract was about 0.33 ng/mL. The results show that there is a relative quantitative relationship between the degree of fermentation and the level of theanine. The findings indicate that non‐fermented green tea and partially fermented yellow tea contained more theanine than dark, black, and woo‐loong tea, which all undergo the fermentation.