Determination of theanine in green tea, green tea-based beverages, and dietary supplements by high performance liquid chromatography using orthophthalaldehyd (OPA) derivation

Theanine is a natural amino acid abundant in green tea and is added to green tea-based beverages and health supplements containing green tea extract. In this study, a method for analyzing theanine using HPLC with o-phthalaldehyde (OPA) derivatization has been developed and validated. Theanine was extracted from the sample using water and then derivatized with OPA reagent, then chromatographically separated on a Xbridge C18 column (250 mm × 4.6 mm; 5 µm), with a mobile phase consisting of 20 mM ammonium acetate and methanol in a gradient program. Fluorescence detector was used with an excitation wavelength of 345 nm and an emission wavelength of 455 nm. The method demonstrated good specificity, recovery and accuracy required by AOAC. The limits of detection and quantification were 3.0 µg/g and 10.0 µg/g, respectively. This method has been applied to analyze the theanine content in tea samples, beverages, and health supplements containing green tea extract at the National Institute for Food Control.