Simple and Quick Analysis of Theanine in Tea by Automatic Pre-Column Derivatization Method

Importance of the Topic

Theanine is a key free amino acid responsible for the umami taste and quality of tea, particularly green tea, and is linked to various physiological benefits such as relaxation and stress reduction.
The increasing interest in health foods has driven the need for rapid and reliable analysis of amino acids in tea.

Objectives and Study Overview

This study aims to establish a simple and fast HPLC method for simultaneous quantification of theanine and 19 other amino acids in tea using automatic pre-column derivatization in the autosampler needle of a Nexera system.
Key goals include validating linearity and repeatability and applying the method to different types of tea.

Methodology

Primary amino acids were derivatized with o-phthalaldehyde and 3-mercaptopropionic acid by co-injection in the autosampler needle.
Separation was performed on a Shim-pack XR-ODS column using a low pressure gradient of acetate buffer, water/acetonitrile, and acetate buffer with EDTA.
Detection was achieved via fluorescence monitoring at excitation 350 nm and emission 450 nm.

Used Instrumentation

• Nexera HPLC system with automatic pretreatment and co-injection autosampler
• Shim-pack XR-ODS column (100 mm x 3 mm I.D., 2.2 μm)
• Fluorescence detector

Main Results and Discussion

All 20 amino acids were baseline separated within 15 minutes.
Calibration curves for theanine, aspartic acid, glutamic acid, serine, glutamine, and arginine demonstrated excellent linearity (R2 > 0.9997) over 0.25–25 μmol/L and repeatability (%RSD) below 1.3%.
Analysis of five tea samples (kabusecha, sencha, bottled green, oolong, black) revealed distinct amino acid profiles, with green teas showing the highest theanine content.

Benefits and Practical Applications of the Method

This approach eliminates complex offline derivatization and additional equipment, enabling streamlined sample preparation and high throughput.
The rapid analysis time and robust performance make it suitable for quality control in tea production and research on bioactive compounds.

Future Trends and Potential Applications

Further automation through online sample preparation could increase throughput.
The method may be extended to other food and beverage matrices or coupled with mass spectrometry for enhanced specificity.
Emerging portable HPLC platforms and microfluidic technologies could enable on-site analysis.

Conclusion

The automatic pre-column derivatization method implemented on a Nexera system offers a fast, reliable, and simple solution for quantifying theanine and amino acids in various tea types.
Its high sensitivity, precision, and ease of use provide significant advantages for routine quality control and research applications.