Simultaneous quantification of DL-amino acids in tea using a robust and sensitive LC- MS/MS method

Significance of the Topic

Free amino acids (FAAs) in tea influence taste, aroma and market value. D-amino acids form under fermentation, heat and pH stress, and their ratio to L-forms can reveal processing and storage history. Reliable profiling of chiral FAAs supports quality control, grading and authentication of high-value teas.

Objectives and Study Overview

This study aimed to develop and validate a rapid, sensitive and robust LC-MS/MS method for simultaneous quantification of 37 DL-amino acids in tea brew. Using UPLC separation with a chiral column and tandem quadrupole detection, the method targets low-level D-amino acids alongside abundant L-forms to assess typical concentrations in oolong tea.

Methodology and Instrumentation

Samples of oolong tea were brewed, filtered and derivatized with aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag Ultra) to enhance detectability and resolve enantiomers. Chromatographic separation employed a Daicel ChiralPak ZWIX+ column (3.0 × 150 mm, 3 µm) at 40 °C with a water/methanol gradient containing 25 mM ammonium formate and 0.1 % formic acid. Detection was performed on a Xevo TQ-S micro Triple Quadrupole Mass Spectrometer in ESI+ mode using multiple reaction monitoring (MRM). Instrument platforms and software included:

• ACQUITY UPLC I-Class PLUS System
• Xevo TQ-S micro MS/MS
• MassLynx V4.2 and TargetLynx for data processing

Main Results and Discussion

All 37 DL-amino acids exhibited excellent chromatographic resolution and linearity (R² > 0.993). Instrument precision at 1 ng/mL showed < 15 % RSD for retention times and peak areas. Limits of detection reached 0.25 ng/mL for most analytes. In brewed oolong tea, L-amino acids ranged from 3 to 3000 µg/g (dry leaf basis), while 12 D-forms were detected at 0.2–18.2 µg/g, approximately 1 000- to 10 000-fold lower than L-enantiomers. These findings align with literature on D-amino acid profiles in fermented teas.

Benefits and Practical Applications

• High sensitivity and selectivity for 37 DL-amino acids in complex matrices
• Full enantiomeric separation using a single chiral column and ACQ derivatization
• Wide dynamic range allowing direct injection of diluted and undiluted samples
• Rapid sample preparation with reduced time and cost, suitable for routine quality control

Future Trends and Potential Uses

Emerging directions include extending this approach to other fermented foods and beverages, integrating automation for high-throughput screening, and exploring alternative derivatization chemistries to further shorten analysis time. Data on D/L ratios could feed into machine-learning models for tea authentication and traceability.

Conclusion

A validated UPLC-MS/MS protocol enables accurate quantification of DL-amino acids in tea with strong linearity, precision and sensitivity. Its simplicity and robustness make it ideal for commercial and research laboratories seeking to monitor processing markers and enforce quality standards.

References

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