Development of Sensitive and Simultaneous Determination Method for Thirty-Seven D/L-Amino Acids by Automatic Pre-column Derivatization with Chiral Thiol Using UHPLC

Significance of the Topic

The separation and quantification of D- and L-amino acids is critical for food quality, flavor profiling and disease biomarker discovery. Traditional LC/MS methods face matrix effects, while multi-dimensional HPLC is time-consuming and complex. A rapid, sensitive UHPLC method with chiral derivatization can streamline analyses in various food and beverage matrices.

Objectives and Study Overview

This work aimed to develop and validate a single-run UHPLC method for simultaneous determination of 37 proteinogenic D/L-amino acids (excluding proline). The study focused on automatic pre-column derivatization with OPA and chiral thiols (N-acetyl-L-cysteine or N-isobutyryl-L-cysteine), optimized chromatographic conditions, and application to beverage samples.

Experimental Methodology

The method combined automatic derivatization and mobile phase blending:

• Sample matrices: two beer types, sake, red wine, white wine, diluted in HCl and filtered.
• Derivatization: OPA/NIBC reagent mixing, 1.5 min reaction inside the autosampler needle.
• Chromatography: Nexera X3 UHPLC, C18 column, gradient elution (phosphate buffer pH 6.9 with acetonitrile/methanol), flow rate 0.22 mL/min, 20 °C column temperature, FL detection Ex 338 nm/Em 455 nm.
• Validation: RSD ≤1.6% (n=6), linearity r2 > 0.999, recovery 84.9–108.6% in spiked beer.

Instrumentation

• UHPLC system: Shimadzu Nexera X3 with autosampler, binary pumps, column oven.
• Column: CERI L-column3 C18 (150 × 2.1 mm, 2.0 µm) with pre-column filter.
• Detector: fluorescence detector FLR.

Main Results and Discussion

• Complete separation of 37 D/L-amino acid diastereomers in 66 min, ~45% faster than previous methods.
• Optimal derivatization with OPA 2 mg/mL and NIBC 1 mg/mL, 1.5 min reaction time.
• High sensitivity and precision across all analytes; contaminants in real samples were well resolved.
• Application to beverage samples revealed D-amino acid proportions ≤6% and consistent profiles with literature.
• PCA discriminated between beer, wine, and sake based on D/L abundance patterns.

Benefits and Practical Applications

• Rapid, robust profiling of D/L-amino acids in food and beverage quality control.
• Avoids expensive MS detection and complex multi-dimensional HPLC setups.
• Automated derivatization ensures reproducible reaction times and high throughput.
• Suitable for QA/QC labs, research on flavor chemistry and biomarker discovery.

Future Trends and Potential Applications

Further miniaturization and integration with online sample preparation could reduce analysis time below one hour. Expansion to non-proteinogenic amino acids or peptide mapping may broaden the method’s utility. Coupling with chemometric tools can support large-scale profiling in functional foods and clinical diagnostics.

Conclusion

A streamlined UHPLC method with automatic OPA/chiral thiol derivatization was established for 37 D/L-amino acids. The approach achieved high sensitivity, precision and rapid separation in complex beverage matrices without costly instrumentation, offering a practical solution for routine profiling and research applications.

References

• Iwata N, Watabe Y, Horie S, Hayakawa Y. Chromatography 2021;42:133–141.
• Jin D, Miyahara T, Oe T, Toyo’oka T. Anal Biochem 1999;269:124–132.
• Ali HS, Pätzold R, Brückner H. Amino Acids 2010;38:951–958.