Automated Analysis of Thirty-seven D/L-amino Acids using Liquid Chromatography with Fluorescence Detection and Its Application to Liquor Samples

Importance of the Topic

Enantiomeric separation of amino acids is critical for understanding their roles in food quality, flavor, preservation and biological systems. While L-amino acids are abundant and well studied, D-amino acids occur at low levels in fermented foods and biological samples and can influence sensory attributes and stability. A rapid, automated approach for comprehensive D/L-amino acid profiling supports food science, quality control and biochemical research.

Objectives and Study Overview

This work describes the development and validation of an automated HPLC method for the simultaneous analysis of thirty-seven proteinogenic D/L-amino acids (excluding proline) using fluorescence detection. Key goals were to achieve complete enantiomeric resolution in a single workflow, minimize manual operation time, ensure high reproducibility, and demonstrate application to real liquor samples (beer, sake, red and white wine).

Methodology and Instrumentation

• Derivatization Strategy: Pre-column reaction with o-phthalaldehyde (OPA) combined with chiral thiols N-acetyl-L-cysteine (NAC) or N-isobutyryl-L-cysteine (NIBC) to form fluorescent diastereomers.
• Automated Pretreatment: Autosampler executes OPA/NAC and OPA/NIBC mixing and injection inside the needle, maintaining constant reaction time for reproducibility.
• Chromatographic Setup: Shimadzu Nexera X3 UHPLC with Shim-pack Scepter 1.9 µm C8 column (150 × 3.0 mm, 35 °C), 0.6 mL/min flow, switching between two gradient programs via low-pressure mobile phase blending.
• Detection: Fluorescence detector RF-20AXS at Ex 350 nm/Em 450 nm.

Main Results and Discussion

• Complete separation of 37 D/L-amino acid pairs achieved in two complementary 60-min runs (total ~120 min).
• Retention time RSD ≤ 0.1 % and peak area RSD ≤ 1.5 % (n = 6) demonstrated excellent reproducibility.
• Calibration linearity over appropriate concentration ranges yielded r² ≥ 0.999 for all analytes.
• Application to beer, sake and wines revealed D-Asp, D-Glu, D-Ser, D-His, D-Ala and D-Leu in all samples. D-Phe was unique to beers; D-Gln and D-Trp appeared only in wines. D-isomer contents were generally low relative to L-forms.

Benefits and Practical Applications

• Automated mobile phase blending and pre-column derivatization reduced manual labor and cut analysis preparation time by over 1 hour per 20 samples.
• Simplified HPLC configuration eliminates need for multi-dimensional setups or mass spectrometry, reducing cost and matrix effects.
• High throughput, reproducibility and sensitivity support food and beverage quality control, fermentation monitoring and amino acid research.

Future Trends and Potential Applications

Further integration with mass spectrometric detection could enable structural confirmation and trace-level quantification in complex matrices. Miniaturized and high-pressure systems may shorten analysis time. Green chemistry approaches to derivatization and mobile phases, as well as AI-driven method optimization, will advance enantiomeric amino acid profiling in pharmaceuticals, clinical diagnostics and environmental studies.

Conclusion

An automated HPLC-FL method using OPA/NAC and OPA/NIBC derivatization on a Nexera X3 platform provides robust, reproducible enantiomeric separation of thirty-seven D/L-amino acids. The workflow reduces labor, avoids complex instrumentation, and is successfully applied to liquor samples, demonstrating its potential for routine QA/QC and research applications.

References

• Iwata N. Automated Analysis of Thirty-seven D/L-amino Acids using Liquid Chromatography with Fluorescence Detection: Application to Liquor Samples. Shimadzu Application News No. L592, June 2021.