Simultaneous Analysis of Amino Acids Using Automatic Pretreatment Function of Integrated HPLC

Importance of Automated Pre-Column Derivatization for Amino Acid Analysis

Rapid and sensitive quantification of amino acids is critical in food, pharmaceutical, and clinical research. Automated pre-column derivatization streamlines sample preparation, reduces reagent consumption, and minimizes human error, while improving throughput and reproducibility in routine HPLC assays.

Objectives and Study Overview

This work demonstrates an integrated HPLC system (LC-2070C) capable of fully automated pre-column derivatization of amino acids. The study aims to achieve simultaneous analysis of 20 proteinogenic amino acids and selected functional amino acids (citrulline, taurine, theanine, GABA) within 20 minutes, evaluating linearity, repeatability, and performance in real samples.

Methodology and Instrumentation

• Instrumentation: Shimadzu LC-2070C with automatic pretreatment unit, Shim-pack XR-ODSII column (100 × 3.0 mm, 2.2 µm), RF-20Axs fluorescence detector (Ch1 Ex.350/Em.450 nm, Ch2 Ex.266/Em.305 nm).
• Derivatization: In-needle mixing of sample with o-phthalaldehyde (OPA)/mercaptopropionic acid for primary amino acids and 9-fluorenylmethyl chloroformate (FMOC) for secondary amino acids.
• Mobile phases: Acetate buffers (pH 5-6), acetonitrile, water; low-pressure gradient over 19.5 min.
• Sample preparation: Direct injection of mixed derivatization reagents, 1 µL injection volume.

Main Results and Discussion

• Separation of 20 proteinogenic amino acids achieved in under 14 min with baseline resolution; functional amino acids separated within 14 min.
• Linearity: r2 ≥ 0.998 for all analytes over 1–50 µmol/L; repeatability: area RSD ≤ 1.3% (n=6 at 12.5 µmol/L).
• Analysis of beer sample: Successful quantification of native amino acid profiles after simple dilution and filtration.

Benefits and Practical Applications

• Minimized sample and reagent volumes through in-needle derivatization.
• Elimination of external reaction vials reduces plastic waste and preparation steps.
• High sensitivity and selectivity suitable for QC in food, beverage, and pharmaceutical laboratories.

Future Trends and Potential Applications

• Expansion to non-proteinogenic and post-translationally modified amino acids.
• Integration with mass spectrometric detection for structural confirmation.
• High-throughput screening in metabolomics and clinical diagnostics.
• Miniaturization and microfluidic platforms for point-of-care testing.

Conclusion

Automated on-line pre-column derivatization with the LC-2070C provides a rapid, reproducible, and environmentally friendly approach for amino acid analysis. By consolidating all steps within the injection needle, it delivers high sensitivity, reduced reagent consumption, and simplified workflows, meeting the demands of modern analytical laboratories.

Reference

• Osaka Y., Tanabe A., Kiyama M. Simultaneous Analysis of Amino Acids Using Automatic Pretreatment Function of Integrated HPLC. Application News, Shimadzu Corporation, First Edition Jan. 2026.