Automated Precolumn Derivatization for the Enantioseparation of Amino Acids Using the Agilent 1290 Infinity II LC

Significance of the Topic

Amino acid enantiomers play distinct biological roles, impacting flavor profiles in food products and serving as biomarkers in biomedical research. Reliable and sensitive methods for enantioseparation enable quality control in food testing, metabolic studies, and safety assessment of fermented products.

Objectives and Study Overview

This study demonstrates a fully automated precolumn derivatization approach for high-resolution enantioseparation of proteinogenic amino acids using the Agilent 1290 Infinity II LC system. The goal was to simplify sample preparation, achieve sensitive fluorescence detection, and apply the method to both standard mixtures and real-world vinegar samples.

Methodology and Used Instrumentation

The analytical workflow combines online derivatization with o-phthalaldehyde (OPA) and N-isobutyryl-L-cysteine (IBLC) followed by reversed-phase UHPLC separation on a Poroshell HPH-C18 column and fluorescence detection. Key modules included:

• Agilent 1290 Infinity II High-Speed Pump
• Agilent 1290 Infinity II Multisampler with 40 µL loop and sample cooler
• Agilent 1290 Infinity II Multicolumn Thermostat
• Agilent 1260 Infinity Fluorescence Detector (Ex 230 nm, Em 450 nm)

Chromatographic conditions featured a binary gradient (50 mM sodium acetate, pH 6.0 vs. acetonitrile/methanol/water 45/45/10), 0.7 mL/min flow, 30 °C column temperature. The injector program was optimized to draw derivatization reagents and sample sequentially, mix, wait, and inject automatically.

Main Results and Discussion

Separation of 26 primary amino acids (excluding proline and cysteine) was achieved in 35 minutes with baseline resolution of D- and L-forms. Precision of retention time was better than 0.1 % RSD, peak area precision under 3.0 % RSD, and detection limits of 0.04–0.15 pmol/µL (signal-to-noise = 3). Application to three vinegar types revealed:

• Kurozu vinegar exhibited the broadest profile and higher levels of both L- and D-amino acids.
• Common rice vinegar and balsamic vinegar showed fewer enantiomers and lower overall concentrations.
• Differences in D-amino acid content reflect raw material and fermentation variability.

Benefits and Practical Applications

This automated derivatization and separation platform offers:

• High throughput with minimal manual handling.
• Enhanced sensitivity via fluorescence detection of OPA/IBLC derivatives.
• Robust quantification for food quality control and metabolic profiling.
• Compatibility with diverse sample matrices such as vinegars, fermented beverages, and biological fluids.

Future Trends and Possibilities

Advances may include integration with mass spectrometry for structural confirmation, expansion to secondary and non-proteinogenic amino acids, and miniaturized formats for on-site analysis. Machine learning algorithms could further optimize separation parameters and interpret complex enantiomeric patterns in food and clinical samples.

Conclusion

The Agilent 1290 Infinity II LC system with automated OPA/IBLC precolumn derivatization provides a sensitive, reproducible, and efficient method for enantioseparation of amino acids. Its demonstrated performance on standard mixtures and real vinegar samples underscores its value for analytical laboratories focused on food testing, quality assurance, and research applications.

Reference

1. Inoue Y; et al. Effect of D-Amino Acids as Taste Modifiers in Fermented Foods. Trace Nutrients Research 2013, 31, 59.
2. Ilisz I; et al. Recent advances in the direct and indirect liquid chromatographic enantioseparation of amino acids and related compounds: a review. J. Pharm. Biomed. Anal. 2012, 69, 28.
3. Brückner H; et al. Liquid chromatographic determination of amino acid enantiomers by derivatization with o-phthaldialdehyde and chiral thiols – Applications with reference to food science. J. Chromatogr. A 1995, 697, 229.