Automation of Sample Derivatization Using the Agilent 1260 Infinity II Prime LC System for Amino Acid Analysis

Significance of the Topic

Amino acid analysis is critical for food quality assessment, biomedical research, and industrial process control. Accurate and reproducible quantification of amino acids supports nutritional evaluation, product consistency, and regulatory compliance.

Objectives and Overview of the Study

This application note presents an automated precolumn derivatization and fast LC-FLD workflow using the Agilent 1260 Infinity II Prime LC System. The goal is to separate and quantify 23 amino acids in beverages such as soft drinks and red wine within a 9-minute run time.

Instrumentation Used

• Agilent 1260 Infinity II Prime LC System
• Flexible Pump (G7104C)
• Multisampler with 100 µL loop and in-loop derivatization capability (G7167A)
• Multicolumn Thermostat (G7116A)
• Fluorescence Detector Spectra FLD (G7121B)

Methodology

• Automated in-loop derivatization using o-phthalaldehyde (OPA) for primary amines and 9-fluorenylmethylchloroformate (FMOC) for secondary amines.
• Borate buffer (pH 10.2) for alkalization, followed by reagent mixing and quenching with injection diluent in ~3.5 minutes per sample.
• Mobile phase A: phosphate buffer; B: acetonitrile/methanol/water (45/45/10 v/v/v).
• Column: Agilent AdvanceBio AAA LC column (3.0 × 100 mm, 2.7 µm) with guard.
• Gradient: 2% B to 57% B over 6.8 minutes, ramp to 100% B, total run time 9 minutes at 1.2 mL/min, column at 40 °C.
• FLD multi-emission monitoring at 315 nm and 455 nm with programmable switching; PMT gain adjusted per sample concentration.

Results and Discussion

• Baseline separation of 23 amino acids in under 9 minutes with retention time RSD < 0.1%.
• Peak area precision better than 1% RSD (n=10) for most analytes, demonstrating high reproducibility.
• Limits of detection down to 0.225 pmol/µL with linear dynamic range from 0.5 to 90 pmol/µL (R² ≥ 0.999).
• Application to soft drink and red wine samples revealed distinct amino acid profiles. PMT gain settings were tuned to accommodate high- and low-level analytes in a single run.

Benefits and Practical Applications

• Fully automated derivatization reduces manual handling and improves throughput.
• Short cycle time (< 10 minutes) enables high sample throughput in routine QC laboratories.
• Sensitive FLD detection without MS simplifies instrumentation and operation.
• Method adaptability via PMT gain control permits analysis of diverse sample matrices and concentration ranges.

Future Trends and Potential

Automation of derivatization is expected to extend to online coupling with high-resolution mass spectrometry and microflow LC. Emerging column chemistries may further reduce analysis times while enhancing resolution. Integration with data processing and chemometric tools will facilitate advanced quality control and real-time monitoring of complex mixtures.

Conclusion

The Agilent 1260 Infinity II Prime LC System with automated in-loop OPA/FMOC derivatization delivers a rapid, robust, and sensitive workflow for amino acid analysis in beverages. The method achieves fast separation, excellent precision, and low detection limits, making it well suited for routine food and beverage testing.

Reference

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• Hitchcock J. et al. Agilent Technologies application note, 2019, publication 5994-0586EN.
• Long D. et al. J. Agric. Food Chem. 2012, 60(17), 4259–4264.