Pre-column Amino Acid Analysis of Hydrolyzed Alternative Proteins

Importance of the Topic

Protein hydrolysate amino acid profiling is critical for evaluating the nutritional quality and functional properties of alternative proteins. Precise quantification of individual amino acids informs product formulation, quality control, and regulatory labeling in food and feed industries.

Study Objectives and Overview

This study demonstrates an automated pre-column derivatization workflow for amino acid analysis of hydrolyzed alternative proteins using the Nexera XR high performance liquid chromatograph with RF-20AXS fluorescence detection. The aim is to accommodate three hydrolysis treatments—acidic, alkaline, and performic oxidation—within a single HPLC platform, employing two analytical methods on one column and using shared reagents.

Methodology and Instrumentation Used

Instrumentation Used

• Nexera XR HPLC system
• Shim-pack XR-ODSII column (100 mm × 3.0 mm I.D., 2.2 µm)
• RF-20AXS fluorescence detector (Ex/Em channels 350/450 nm and 266/305 nm)

Derivatization and Separation

• Automated mixing of borate buffer, MPA/OPA reagent, and FMOC reagent in the autosampler needle
• Analytical conditions 1: acetate buffer gradient for amino acids from acid and alkaline hydrolysis
• Analytical conditions 2: citrate buffer gradient for cysteic acid and methionine sulfone

Hydrolysis Protocols

• 6 M HCl hydrolysis for general amino acids
• 4.2 M NaOH hydrolysis for tryptophan preservation
• Performic acid oxidation followed by acid hydrolysis for sulfur-containing residues

Main Results and Discussion

Standard mixtures of 18 amino acids plus cysteic acid and methionine sulfone achieved baseline separation within 12 minutes under each method. Calibration curves for cysteic acid and methionine sulfone exhibited coefficients of determination above 0.999. Analysis of cricket powder and soybean meat after each hydrolysis protocol yielded reproducible chromatograms. Repeatability tests (n=6) showed peak area relative standard deviations below 3% for all analyzed amino acids in both matrices.

Benefits and Practical Applications

• Fully automated sample derivatization reduces manual handling, operator error, and preparation time
• Use of a single column and common reagents simplifies instrument setup and maintenance
• Comprehensive coverage of nutritional and oxidized amino acids supports formulation and QA/QC of alternative protein products
• High throughput and robust reproducibility facilitate routine laboratory workflows

Future Trends and Opportunities

Integration of automated hydrolysis pathways with online sample preparation and mass spectrometry detection may enhance sensitivity, broaden analyte scope, and reduce total analysis time. Coupling with data analytics and AI-driven interpretation will further streamline amino acid profiling in emerging protein sources.

Conclusion

The Nexera XR automated pre-column derivatization method delivers efficient, reproducible, and high-throughput amino acid analysis across multiple hydrolysis treatments. This approach addresses the growing need for reliable nutritional evaluation of alternative proteins in research, food development, and quality assurance.

References

1. Good Food Institute, Defining alternative proteins
2. Shimadzu Application News, High-Speed Simultaneous Analysis of Amino Acids by Pre-column Derivatization Using Automatic Pretreatment Function, 01-00441-EN