ANALYTICAL SOLUTIONS FOR THE ANALYSIS OF BOUND AND FREE AMINO ACIDS IN FOODS AND FEEDS

Importance of Topic

The precise analysis of bound and free amino acids in foods and feeds is critical for assessing nutritional value, detecting product adulteration and optimizing fermentation or feed formulations. Quantitative amino acid profiles inform dietary balance, highlight growth-limiting sulfur amino acids and serve regulatory and QA/QC purposes.

Objectives and Study Overview

This study evaluates a complete UPLC Amino Acid Analysis Solution for simultaneous quantitation of free and protein-bound amino acids in diverse feed matrices. It verifies method accuracy against a pure protein reference, examines robustness across multiple feed types and instrument configurations, and demonstrates reliable measurement of sulfur-containing residues.

Methodology and Derivatization

Sample Preparation and Hydrolysis:

• Bovine serum albumin (BSA) hydrolysate (1.0 mg/mL) diluted and derivatized to confirm method accuracy.
• Four hydrolyzed feed types (swine diet, poultry diet, whole soybean, soybean meal) prepared at 1.0 mg/mL under 0.1 M HCl.
• Acid hydrolysis conducted with and without performic acid oxidation to stabilize cystine and methionine.

Derivatization Protocol:

1. Neutralize excess acid with 0.1 M NaOH.
2. Add borate buffer, sample and AQC reagent; heat at 55 °C for 10 minutes.
3. Allow derivatives to stabilize at room temperature for up to one week.

Used Instrumentation

• LC System: Waters ACQUITY UPLC.
• Column: AccQ·Tag Ultra, 2.1×100 mm, 1.7 µm at 55 °C.
• Sample compartment: 20 °C; flow rate: 700 µL/min.
• Mobile phases: diluted AccQ·Tag Ultra Eluent A and Eluent B per system guide.
• Detection: UV at 260 nm; injection 1 µL partial loop.

Results and Discussion

Accuracy and Precision:

• BSA hydrolysate measurements matched expected AA composition within ±4% deviation.
• Overall method variability under 1% RSD across 75 analyses over five days.

Feed Matrix Performance:

• Chromatograms of all four hydrolyzed feed types showed consistent retention times and no interfering peaks.
• Quantitative results for 17 amino acids, including cystine (as cysteic acid) and methionine sulfone, demonstrated reproducible weight-percent values with RSD <1%.
• Feed composition trends reflected expected differences, such as higher lysine in soy samples and altered leucine/isoleucine ratios in mixed feeds.

Benefits and Practical Applications

The integrated UPLC Amino Acid Analysis Solution offers:

• Rapid 9.5-minute run time for complete amino acid profiles.
• Reliable measurement of both free and bound amino acids in complex food and feed matrices.
• Accurate quantitation of sulfur-containing residues through paired oxidized/unoxidized analyses.
• High throughput and reproducibility for routine QA/QC and research laboratories.

Future Trends and Applications

Emerging opportunities include:

• Automation and coupling with robotics for large-scale nutritional screening.
• Integration with online monitoring for real-time process control in fermentation and feed production.
• Data integration into AI-driven platforms for predictive nutritional modeling and supply chain validation.

Conclusion

The Waters UPLC Amino Acid Analysis Solution, combining AccQ·Tag derivatization and ACQUITY UPLC separation, delivers accurate, precise and high-throughput profiling of free and bound amino acids in diverse feed matrices, including reliable analysis of sulfur-containing residues. Its robustness and reproducibility make it well suited for nutritional assessment, product authentication and QA/QC workflows.

References

• AOAC Official Method 994.12, Amino Acids in Foods.
• Waters UPLC Amino Acid Analysis Solution Guide (P/N 71500129702).
• NIST Reference Material 2389, Amino Acids in 0.1 mol/L HCl.