Analysis of Amino Acids in Plant-Based Proteins and Pet Foods – Modification of AOAC 2018.06 to Fit for Novel Foods and Ingredients

Importance of the Topic

Amino acid profiling is essential for nutritional labeling, quality control and adulteration detection in both pet foods and plant-derived proteins. The analysis of hydroxyproline serves as a critical indicator of animal-derived components, such as gelatin and collagen, which helps ensure compliance with dietary requirements including halal and kosher standards. Accurate quantification of this marker alongside standard amino acids supports consumer safety and integrity in the food industry.

Objectives and Study Overview

This study aims to adapt the AOAC International Official Method 2018.06, originally designed for infant formula and dairy products, to accommodate the analysis of hydroxyproline and other amino acids in pet foods and novel plant-based protein ingredients. The project involved optimizing chromatographic conditions to achieve sufficient retention and resolution for all target analytes.

Methodology and Instrumentation

• Sample Collection and Preparation:
  • Pet foods: dry and wet dog/cat formulations, cat treats, chicken feed (5.0 g mixed with water, homogenized).
  • Plant proteins: pea, brown rice, pumpkin seed, soy powders (1.0 g mixed with water).
  • Acid hydrolysis, neutralization, and AQC derivatization following AOAC 2018.06 protocol.
• Chromatographic Conditions:
  • Column: ACQUITY UPLC BEH C18, 1.7 µm, 2.1 × 150 mm.
  • Mobile Phases: AccQ∙Tag Ultra Eluent A/water (1:20) and AccQ∙Tag Eluent B.
  • Flow Rate: 0.4 mL/min; Column Temperature: 50 °C; Detection at 260 nm.
  • Injection: 1 µL partial loop with overfill; gradient optimized for hydroxyproline retention.
• Instrumentation:
  • ACQUITY Premier UPLC System with PDA Detector (Waters Corporation).
  • Empower CDS software for data acquisition and processing.

Main Results and Discussion

• Chromatographic Optimization:
  • Adjusted gradient program to achieve baseline separation of hydroxyproline and critical amino acid pairs (CysX/Met, Asp/MetSO₂, Ser/Arg) with resolution >2.0.
• Analytical Performance:
  • Repeatability and intermediate reproducibility for retention times and resolution met AOAC performance criteria.
  • Sensitivity and linearity were maintained across the concentration range relevant for pet foods and plant proteins.
• Sample Analysis:
  • Successfully quantified a full amino acid profile, including hydroxyproline, in six commercial pet foods and four plant-based protein powders.

Benefits and Practical Applications

This modified method provides a robust and practical tool for routine quality control laboratories to:

• Verify nutritional claims in pet food formulations.
• Detect adulteration by monitoring hydroxyproline levels.
• Ensure compliance with dietary restrictions such as halal and kosher labeling.

Future Trends and Applications

• Expansion to other novel protein sources, including insect and microbial ingredients.
• Integration with mass spectrometry detection to improve specificity and sensitivity.
• Automation of sample preparation for high-throughput screening in industrial settings.

Conclusion

Modifying the AOAC 2018.06 method enabled comprehensive amino acid analysis, including hydroxyproline, in pet foods and plant-based proteins. The optimized protocol delivers reliable separation, high reproducibility, and meets performance criteria, making it a valuable approach for the food industry.

References

• Yang J., Rainville P., Harden S. Analysis of Amino Acids in Plant-Based Proteins and Pet Foods – Modification of AOAC 2018.06, Waters Corporation, 2025.