ANALYSIS OF AMINO ACIDS IN PLANT-BASED PROTEINS AND PET FOODS – MODIFICATION OF AOAC 2018.06 TO FIT FOR NOVEL FOODS AND INGREDIENTS

Significance of the Topic

The accurate determination of amino acid profiles in pet foods and plant-based protein ingredients is critical for ensuring nutritional quality, detecting adulteration with animal-derived hydrolyzed products, and verifying compliance with dietary standards (halal, kosher). Hydroxyproline serves as a key marker for gelatin or collagen presence, making its reliable quantification essential in modern food quality control.

Objectives and Study Overview

This study aimed to adapt and optimize the AOAC International Official Method 2018.06—originally designed for dairy and infant formula—to provide robust and comprehensive analysis of 22 amino acids, including hydroxyproline, in a range of pet foods and plant-based protein powders. The work included method development, performance validation, and application to real samples.

Methodology and Instrumentation

Sample Preparation

• Pet foods: Dry and wet dog/cat foods, treats, and chicken feed were homogenized with water to form test mixtures.
• Plant proteins: Pea, brown rice, pumpkin seed, and soy powders were similarly mixed and homogenized.
• 800 mg aliquots of each homogenate underwent acid hydrolysis, neutralization, and derivatization with 6-aminoquinoline succinic carbamate (AQC) following AOAC 2018.06 protocols.

Chromatographic Conditions and Instrumentation

• System: Waters ACQUITY Premier UPLC with photodiode array detector (260 nm).
• Column: ACQUITY UPLC BEH C18, 1.7 μm, 2.1 × 150 mm, with 0.2 μm inline filter; temperature set at 50 °C.
• Mobile phases: A (AccQ∙Tag Ultra Eluent A concentrate/water, 1:20 v/v), B (AccQ∙Tag Eluent B); flow rate 0.4 mL/min.
• Injection: 1 μL partial loop with needle overfill, optimized needle wash solvents.
• Gradient: Modified elution profile to improve retention and resolution of hydroxyproline alongside standard amino acids.

Main Results and Discussion

Chromatography Optimization

• Adjusted gradient slopes and hold times yielded baseline separation for all 22 amino acids, including hydroxyproline, which was poorly retained under the original AOAC conditions.
• System suitability criteria (resolution, retention time repeatability) met or exceeded USP thresholds across robustness tests.

Method Performance

• Repeatability and intermediate reproducibility for retention times and resolution demonstrated relative standard deviations below 2%.
• Linearity, sensitivity, and resolution parameters satisfied regulatory requirements for food analysis.

Sample Analysis

• Amino acid profiles of six commercial pet foods and four plant protein powders were successfully obtained, revealing characteristic compositions and verifying method applicability.

Practical Applications and Benefits

This modified method enables:

• Reliable quantification of hydroxyproline to detect animal-derived gelatin or collagen adulteration in novel foods.
• Comprehensive nutritional profiling for pet food formulation and quality assurance.
• Regulatory compliance assessment for halal, kosher, and other dietary standards.

Future Trends and Applications

Emerging directions include:

• Integration of tandem mass spectrometry detection to enhance specificity and sensitivity.
• Automation and high-throughput workflows for large-scale screening in food quality laboratories.
• Extension of the method to alternative protein sources (cultured meats, insect proteins) and modified amino acids.

Conclusion

The optimized adaptation of AOAC 2018.06 provides a robust, reproducible, and comprehensive approach for amino acid analysis in pet foods and plant-based proteins. It effectively resolves hydroxyproline and meets stringent performance criteria, supporting both routine quality control and specialized dietary compliance testing.

References

Yang J., Rainville P., Harden S. Analysis of Amino Acids in Plant-Based Proteins and Pet Foods: Modification of AOAC 2018.06 to Fit for Novel Foods and Ingredients. Waters Corporation; 2025.