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 verifying protein quality and detecting adulteration in foods. Plant-based proteins and pet foods pose unique analytical challenges due to variable matrices and the presence of marker amino acids such as hydroxyproline. Reliable quantification of all amino acids supports nutritional labeling, authenticity testing, and regulatory compliance in infant formulas, adult nutrition, halal/kosher verification, and novel food ingredient assessment.

Objectives and overview

This study aimed to adapt the AOAC International Official Method 2018.06—originally developed for infant formula and dairy products—for the analysis of amino acids in pet foods and plant-based proteins. Key goals included improving retention and resolution of hydroxyproline (HyPro), expanding the amino acid target list, and confirming method robustness and reproducibility across diverse samples.

Methodology and instrumentation

The modified workflow retained the core steps of acid hydrolysis, neutralization, and AQC pre-column derivatization from AOAC 2018.06. Sample types included dry and wet dog and cat foods, chicken feed, and powders of pea, brown rice, pumpkin seed, and soy proteins. Portions of homogenized sample mixtures underwent hydrolysis before derivatization.

• LC system: ACQUITY Premier UPLC with PDA detector set at 260 nm.
• Column: ACQUITY UPLC BEH C18 or AccQ∙Tag Ultra C18, 2.1 × 150 mm, 1.7 μm.
• Mobile phases: AccQ∙Tag Ultra eluents A and B, with customized gradient program.
• Flow rate: 0.4 mL/min; column temperature: 50 °C; injection: 1 µL PLNO mode.

Main results and discussion

Gradient and mobile phase adjustments successfully resolved hydroxyproline alongside 22 other amino acids. Method optimization achieved USP resolution criteria (lower bounds > 2.0) for critical pairs such as CysX/Met and Ser/Arg. Repeatability and intermediate reproducibility of retention times met stringent criteria, and linear calibration curves demonstrated high sensitivity.

• Hydroxyproline eluted with baseline separation, enabling detection in pet foods and plant proteins.
• Chromatograms of six pet food formulations and four plant protein powders showed consistent peak resolution and minimal interference.
• Robustness tests confirmed method tolerance to slight variations in flow rate, temperature, and mobile-phase composition.

Benefits and practical applications

The modified AOAC method provides a reliable approach for comprehensive amino acid analysis in complex food matrices. Laboratories can leverage the protocol to:

• Authenticate protein sources and detect adulteration in pet foods and novel plant-based ingredients.
• Support nutritional claims and compliance with regulatory standards.
• Monitor marker amino acids for halal, kosher, or vegetarian food verification.

Future trends and potential applications

As the market for alternative proteins grows, further trends and opportunities include:

• Automation of sample preparation to increase throughput in QA/QC environments.
• Integration with mass spectrometry detectors for enhanced specificity and trace-level detection.
• Extension to emerging protein sources such as insect, algal, or cell-cultured ingredients.
• Data-driven method optimization using chemometric tools to adapt protocols for novel matrices.

Conclusion

The tailored modification of AOAC 2018.06 successfully broadened its applicability to pet foods and plant-based proteins, including quantification of hydroxyproline. Excellent chromatographic performance, reliability, and reproducibility demonstrate that the method is fit for routine analysis of amino acid profiles in diverse food matrices.