UPLC™-UV Analysis of Amino Acids in Dairy Products – Implementing an International Standard on the ACQUITY™ Premier System

Significance of the Topic

Amino acid profiling in dairy products is critical for nutritional evaluation, quality control, and detection of adulteration. High-throughput, reliable methods enhance laboratory efficiency and ensure compliance with international standards.

Objectives and Study Overview

This study aimed to implement AOAC Official Method 2018.06 on a Waters ACQUITY Premier UPLC system, optimizing chromatographic conditions and sample preparation to achieve robust separation and accurate quantification of amino acids in various dairy matrices.

Methodology and Instrumentation

Amino acids were extracted by acid hydrolysis with DDP and phenol, followed by neutralization and pre-column derivatization using AccQ·Tag Ultra reagents. Reversed-phase UPLC separation was performed at 260 nm on an ACQUITY Premier System equipped with a Binary Solvent Manager, Fixed Loop Sample Manager, ACQUITY UPLC BEH C18 (1.7 μm, 2.1×150 mm) column, inline filter, and PDA eLambda detector. Data acquisition and processing used Empower 3 CDS.

Main Results and Discussion

• Adapted mobile phase A preparation using Class A volumetric flask and modified eluent ratio (100:600) ensured consistent retention times for early-eluting amino acids.
• Injection mode (PLNO) with sixfold overfill, inline filtration, and optimized derivatization recipe improved peak shape and resolution.
• Column temperature set at 50 °C provided optimal selectivity and reproducibility; slight ±2 °C variations significantly affected performance.
• Calibration curves for 17 amino acids exhibited R² > 0.997; estimated LOQs ranged 0.02–0.16 μM.
• Repeatability (intra-day RSD) for retention time was <3% (early eluters) and <1% (others); tailing factors ≤1.11; resolution >2.0 for all relevant pairs.
• Intermediate precision (inter-day, column batches) demonstrated RSD <5% for early eluters and <1.7% for others; resolution remained robust across conditions.
• Baseline separation achieved for amino acids in bovine and goat milk powder, infant formula, whey, and casein powders with RSDs <4.8% in triplicate analyses.

Benefits and Practical Applications

• Faster analysis without time-consuming performic acid oxidation step.
• Simultaneous quantification of tyrosine alongside other amino acids.
• Compliance with AOAC, ISO, IDF, and AACC standards ensures method validity.
• Suitable for routine QA/QC in dairy and nutritional product laboratories.

Future Trends and Perspectives

Advances may include integration of mass spectrometric detection for enhanced specificity, further miniaturization and automation of sample preparation, and application of machine learning for data interpretation. Ongoing improvements in column chemistries and system designs will support higher throughput and expanded analyte coverage.

Conclusion

Implementation of AOAC Method 2018.06 on the ACQUITY Premier System with minor methodological adaptations delivered reliable, sensitive, and reproducible amino acid profiles in diverse dairy matrices. This optimized workflow offers a robust solution for high-quality nutritional analysis.

Reference

1. Jaudzems G., Fuerer C. (2022) Determination of total amino acids in infant formulas, adult nutritionals, dairy, and cereal matrices by UHPLC–UV: Interlaboratory Validation Study, Final Action 2018.06. Journal of AOAC International, 105(6):1625–1639.
2. International Organization for Standardization (2022) Milk and milk products – Determination of amino acids in infant and adult/paediatric nutritional formulas and dairy products (ISO 4214:2022).
3. Cereals and Grains Association (2022) Total Amino Acids by UHPLC-UV (AACC 07‐50.01).
4. Waters Corporation (2007) UPLC Amino Acid Analysis Solution System Guide, Rev B, 71500129702.
5. Waters Corporation (2018) Amino Acid Analysis Application Notebook, 720006130.
6. Waters Corporation (2024) AccQ·Tag Ultra Derivatization Kit Care and Use Manual, Rev A, 715001331.
7. Cohen S. (1988) Analysis of Sulfur Containing Amino Acids III. Alkylation of Cysteine. Waters Lab Highlights LAH0379.
8. International Council for Harmonisation (2005) Q2(R1) Validation of Analytical Procedures: Text and Methodology.
9. For Waters ACQUITY Systems used in the multi-lab study refer to supplemental Table 1 in Jaudzems & Fuerer (2022).