Determination of 17 AQC derivatized Amino acids in baby food samples

Importance of the Topic

Amino acid analysis plays a critical role in evaluating the nutritional quality and safety of food products, particularly in infant nutrition where precise amino acid profiles ensure dietary compliance and support healthy development. Rapid, high-throughput methods are essential for meeting industrial, regulatory, and research demands.

Objectives and Overview

This study presents a robust UHPLC method for the simultaneous determination of 17 proteinogenic amino acids in hydrolyzed baby food samples. Pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is combined with a fast reversed-phase separation and dual detection (UV and fluorescence) to achieve rapid and sensitive quantification.

Methodology and Instrumentation

AQC derivatization was performed by mixing sample or standard with borate buffer and AQC reagent followed by incubation at 50 °C for several minutes to form stable urea derivatives. Chromatographic separation used a 100 × 2 mm, 2 µm C18 column (Bluespher® 100) at 45 °C. A binary gradient of 50 mM sodium acetate (pH 5.75) and a mixture of acetate buffer/acetonitrile (30:70, pH 6) at 0.8 mL/min enabled baseline resolution of all analytes within 8 minutes. Detection was performed with a PDA-1 UV detector at 245 nm (2 µL flow cell) and an RF-20A xs fluorescence detector (Ex 250 nm, Em 395 nm).

Main Results and Discussion

Complete baseline separation of 17 AQC-derivatized amino acids was achieved in under 8 minutes. Retention time precision was below 0.5% RSD and peak area precision below 2.5% RSD over five replicates. Limits of detection reached ~0.4 pmol (UV) and ~0.004 pmol (fluorescence). Lysine exhibited mono- and di-derivatized peaks, reflecting its two reactive sites. An unidentified by-product was observed but did not interfere with target analytes. Hydrolyzed baby food analysis confirmed quantification of all amino acids except cystine with high accuracy.

Benefits and Practical Applications

• Rapid analysis (< 8 min) increases sample throughput and reduces instrument idle time
• Stable AQC derivatives allow batch processing and delayed measurement
• UV detection is sufficient for food-level concentrations; fluorescence enhances sensitivity for trace analyses
• Automatable derivatization streamlines workflow in high-volume laboratories

Future Trends and Applications

Future developments may include coupling with mass spectrometry for structural confirmation, micro-UHPLC for reduced solvent use, and extension to clinical diagnostics, peptide mapping, and complex biological matrices. Enhanced automation of sample preparation and data analysis will further improve throughput and robustness.

Conclusion

The AQC-based UHPLC method offers rapid, sensitive, and reproducible quantification of 17 proteinogenic amino acids in baby food. Short run times, stable derivatives, and flexible detection modes make this approach well suited for routine quality control in the food industry and adaptable to clinical and research laboratories.

Reference

• P. Hernandez-Orte, J. Cacho, V. Ferreira, J. Agric. Food Chem. 50:2891 (2002)
• S. Moore, W. H. Stein, J. Biol. Chem. 176:367–388 (1948)
• I. Davidson, Methods Mol. Biol. 211:111–122 (2002)
• W. D. Hil, F. H. Walters, T. D. Wilson, J. D. Stuart, Anal. Chem. 51:138–141 (1979)
• S. A. Cohen, D. P. Michaud, Anal. Biochem. 211:279–287 (1993)
• M. P. Bartolomeo, F. Maisano, J. Biomol. Tech. 17(2):131–137 (2006)