Determination of Amino acids with OPA

Importance of the Topic

Accurate determination of amino acids is crucial for understanding metabolic pathways, quality control in pharmaceuticals and nutritional analysis. OPA pre-column derivatization enhances detection sensitivity and selectivity in HPLC workflows.

Objectives and Study Overview

This application note presents a validated reversed-phase HPLC method (Method VPH0010J) with OPA derivatization for the separation and quantification of 16 standard amino acids within a 20-minute runtime.

Methodology and Instrumentation

• Column: ProntoSIL 120-3-C18 H, 250 x 3.0 mm ID
• Mobile phases: A) 20 mM sodium acetate in water/acetonitrile (97:3), B) 20 mM sodium acetate in water/acetonitrile (50:50)
• Gradient: 5–28% B (0–144 s), 28–45% B (145–560 s), 45–82% B (561–896 s), 82–90% B (897–1215 s)
• Flow rate: 0.6 mL/min; Temperature: 30 °C; Injection volume: 1 μL
• Detection: Fluorescence (Ex 330 nm, Em 450 nm) and UV at 330 nm

Main Results and Discussion

• Baseline separation of all 16 amino acids achieved in under 20 minutes.
• Strong fluorescence response with low background noise enabled clear peak identification.
• Effective separation from polar (Asp, Glu) to hydrophobic (Leu, Lys) residues demonstrated method versatility.

Benefits and Practical Applications

• High-throughput compatible analysis for clinical and QC laboratories.
• Sensitive detection suitable for trace-level amino acid profiling in biological, food and environmental samples.
• Dual detection modes provide confirmatory data for result validation.

Future Trends and Opportunities

• Coupling with mass spectrometry for enhanced structural characterization and quantitation.
• Automated derivatization workflows to improve reproducibility and throughput.
• Method extension to atypical amino acids and related metabolites.

Conclusion

OPA pre-column derivatization in combination with reversed-phase HPLC delivers a robust, sensitive and rapid platform for comprehensive amino acid analysis, meeting the demands of diverse analytical environments.

Reference

No references provided in the source document.