Analysis of Foods Using Nexera™ Post-Column Amino Acid Analysis System

Importance of Post-Column Amino Acid Analysis

Post-column amino acid analysis with o-phthalaldehyde (OPA) derivatization plays a pivotal role in fields ranging from clinical research to food quality control and pharmaceutical development. Its resilience to complex sample matrices and broad applicability make it an indispensable tool for laboratories requiring high sensitivity and selectivity in amino acid profiling.

Study Overview

This application report introduces the Nexera™ post-column amino acid analysis system, highlighting the system’s capabilities for quantitative determination of proteinogenic and free amino acids under various separation modes (Na type and Li type). The objectives are to demonstrate analytical conditions, pretreatment protocols, and performance on a range of food and beverage matrices.

Methodology and Instrumentation

The core technique combines cation exchange HPLC with post-column OPA derivatization and fluorescence detection (Ex 350 nm / Em 450 nm). Two separation modes are employed:

• Na type: Optimized for proteinogenic amino acids (~20 compounds), with high resolution, high speed, and tryptophan-inclusive programs.
• Li type: Designed for free amino acids (~39 compounds), offering extended coverage via lithium citrate mobile phases.

Key instrumentation and consumables:

• HPLC system: Nexera™ UHPLC platform
• Columns: Shim-pack™ Amino-Na (100 × 6.0 mm, 5 µm) and Shim-pack™ Amino-Li (100 × 6.0 mm, 5 µm)
• Ammonia trap: Shim-pack ISC-30/S0504 (Na and Li variants)
• Mobile phases: Dedicated amino acid kits (Na and Li type)
• Reaction reagents: OPA-based reagent kits
• Detection: Post-column fluorescence module

Pretreatment protocols vary with sample type, including acid hydrolysis (HCl or NaOH), oxidation (performic acid), ultrafiltration, and automated dilution steps to ensure matrix removal and quantitative recoveries.

Main Results and Discussion

Na Type Analysis:

• High resolution mode achieved baseline separation of 18–20 proteinogenic amino acids within 60–70 minutes. Essential amino acids were clearly resolved in hydrolyzed matrices such as soybeans, protein powders, and baby formula.
• High speed mode reduced run times to 45 minutes, suitable for simple matrices like sports drinks, beer, and yogurt, while maintaining quantitative precision for key amino acids (Val, Leu, Ile, Arg).
• Tryptophan-inclusive mode employed alkali hydrolysis to preserve labile tryptophan, allowing simultaneous quantification of 20 amino acids including GABA and ammonia.
• Performic acid oxidation-hydrolysis enabled conversion of cysteine and methionine to cysteic acid and methionine sulfone, achieving clear quantification of sulfur-containing residues.

Li Type Analysis:

• Extended profiling of ~39 free amino acids was demonstrated with a 150-minute gradient, resolving unique species such as phospho-amino acids, methylated histidines, carnosine, and anserine in standard and complex matrices (ham, black vinegar, broccoli sprouts).
• High resolution was retained despite the presence of abundant interfering compounds, underscoring the method’s robustness for biochemical and food research.

Benefits and Practical Applications

The Nexera post-column system delivers:

• High sensitivity and linearity for low-abundance amino acids (e.g., proline, GABA).
• Resistance to sample matrix effects, as derivatization occurs post-separation.
• Flexible workflows using preconfigured reagent and mobile phase kits, reducing method development time.
• Applicability across nutritional analysis, clinical diagnostics, pharmaceutical QC, and food authenticity testing.

Future Trends and Potential Applications

Emerging directions include:

• Integration with mass spectrometry detectors for confirmatory analysis and structural elucidation.
• Miniaturized columns and ultrafast gradients to further reduce analysis times.
• Automated sample preparation modules for high-throughput screening.
• Application of chemometric and AI-driven data analysis to interpret complex amino acid profiles in metabolomics and precision nutrition.

Conclusion

The Nexera post-column amino acid analysis system offers versatile, high-performance workflows for both proteinogenic and free amino acid quantification. Its combination of robust separation chemistry, sensitive fluorescence detection, and standardized kits streamlines routine and advanced amino acid analyses across diverse sample types.

References

• Application News No. L568, Shimadzu Corporation (First Edition: Oct. 2020)
• Application News No. L529B, Shimadzu Corporation