Analysis of Histamine and Tyramine Using Prominence Amino Acid Analysis System

Significance of the Topic

The monitoring of biogenic amines such as histamine and tyramine is crucial for ensuring food safety and quality. These non-volatile amines form during microbial decomposition of amino acids and can trigger food poisoning symptoms and migraines. Regulatory limits for histamine in fish products have been established by Codex and regional authorities, while tyramine is known to exacerbate histamine toxicity. Rapid and reliable analysis methods are therefore essential in food industry laboratories.

Objectives and Study Overview

This application note demonstrates a robust analytical protocol for simultaneous quantification of histamine and tyramine using the Shimadzu Prominence Amino Acid Analysis System. The goal is to achieve high sensitivity and repeatability with minimal sample preparation, employing post-column derivatization and gradient elution to streamline routine testing of processed foods, fish products, and beverages.

Methodology

• Sample Preparation: Simple ten-fold dilution in pH 2.2 sodium citrate buffer followed by 0.2 µm filtration.
• Derivatization: Post-column reaction with ortho-phthalaldehyde (OPA) reagent stream to generate fluorescent derivatives.
• Chromatographic Conditions:
  • Column: Shim-pack Amino-Na (100 mm × 6.0 mm I.D.)
  • Gradient Program: Mobile Phase B from 80% to 65% over 15 min, hold, then re-equilibration to 80% by 20.01 min, total run 25 min.
  • Flow Rate: 0.6 mL/min at 60 °C.
• Detection: RF-20Axs fluorescence detector, excitation 350 nm, emission 450 nm.

Used Instrumentation

• Prominence HPLC with post-column OPA derivatization module
• Shim-pack Amino-Na analytical column
• Shim-pack ISC-30 ammonia trap column
• Amino Acid Mobile Phase Kits (Na type B and C)
• Amino Acid Reagent Kit for OPA derivatization
• RF-20Axs fluorescence detector

Main Results and Discussion

• Standard Analysis: Clear baseline resolution of histamine and tyramine at 10 mg/L each, retention times approx. 9.97 min and 11.87 min.
• Linearity: Achieved excellent calibration over 0.1–100 mg/L for both analytes with R2 > 0.9998.
• Repeatability: At 1 mg/L, peak area and retention time RSDs were below 1.1% and 0.2%, respectively, demonstrating method precision well within regulatory needs.
• Food Samples: Reliable quantification in fish sauce, soy sauce, red and white wines spiked at 50 mg/L levels, confirming applicability to complex matrices.

Benefits and Practical Applications

• Minimal sample pretreatment reduces analysis time and labor.
• High sensitivity and wide linear range meet regulatory and quality-control requirements.
• Robust repeatability ensures consistent performance in routine testing.
• Post-column derivatization avoids complex pre-column reagent handling.

Future Trends and Applications

• Integration of automated sample handling and data processing workflows.
• Expansion to broader panels of biogenic amines for comprehensive food safety profiling.
• Coupling with mass spectrometric detection for structural confirmation and lower detection limits.
• Development of universal reagent kits with extended reagent stability and simplified logistics.

Conclusion

This study presents a streamlined HPLC-FLD method for accurate determination of histamine and tyramine in various food matrices. The approach offers high throughput, strong method performance, and straightforward instrumentation, positioning it as an effective tool for routine food safety laboratories.

Reference

No external literature references were cited in this application note.