Analysis of Nucleic Acid Related Substances in Fish Meat and Automatic Calculation of Freshness (K Value) Using Multi-Data Report Function

Significance of the Topic

Rapid evaluation of fish muscle freshness is critical for food safety and quality control. Fish tissues degrade faster than terrestrial meats due to high water content and softer texture, leading to spoilage and potential histamine-related allergies. The K-value, based on ATP breakdown products, offers a quantitative freshness index that is simple, reliable, and automatable.

Objectives and Overview of the Study

This work aims to demonstrate a streamlined HPLC method for quantifying ATP-related compounds in fish muscle and calculating the K-value. Tuna samples were analyzed over storage intervals to evaluate freshness decay. A multi-data report function was applied to automate result compilation and visualization.

Methodology and Instrumentation

Sample Preparation:

• Homogenize 2 g of tuna muscle.
• Extract sequentially with 10 mL 10 % perchloric acid and two aliquots of 10 mL 5 % perchloric acid.
• Neutralize combined supernatants with stepwise KOH additions at 4 °C.
• Filter extract and inject 10 µL into HPLC.

Chromatographic Conditions:

• System: Nexera™ HPLC with LabSolutions™ DB/CS.
• Column: Shim-pack™ GIST 3 µm C18 AQ (100 × 3.0 mm, 3 µm) at 30 °C.
• Mobile phase: gradient of A (water/acetonitrile 100/1 with phosphoric acid and triethylamine) and B (water/acetonitrile 80/20 with same additives).
• Flow rate: 0.8 mL/min; PDA detection at 260 nm.

Calibration and Reproducibility:

• Six ATP-related compounds (Hx, HxR, IMP, AMP, ADP, ATP) were calibrated over 1–600 µmol/L with R² ≥ 0.9999.
• Retention time and peak area RSDs were below 1 %.

Column Durability:

• No pressure increase after 300 injections.
• Theoretical plate number and symmetry showed < 10 % change.

Multi-Data Report Function:
Automated report generation in spreadsheet format enabled rapid monitoring of K-value changes over storage time.

Main Results and Discussion

Freshness trends for albacore and yellowfin tuna were tracked over 0–3 days at refrigeration:

• Albacore K-value rose from 53.8 % to 61.5 %.
• Yellowfin K-value increased from 2.5 % (sashimi grade) to 20.9 %.

These changes correlated with ATP degradation pathways (ATP→ADP→AMP→IMP→HxR→Hx), confirming K-value sensitivity to storage conditions.

Benefits and Practical Applications

• Simple sample preparation without derivatization.
• High throughput analysis with gradient elution and column washing.
• Automated data handling reduces operator workload.
• Adaptable to quality control in fisheries, processing plants, and research.

Future Trends and Opportunities

Advances in miniaturized HPLC, on-line extraction, and integration with LIMS can further streamline freshness testing. Application to other seafood species and real-time monitoring during cold chain transport are emerging directions. Integration with machine learning models may enhance prediction of shelf life and spoilage kinetics.

Conclusion

The presented HPLC method provides a rapid, reproducible, and automated approach for freshness assessment in fish via K-value determination. Its robustness and high throughput make it suitable for routine quality control and research applications.

References

• Usui Shigeru, Watanabe Etsuo. Comparison of freshness changes in fresh and frozen black marlin via K-value, 2012. (Japanese only)