Quick Estimation of the Freshness and the Level of Putrefaction in Fish Meat Using Nexera Dual Injection System

Significance of the Topic

The delicate structure and high water content of fish muscle accelerate spoilage, posing food safety risks. Quantitative freshness indices such as the K value, based on ATP degradation, and histamine levels, a heat-stable putrefaction marker, are critical for ensuring product quality and consumer health.

Objectives and Study Overview

This work introduces a rapid, simultaneous HPLC method to determine fish freshness (K value) and putrefaction (histamine) alongside nutritional amino acids. By leveraging a dual injection system, distinct chromatographic conditions run in parallel within a single instrument, enhancing efficiency and data management.

Methodology and Instrumentation

Samples underwent deproteinization, perchloric acid extraction, and pH adjustment. Amino acids and histamine were derivatized automatically pre‐column using o-phthalaldehyde and FMOC reagents. Key instrumentation included

• Nexera dual injection HPLC system
• Photodiode array detector for ATP compounds
• Fluorescence detector with two excitation/emission channels for amino acids and histamine
• Shim-pack GIST C18 AQ column for nucleotides; Shim-pack Velox C18 column for amino acids
• SIL-40 autosampler with dedicated sample racks and low-adsorption TORAST-H glass vials

Main Results and Discussion

Calibration curves for all 31 analytes exhibited excellent linearity (r2 ≥ 0.999). Perchloric acid extraction achieved stable recoveries for histamine (92–98%) and histidine. Spike recovery averaged 99.8%. Analysis of yellowfin and albacore tuna stored at 4 °C and 25 °C revealed:

• After one day at 4 °C, K value increased moderately; histamine remained undetectable.
• After one day at 25 °C, a sharp rise in K value occurred; histamine was still absent.
• After six days at 4 °C, albacore tuna reached spoilage‐level K values and histamine concentration of 2.1 mg/100 g, below the Codex limit.

The method effectively separated histamine from structurally similar amino acids, ensuring accurate quantification.

Benefits and Practical Applications

The dual injection strategy consolidates two analyses into one run, reducing turnaround time and generating a single data file per sample for streamlined record keeping. It supports quality control in fisheries, seafood processing, and regulatory testing.

Future Trends and Potential Applications

Future developments may include integration with high-throughput autosamplers, expansion to other spoilage markers and seafood species, and real-time monitoring via IoT-enabled sensors and advanced data analytics to enhance supply chain traceability.

Conclusion

A robust HPLC method using a dual injection system enables simultaneous quantification of ATP degradation products, histamine, and amino acids in fish meat. This unified approach improves analytical throughput and ensures reliable assessment of freshness and spoilage.

References

• Usui K., Watanabe E., Comparison of changes in freshness in fresh and frozen black marlin using the K-value, Bulletin No. 5 of the Kanagawa Prefectural Fisheries Technology Center, pages 11–14, 2012