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

Importance of the Topic

Fish meat is highly prone to spoilage due to its delicate muscle structure and high water content compared to terrestrial meats. Ensuring accurate and rapid assessment of fish freshness is vital for consumer safety and quality control. The K value, based on ATP breakdown, provides a numerical freshness index, while histamine accumulation signals putrefaction and potential foodborne allergic risk. A unified analytical approach for these markers alongside key amino acids can greatly streamline seafood quality assessment.

Objectives and Study Overview

This study aimed to develop and validate a simultaneous high‐performance liquid chromatography (HPLC) method using a dual injection system (Nexera) to quantify ATP‐related compounds for K value calculation, histamine, and a panel of twenty‐four amino acids in fish meat. The goal was to achieve rapid throughput, robust performance, and integrated data management for routine food safety inspection.

Methodology and Used Instrumentation

• HPLC System: Nexera dual injection with two independent analytical flow paths.
• Detectors: Photodiode array (PDA) at 260 nm for ATP‐related compounds; fluorescence detection for amino acids and histamine (Ex/Em: 350/450 nm and 266/305 nm).
• Columns: Shim-pack GIST C18 AQ (100 × 3.0 mm, 3 µm) for nucleotides; Shim-pack Velox C18 (100 × 3.0 mm, 2.7 µm) for amino acids.
• Sample Preparation: Deproteinization, perchloric acid extraction, pH adjustment; automatic pre‐column derivatization with o-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) in low-adsorption TORAST-H glass vials.
• Chromatographic Conditions: Gradient elution for ATP‐related analytes; isocratic program for amino acids. Column temperatures set at 30 °C and 35 °C, flow rate 0.8 mL/min, injection volumes 10 µL and 1 µL respectively.

Main Results and Discussion

• Extraction Efficiency: Perchloric acid extraction yielded over 92 % recovery for histamine and histidine versus water.
• Recovery and Precision: Spiking tuna samples at the Codex histamine limit (10 mg/100 g) produced average recoveries of 99.8 % (RSD 2.1 %).
• Linearity: Calibration curves for 31 target compounds exhibited excellent linearity (r² ≥ 0.999) across relevant concentration ranges.
• Application to Tuna Samples: Yellowfin tuna stored at 4 °C and 25 °C showed incremental increases in K value over time; histamine remained undetectable within 24 h. Albacore tuna refrigerated for six days reached a K value indicative of putrefaction (70.4 %) and contained 2.1 mg/100 g histamine (below regulatory limit).
• Chromatographic Separation: Histamine was successfully resolved from structurally similar amino acids (e.g., histidine, alanine, taurine), ensuring accurate quantification.

Benefits and Practical Applications

• Simultaneous multi-analyte determination reduces analysis time, labor, and sample handling.
• Integrated data output from dual injection consolidates results into a single file for streamlined reporting and traceability.
• Automated derivatization protocol enhances reproducibility and maintains consistent analysis intervals.
• Method is well suited for routine quality assurance, regulatory testing, and rapid inspection in seafood processing environments.

Future Trends and Potential Applications

• Extension of dual injection techniques to other food matrices (meat, dairy, beverages) and spoilage or quality markers.
• Coupling with mass spectrometry for increased selectivity, sensitivity, and structural confirmation.
• Development of portable, high-throughput systems for on-site freshness monitoring.
• Integration of predictive software models combining K value, histamine levels, and other biochemical indicators to forecast spoilage dynamics.

Conclusion

The Nexera dual injection HPLC method enables robust, simultaneous quantification of ATP‐derived freshness indices, histamine levels, and amino acid profiles in fish meat. This approach offers enhanced throughput, reliable performance, and simplified data management, supporting improved food safety and quality control in the seafood industry.

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. 2012;11–14.