Determination of Paralytic Shellfish Toxins and Tetrodotoxin in Shellfish using HILIC/MS/MS

Significance of the Topic

The accumulation of paralytic shellfish toxins (PST) and tetrodotoxin (TTX) in bivalve mollusks poses significant public health risks and is subject to strict regulatory limits in many regions, including an action level of 800 µg STX equivalents per kg in the EU. Conventional detection techniques such as mouse bioassay and precolumn oxidation with fluorescence detection are laborious, multistage, and sometimes fail to detect all congeners. A streamlined hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS) method offers a rapid, sensitive, and comprehensive alternative for routine monitoring.

Objectives and Overview of the Study

This study describes the development and single-laboratory validation of an HILIC/MS/MS approach for the simultaneous determination of 25 PST analogs and TTX in shellfish tissue. Using an Agilent 1290 Infinity II LC system coupled to a 6495B triple quadrupole MS, the method aims to reduce sample preparation complexity, shorten run times to 11 minutes, and deliver the sensitivity, precision, and accuracy required for regulatory compliance and high-throughput analysis.

Methodology and Instrumentation

Sample Preparation:

• Extract 5 g homogenized shellfish tissue with 1% acetic acid (1:1, w/v) by vortex mixing and heat treatment.
• Centrifuge, adjust supernatant with ammonia, and perform SPE cleanup on a graphitic carbon cartridge.
• Elute toxins with 20% acetonitrile/0.25% acetic acid, then dilute for HILIC injection.

Instrumentation Used:

• Agilent 1290 Infinity II LC with amide HILIC column (2.1×150 mm, 1.7 µm) and precolumn.
• Agilent 6495B Triple Quadrupole LC/MS with Jet Stream ESI, fast polarity switching, and dynamic MRM.
• Mobile phases comprising water/formic acid/ammonium hydroxide and acetonitrile/water/formic acid gradients.
• MassHunter software for acquisition and quantitation.

Main Results and Discussion

The method achieved baseline separation of critical epimeric toxin pairs and delivered linear calibration curves (R² > 0.994) over relevant ranges. Limits of detection (0.02–0.37 nmol/L) and quantitation (0.08–1.22 nmol/L) translate to sub-µg STX equivalents per kg of tissue. Precision metrics met criteria (within-batch RSD 4–17%; between-batch over one week RSD 8–23%) and long-term reproducibility over one year (RSD 16–27%). Accuracy assessed against a certified reference material ranged from 86% to 116%.

Benefits and Practical Applications

• Simultaneous analysis of PST and TTX in a single 11-minute run.
• High specificity via dual MRM transitions per analyte.
• One-step extraction and straightforward SPE reduce analyst time and consumables.
• Suitable for routine QA/QC and regulatory laboratories with cost-effective operation.

Future Trends and Opportunities

Opportunities include automating SPE cleanup with robotics, expanding the method to other emerging marine biotoxins, integrating high-resolution MS for non-targeted screening, developing online SPE-HILIC interfaces, and global harmonization of toxicity equivalency factors. Miniaturized columns and faster gradients can further enhance throughput.

Conclusion

The validated HILIC/MS/MS method enables rapid, sensitive, and accurate quantitation of 25 PST analogs and TTX in shellfish, outperforming traditional assays in simplicity and throughput while meeting stringent regulatory standards.

References

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