Determination of Lipophilic Marine Biotoxins in Molluscs by LC-MS/MS using Offline Extraction

Significance of the Topic

The accumulation of lipophilic marine biotoxins in bivalve molluscs poses a serious risk to food safety and public health. Outbreaks of toxic microalgal blooms have led to human poisoning via contaminated shellfish, resulting in gastrointestinal and neurological symptoms. European regulations set strict limits for these toxins to protect consumers and mandate reliable analytical methods for routine monitoring.

Objectives and Study Overview

This study aims to validate an EU-harmonized offline extraction procedure coupled with LC-MS/MS for quantifying key lipophilic toxins—okadaic acid (OA), dinophysistoxins (DTX-1, DTX-2), pectenotoxin-2 (PTX-2), azaspiracids (AZA-1, 2, 3) and yessotoxin (YTX)—in mussel samples. The goal is to demonstrate method performance according to regulatory requirements.

Methodology and Instrumentation

Sample Preparation and Cleanup:

• Homogenize 2 g mussel tissue with two sequential extractions in 9 mL methanol
• Combine extracts and purify via C18 SPE cartridge with 0.45 μm syringe filter

Chromatography:

• Thermo Scientific Accela UHPLC with Hypersil GOLD™ column (50×2.1 mm, 1.9 μm)
• Gradient elution from 30% to 90% acetonitrile in 3 min at 200 μL/min

Mass Spectrometry:

• Thermo Scientific TSQ Quantum Ultra triple quadrupole with H-ESI II source
• Selected Reaction Monitoring (SRM) for optimized transitions

Main Results and Discussion

Calibration curves showed excellent linearity (r2 > 0.99) across 2–50 μg/kg. Method repeatability (inter-day, n=10) yielded CV% below 20% for all analytes. Estimated LODs ranged from 0.56 to 5.66 μg/kg and LOQs from 1.11 to 10.45 μg/kg. Recoveries after SPE cleanup were 93–108%, confirming accuracy. Chromatographic separation achieved baseline resolution within a 15-minute run.

Benefits and Practical Applications

This LC-MS/MS method offers high sensitivity, specificity, and throughput for regulatory compliance. The simple offline extraction and robust cleanup support routine monitoring in QA/QC and environmental laboratories.

Future Trends and Opportunities

Advances may include automated sample preparation, miniaturized extraction techniques, expansion to emerging toxins, and integration of high-resolution mass spectrometry. Collaborative ring trials could further harmonize interlaboratory performance.

Conclusion

The validated offline extraction LC-MS/MS protocol is selective, sensitive, and reproducible for quantifying EU-regulated lipophilic marine biotoxins in bivalve molluscs, supporting enhanced food safety monitoring.

References

1. Regulation (EC) No 853/2004 of the European Parliament and of the Council, Official Journal L139, 2004.
2. Commission Regulation (EU) No 15/2011, Official Journal L6, 2011.
3. Commission Regulation (EC) No 2074/2005, Official Journal L388, 2005.
4. EU-RL-MB, AESAN, EU-Harmonised SOP for Lipophilic Marine Biotoxins by LC-MS/MS – Version 3, 2011.
5. UNICHIM Manual No. 179/0, Guidelines for Analytical Method Validation, 1999.