The Determination Method of the Lipophilic Marine Biotoxins in Bivalve Subject to EU Regulations Using a Triple Quadrupole Mass Spectromete

Importance of the Topic

Shellfish can accumulate lipophilic marine biotoxins produced by dinoflagellates leading to serious human health hazards. Regular monitoring of these toxins in bivalve molluscs is critical to ensure food safety and to meet stringent regulatory limits set by the EU and other authorities.

Objectives and Study Overview

This study presents a validated LCMS MS method that enables the simultaneous quantification of five key lipophilic marine biotoxins in scallop samples. The method follows the EU harmonised procedures and aims to improve sensitivity, simplify sample preparation, and support compliance with maximum allowable levels.

Methodology and Instrumentation

Sample Preparation

• Extraction of scallop tissue with and without alkaline hydrolysis to measure free and total amounts of okadaic acid and dinophysistoxins
• Use of an autosampler preprocessing function to automatically create matrix matched calibration curves and perform standard addition

Chromatographic Conditions

• High pH resistant ODS column 100 mm × 2.1 mm 3.5 μm
• Mobile phase A 2 mM ammonium bicarbonate aqueous solution pH 11
• Mobile phase B acetonitrile 2 mM ammonium bicarbonate aqueous solution pH 11 in a 9 to 1 ratio
• Flow rate 0.3 mL per min column temperature 40 °C injection volume 5 μL

Mass Spectrometry Conditions

• Triple quadrupole MS with electrospray ionisation in positive and negative modes
• Interface voltages set to plus 4 kV and minus 3 kV
• Nebulising gas 2.5 L per min drying gas 15 L per min
• Collision induced dissociation pressure 270 kPa probe position plus 2 mm
• Multi reaction monitoring transitions for okadaic acid dinophysistoxin-1 pectenotoxin-2 azaspiracid-1 and yessotoxin were optimised for high selectivity and sensitivity

Main Results and Discussion

Calibration curves for all five toxins exhibited excellent linearity with coefficients of determination above 0.9997. Limits of quantification were in the low ng per mL range. Recovery rates for spiked scallop extracts ranged from 95 to 105 for okadaic acid dinophysistoxin-1 and yessotoxin regardless of hydrolysis. Pectenotoxin-2 and azaspiracid-1 were effectively removed by the hydrolysis step as expected. The method delivered consistent performance and enabled simultaneous determination within a single analytical run.

Benefits and Practical Applications

• Complies with EU Regulation EC853/2004 and subsequent updates for lipophilic marine biotoxins
• Simultaneous detection of five toxin groups reduces analysis time and labour
• High sensitivity supports food safety monitoring at regulatory limits
• Automated matrix matched calibration enhances accuracy and throughput

Future Trends and Potential Applications

Adaptation of this workflow to additional toxin classes and shellfish species could broaden surveillance capabilities. Integration with high resolution mass spectrometry may offer further improvements in identification and quantification. Advances in online sample preparation and rapid screening are expected to support real time monitoring in aquaculture and processing environments.

Conclusion

The described LCMS MS method fulfils regulatory requirements for simultaneous analysis of key lipophilic marine biotoxins in scallop samples. Its robust performance sensitivity and automation features make it well suited for routine food safety laboratories and regulatory reference institutions.

Reference

EU Harmonised Standard Operating Procedure for determination of Lipophilic marine biotoxins in molluscs by LC MS MS Version 5 January 2015