UPLC/MS/MS Method for the Routine Quantification of Regulated and Non-Regulated Lipophilic Marine Biotoxins in Shellfish

Importance of the Topic

Lipophilic marine biotoxins in shellfish pose human health risks such as diarrhetic shellfish poisoning. Rapid and reliable detection is essential to ensure food safety, support regulatory compliance in the European Union, and replace animal bioassays with robust instrumental methods.

Objectives and Study Overview

This study aimed to develop a five minute UPLC MS/MS method to quantify both regulated and non regulated lipophilic marine toxins in shellfish. By coupling ultra performance liquid chromatography with tandem quadrupole mass spectrometry, the goal was to achieve a four fold reduction in analysis time, maintain sensitivity at regulated detection limits, and incorporate emerging cyclic imines for EFSA interest.

Methodology and Instrumentation

Treatment and extraction

• One gram of homogenized shellfish tissue extracted three times with methanol, centrifuged, combined, and brought to volume.
• Optional alkaline hydrolysis for DTX3 esters with sodium hydroxide and neutralization with acid.

Chromatographic and mass spectrometric conditions

• ACQUITY UPLC BEH C18 column, five minute gradient at 0.6 mL per minute and 40 °C.
• Mobile phases of water and acetonitrile with 6.7 mM ammonium hydroxide.
• Xevo TQ S tandem quadrupole operated in alternating positive and negative electrospray modes with multiple reaction monitoring transitions.

Software and data processing

• TargetLynx for automated data reduction and regulatory flagging.
• QuanPedia database for method protocol sharing.

Main Results and Discussion

Analytical performance

• Analysis time reduced from 20 to 5 minutes without loss of resolution.
• Sensitivity sufficient to detect toxins at 0.125 times EU validation levels with signal to noise above three.
• Linearity with R2 above 0.99 for all compounds.

Validation outcomes

• Recoveries ranged from 91 to 104 percent except for pinnatoxin E due to methanol esterification.
• Repeatability (RSDr) generally below 10 percent; reproducibility within regulatory expectations.
• Decision limits (CCα) close to permitted levels enabling positive compliance decisions.

Inclusion of non regulated toxins

• Cyclic imines screened using structural analog MRM transitions.
• Method adaptable to new standards when available.

Benefits and Practical Applications

• Replaces animal bioassays with ethical, sensitive chemical analysis.
• Supports routine EU monitoring programs for regulated toxins.
• Enables early screening of emerging non regulated toxins.
• High throughput fits quality control and research laboratories.

Future Trends and Potential Applications

• Expansion of MRM libraries with new toxin standards.
• Integration into regional surveillance networks for real time monitoring.
• Automated sample preparation and data review workflows.
• Potential for high resolution MS to identify novel toxin analogs.

Conclusion

The UPLC MS/MS method delivers a rapid, robust, and sensitive platform for routine quantification of lipophilic marine biotoxins in shellfish. It meets regulatory requirements, reduces analysis time by four fold, and offers flexibility to include emerging toxins, thus enhancing food safety and laboratory productivity.

Reference

1. European Commission Laying down implementing measures under Regulation EC No 853/2004 Off J Eur Commun L 2005;338:40-41.
2. European Commission Commission Regulation EU No 15/2011 of 10 January 2011 amending Regulation EC No 2074/2005 Off J Eur Commun 2011;L6:3-6.
3. Gerssen A Mulder PPJ McElhinney MA De Boer J Liquid chromatography tandem mass spectrometry method for the detection of marine lipophilic toxins under alkaline conditions J Chromatogr A 2009;1216:1421-1430.