Determination of Paralytic Shellfish Toxins and Tetrodotoxins in Shellfish by Ultra-High Performance Hydrophilic-Interaction Liquid Chromatography-Tandem Quadrupole Mass Spectrometry

Importance of the Topic

Paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) are potent neurotoxins that accumulate in shellfish, posing significant public health risks. Regulatory limits for PSTs in the EU are typically set at 800 µg saxitoxin equivalents per kilogram of shellfish meat, while recent concerns over TTX have led to guidance levels around 44 µg/kg. Routine monitoring requires methods that combine high sensitivity, specificity, speed, and reproducibility.

Study Objectives and Overview

This application note describes the development and validation of a rapid, single-shot analytical workflow to quantify PSTs and TTX in a variety of shellfish matrices using ultra-high performance hydrophilic-interaction liquid chromatography coupled with tandem quadrupole mass spectrometry (UHPLC-HILIC-MS/MS). The goals were to meet official control requirements, achieve limits of detection well below regulatory thresholds, and enable simultaneous analysis of multiple toxin analogues in a single run.

Methodology and Instrumentation

The procedure begins with a dispersive extraction of 5 g homogenized shellfish tissue in 1% acetic acid heated briefly. After centrifugation and neutralization, samples undergo solid-phase extraction cleanup using graphitized carbon cartridges. The cleaned extract is diluted with acetonitrile and injected (2 µL) onto an ACQUITY UPLC BEH Amide column (2.1 × 150 mm, 1.7 µm) at 60 °C. A gradient of water/formic acid/ammonia and acetonitrile/water/formic acid is delivered by the ACQUITY UPLC I-Class PLUS system. Detection employs a Xevo TQ-S mass spectrometer in both positive and negative electrospray modes with optimized multiple reaction monitoring transitions for 19 PST analogues and TTX. Data acquisition and processing use MassLynx v4.2 and TargetLynx XS Application Manager.

Used Instrumentation

• ACQUITY UPLC I-Class PLUS System with FTN Sample Manager
• ACQUITY UPLC BEH Amide Column and VanGuard Pre-column
• Xevo TQ-S Tandem Quadrupole Mass Spectrometer
• MassLynx Software v4.2 and TargetLynx XS
• Supelclean ENVI-Carb SPE Cartridges (250 mg/3 mL)

Key Results and Discussion

Method validation covered 12 shellfish species (mussels, oysters, clams, cockles, scallops) and focused on low, medium, and high spiked levels. Calibration curves in matrix showed linearity (r² > 0.996). Limits of detection for PST analogues ranged from 0.03 to 2.25 µg STX eq/kg, and for TTX 0.25 µg/kg. Reporting limits were <1% of the EU maximum permitted level for most toxins. Recoveries of individual analogues averaged 65–125%, with total PST recoveries of 97–100% across concentrations. Repeatability and reproducibility (HorRat < 2.0) met official control criteria. Long-term analysis of a certified reference material showed overall recoveries of 85–127% for PST analogues and 97% for total toxin content.

Benefits and Practical Applications

• Single-run quantitation of PSTs and TTX simplifies laboratory workflows and reduces analysis time to 11 minutes per injection.
• High sensitivity supports screening far below regulatory limits, improving consumer safety.
• Graphitized carbon SPE effectively removes matrix interferences, enhancing MS/MS performance.
• Flexible chromatographic gradient and flow rates allow method transfer to similar instrumentation platforms.

Future Trends and Potential Applications

Emerging needs include the expansion of monitored toxin panels as new analogues are discovered, further inter-laboratory studies for full collaborative validation, and incorporation of high-resolution MS detection to improve confirmatory power. Miniaturized or automated sample preparation approaches may further increase throughput and reduce solvent consumption in routine testing labs.

Conclusion

The described UHPLC-HILIC-MS/MS method delivers a robust, sensitive, and cost-effective solution for simultaneous determination of PSTs and TTX in shellfish. It meets stringent regulatory requirements for accuracy, precision, and reporting limits, while streamlining sample preparation and analysis time. This approach supports both routine monitoring and rapid response to toxin outbreaks in shellfish production areas.

Reference

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