High Sensitivity Analysis of Diarrhetic Shellfish Poisoning (DSP) Toxins Using Liquid Chromatography Tandem Mass Spectrometry

Significance of the Topic

Diarrhetic shellfish poisoning (DSP) toxins such as okadaic acid (OA) and dinophysistoxins (DTXs) represent a serious food safety issue worldwide. Traditional mouse bioassays (MBA) lack sensitivity and ethical acceptability. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) offers enhanced precision and sensitivity, supporting regulatory monitoring and protecting public health.

Objectives and Study Overview

This study aimed to develop and validate a high-sensitivity LC/MS/MS method for simultaneous screening of six DSP toxins (OA, DTX1, PTX1, PTX2, PTX6, YTX) and for accurate quantification of OA and DTX1 in shellfish. The approach aligns with Japan’s 2015 regulatory update replacing MBA with LC/MS/MS.

Methodology and Instrumentation

Sample Preparation

• Extraction based on EU-RL-MB SOP Ver.4: homogenization in methanol, centrifugation, dilution, filtration.
• Hydrolysis step to measure total OA and DTX1: alkaline treatment, heating, neutralization, filtration.

Used Instrumentation

• UHPLC system: Nexera with ODS column (50 x 2.1 mm, 2 µm).
• Mobile phases: 2 mM ammonium formate/50 mM formic acid in water (A) and acetonitrile/water (95/5) with same additives (B); gradient from 30 to 100 % B.
• Triple quadrupole MS: LCMS-8050, ESI negative, MRM transitions for each toxin (e.g. OA m/z 803.5>255.2).

Main Results and Discussion

Screening performance demonstrated clear MRM peaks for all six toxins in contaminated scallop extracts. Calibration for OA and DTX1 was linear from 0.04 to 20 ppb (r2 > 0.999), with limits of quantification at 0.005 ppb (OA) and 0.01 ppb (DTX1). Repeatability at 0.1 ppb showed RSD ≤ 8.1 %. Matrix effects were evaluated by spiking experiments; 10- and 100-fold dilutions mitigated suppression, yielding recoveries of 96–119 % for oysters and 110–295 % for scallops, within acceptable QC drift (<25 %). Actual market samples contained low levels of OA equivalents (56.2 µg/kg in scallop), well under Japan’s limit (160 µg/kg).

Benefits and Practical Applications

The developed LC/MS/MS workflow offers rapid, animal-free testing with superior sensitivity over MBA. Simplified sample preparation reduces labor, while robust quantification supports routine monitoring in food safety laboratories and compliance with international regulations.

Future Trends and Possibilities of Use

Advances may include automated solid-phase extraction for matrix cleanup, expansion to emerging toxin analogues, coupling with high-resolution MS for untargeted screening, and integration into high-throughput platforms. Wider regulatory adoption and method standardization will further strengthen seafood safety surveillance.

Conclusion

LC/MS/MS using the Nexera-LCMS-8050 system provides a powerful, validated method for DSP toxin analysis. It meets EU and Japanese guidelines, delivers high sensitivity and precision, and enables reliable monitoring of shellfish, replacing the traditional MBA.

Reference

• EU-Harmonised Standard Operating Procedure for Determination of Lipophilic Marine Biotoxins in Molluscs by LC-MS/MS Ver.4
• T. Suzuki and M. A. Quilliam, Analytical Sciences 27 (2011) 571–584