Analysis of Diarrhetic Shellfish Toxins (Okadaic Acid Group) Using Triple Quadrupole LC/MS/MS

Significance of the topic

Diarrhetic shellfish toxins (DSTs) are a serious food-safety concern linked to okadaic acid (OA) and related dinophysistoxins. Regulatory limits have been introduced to protect consumers from outbreaks of diarrhetic shellfish poisoning. Reliable analytical methods are essential to monitor toxin levels efficiently.

Goals and study overview

This work describes the development and validation of a triple quadrupole LC/MS/MS protocol to quantify OA, DTX1, and DTX2 in shellfish. The method replaces the traditional mouse bioassay and addresses regulatory requirements for a 0.16 mg OA-equivalent/kg limit.

Methodology and instrumentation

The procedure involves sample extraction, alkaline hydrolysis, hexane cleanup, and solid-phase extraction. Chromatographic separation uses a Shim-pack Scepter C18 column with a gradient of ammonium formate/formic acid aqueous solution and acetonitrile. Detection is by ESI in negative mode on a Shimadzu LCMS-8060 triple quadrupole mass spectrometer monitoring specific MRM transitions. Key parameters include a 40 °C column temperature, 0.2 mL/min flow rate, and a 5 µL injection volume.

Main results and discussion

The method delivered excellent repeatability (retention time RSD ≤0.05%, peak area RSD ≤3%) at 1 ppb standards. Calibration curves across 0.1–10 ppb showed linearity with r2 ≥0.999. Standard addition experiments on scallop midgut gland CRM yielded recoveries of 85–93% and area RSD below 2.3%, demonstrating effective matrix compensation.

Benefits and practical applications

• High sensitivity and specificity for OA-group toxins
• Rapid turnaround compatible with routine monitoring
• Elimination of animal testing in line with regulatory updates
• Quantitative results expressed as OA equivalents for risk assessment

Future trends and potential applications

Emerging directions include expanded multiplexing of lipophilic toxins, automation of sample preparation, high-throughput platforms, and integration with laboratory information management systems. Method adaptation to portable MS systems may enable field screening.

Conclusion

The validated triple quadrupole LC/MS/MS method provides a robust, accurate, and regulatory-compliant approach to quantify diarrhetic shellfish toxins. Implementation in food-safety laboratories will enhance monitoring efficiency and consumer protection.

References

• Ministry of Health, Labour and Welfare Notice 0306 No. 2, March 6, 2015
• Notice 0308 No. 2 and No. 9, March 8, 2017
• NMIJ/AIST certified reference materials for OA and DTX1
• NRC Canada CRM-DTX2 reference standard