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

Significance of the Topic

Reliable detection of diarrhetic shellfish toxins (DSTs), including okadaic acid and related dinophysistoxins, is critical to protect public health and comply with regulatory limits. Advanced instrumental methods offer rapid, sensitive alternatives to animal bioassays, improving food safety monitoring.

Objectives and Study Overview

This work presents a triple quadrupole LC-MS/MS method for quantifying the okadaic acid group (OA, DTX1, DTX2) in shellfish. It aligns with Japanese Food Sanitation Act requirements, utilizes certified reference materials, and replaces the traditional mouse toxicity test for DSTs.

Methodology and Instrumentation Used

• Sample Preparation: Methanol extraction, alkaline hydrolysis (76 °C, 40 min), acid neutralization, hexane cleanup, and polymer SPE purification.
• Instrumentation:
  • Shimadzu LCMS-8060 triple quadrupole with negative-mode ESI.
  • Shim-pack Scepter C18 column (100 × 2.0 mm, 1.9 µm) at 40 °C, 0.2 mL/min flow.
  • Mobile phases: A) 2 mmol/L ammonium formate with 50 mmol/L formic acid in water; B) acetonitrile/water (95/5) with same additives; gradient from 40 % to 100 % B (0–7.5 min) and back to 40 % (12.5–17.5 min); use front-cut valve (6–10 min).
  • MRM transitions: OA 803.30→255.10, 803.30→113.10; DTX2 same; DTX1 817.30→255.10, 817.30→113.10.
• Calibration: 0.1–10 ppb linear range with r² ≥ 0.999.
• Standard Addition: Applied to scallop midgut extracts at 10, 20, 50 ppb.

Main Results and Discussion

• Repeatability: Retention time RSD < 0.05 %; peak area RSD < 3 % at 1 ppb.
• Linearity: r² ≥ 0.999 for OA, DTX1, DTX2 over 0.1–10 ppb.
• Matrix Effects: Standard addition recoveries of 93 % (OA) and 85 % (DTX1) in scallop midgut; area RSDs of 2.29 % and 0.57 %, respectively.
• Regulatory Compliance: Method meets Japanese Food Sanitation Act specifications for OA-equivalent quantitation.

Benefits and Practical Applications

• Replaces variable, labor-intensive animal tests with a robust instrumental approach.
• High sensitivity and specificity for DST analysis in complex matrices.
• Conversion to OA-equivalents via defined toxicity equivalence factors.
• Suitable for routine monitoring in regulatory and QA/QC laboratories.

Future Trends and Potential Applications

• Extension to additional marine biotoxin groups if regulatory thresholds evolve.
• Integration of high-resolution MS or automated sample handling for higher throughput.
• Development of multi-toxin screening panels for simultaneous analysis.
• Adoption of portable MS platforms for on-site shellfish safety testing.

Conclusion

The validated LC-MS/MS method delivers accurate, precise, and sensitive analysis of okadaic acid group toxins, offering strong repeatability, excellent linearity, and reliable recoveries. It supports food safety compliance, expedites laboratory workflow, and obviates the need for animal assays.

References

• Ministry of Health, Labour and Welfare (Japan). Notice 0306 No. 2, March 6, 2015.
• Ministry of Health, Labour and Welfare (Japan). Notice 0308 No. 2 and No. 9, March 8, 2017.
• National Metrology Institute of Japan/AIST. NMIJ CRM 7520-a certified reference material.
• National Research Council Canada. CRM-DTX2 certified reference material.