Development of Sensitive and Selective Methods for Identifcation of Marine Toxins by Liquid Chromatography Tandem Mass Spectrometry

Importance of the topic

Marine toxins represent a significant risk for public health and seafood safety worldwide. Traditional mouse bioassays are ethically contested and lack the sensitivity and specificity required for modern monitoring programs. The development of liquid chromatography tandem mass spectrometry methods offers a rapid, accurate, and animal‐free alternative capable of detecting multiple toxin classes in a single run.

Objectives and overview of the study

This study aimed to establish and optimize LC‐MS/MS conditions for the simultaneous identification of three major groups of marine toxins: diarrhetic shellfish poisoning toxins (okadaic acid, DTX1, DTX2), ciguatera fish poisoning toxin (ciguatoxin 3C), and globefish poisoning toxin (tetrodotoxin). Key goals included evaluating column chemistries and chromatographic modes to accommodate both hydrophobic and hydrophilic analytes, and assessing solid‐phase extraction pretreatment for sample cleanup.

Methodology and instrumentation

UHPLC system: Nexera X2 (Shimadzu). Mass spectrometer: LCMS‐8050 triple quadrupole with heated electrospray ionization (ESI) and multiple reaction monitoring (MRM). Column evaluations:

• Multi‐mode ODS (Scherzo SM‐C18)
• Reversed phase ODS (L‐column2 ODS) under acidic and neutral mobile phases
• HILIC mode (InertSustain Amide PEEK) for tetrodotoxin

Mobile phases employed formic acid or ammonium formate buffers in water and acetonitrile. SPE cartridges (ISOLUTE C18(EC) and EVOLUTE EXPRESS ABN) were tested for recovery of okadaic acid and DTX1.

Main results and discussion

The multimode ODS column provided the best overall retention and resolution for all five target toxins in a single run of 17.5 minutes. Ciguatoxin ionization favored sodium adducts in positive mode, while DSP toxins were detected as deprotonated molecules in negative mode. Tetrodotoxin exhibited poor retention in reversed phase but showed sharp peaks and high sensitivity in HILIC mode. Limits of detection reached as low as 0.01 ppb for TTX and DTX1 under optimized conditions. SPE recovery for okadaic acid and DTX1 ranged from 66 % to 83 %, demonstrating effective cleanup.

Benefits and practical applications

This approach enables simultaneous, high‐sensitivity screening of multiple toxin classes, reducing analysis time and eliminating the need for animal assays. The method supports regulatory compliance and can be integrated into routine quality control workflows in seafood laboratories.

Future trends and possibilities

Further expansion to additional paralytic shellfish poisoning analogs is under investigation. Automation of sample preparation and matrix effect evaluation using advanced SPE formats will enhance throughput. Emerging technologies such as high‐resolution MS and portable LC‐MS systems may allow on‐site monitoring and real‐time risk assessment.

Conclusion

The proposed LC‐MS/MS protocols deliver a sensitive, selective, and ethically sound solution for marine toxin surveillance. The combination of multimode ODS and HILIC separations, coupled with effective SPE cleanup, forms a robust platform for comprehensive seafood safety testing.

Reference

• AOAC Official Methods of Analysis 2005.06 and 2011.02
• CODEX STAN 292-2008