High sensitivity and separation detection method for ciguatoxin analogues using Li adduct ion by LC-MS/MS

Importance of the topic

Ciguatera fish poisoning is the most widespread seafood‐borne illness worldwide and is caused by ciguatoxins (CTXs), potent polyether neurotoxins produced by benthic dinoflagellates. These toxins occur at trace levels in fish tissue and can induce severe neurotoxic effects at concentrations as low as 0.2 µg/kg. Regulatory agencies including the US FDA and EFSA recommend a control level of 0.01 µg CTX1B equivalent per kilogram of fish. Achieving reliable detection at or below this level is critical for food safety monitoring and public health protection.

Study objectives and overview

This study aimed to develop and validate a high‐sensitivity LC‐MS/MS method for simultaneous detection and separation of nine CTX analogues. The central innovation was exploiting lithium adduct formation ([M+Li]+) in lieu of traditional sodium adducts ([M+Na]+) by adding trace Li+ to the mobile phase. Method performance was evaluated in terms of chromatographic resolution, ionization efficiency, collision energy optimization, limit of quantification (LOQ), accuracy, and reproducibility.

Methodology and Instrumentation

Sample preparation involved spiking fish tissue extracts with certified CTX reference materials. Chromatography was performed on a Shimadzu Nexera X3 UHPLC system coupled to a Shimadzu LCMS‐8060NX triple‐quadrupole mass spectrometer equipped with an electrospray ionization source in positive mode. The mobile phase consisted of acetonitrile and water with 0.01 % formic acid plus 0.1 mM LiCl or NaCl. Trace alkali addition promoted selective formation of [M+Li]+ or [M+Na]+ ions. Multiple reaction monitoring (MRM) transitions were optimized by adjusting collision energy to maximize signal‐to‐noise ratios for each adduct species.

Main Results and Discussion

Ion adduct formation studies demonstrated that Li+ addition suppressed competing sodium adducts and significantly enhanced sensitivity for CTX1B and other analogues. Optimized collision energies improved response and reproducibility (S/N ratio > 2000 for key transitions). Chromatographic comparisons showed that Li‐based mobile phases delivered complete baseline separation of all nine CTX analogues within a 12-minute gradient, whereas MeOH-based or Na+-based methods exhibited coelutions. Calibration curves using quantitative NMR‐certified standards were linear (R2 > 0.999) over the range of interest. LOQs in fish extracts, expressed as µg/kg, ranged from 0.0004 to 0.0012, well below the 0.01 µg/kg regulatory threshold. Three‐point replicate analyses yielded accuracy within ±5 % and relative standard deviations below 8 %.

Benefits and Practical Applications

• The method achieves detection limits an order of magnitude below regulatory action levels, enabling reliable screening of seafood for CTX contamination.
• Implementation on standard LC‐MS/MS instrumentation makes it accessible to both research and routine food testing laboratories.
• Complete separation of multiple CTX analogues supports profiling of toxin variants in environmental and seafood samples.

Future Trends and Potential Applications

Further development could include extension to other marine toxin classes using analogous cationization strategies, integration with high-resolution mass spectrometry for structural confirmation, and automated data processing with machine learning to expedite routine analyses. Miniaturized or field-deployable versions may provide rapid on-site testing in remote harvesting regions.

Conclusion

A novel LC-MS/MS approach using lithium adduct ionization has been established for high-sensitivity, high-resolution detection of nine ciguatoxin analogues. The method meets stringent regulatory LOQs, demonstrates robust accuracy and precision, and can be widely adopted for seafood safety monitoring.

Reference

• Yogi K., Oshiro N., Inafuku Y., Hirama M., Yasumoto T. Detailed LC-MS/MS analysis of ciguatoxins revealing distinct regional and species characteristics in fish and causative alga from the Pacific. Anal. Chem. 2011, 83, 8886–8891.
• Kato T., Yasumoto T. Quantification of Representative Ciguatoxins in the Pacific Using Quantitative Nuclear Magnetic Resonance Spectroscopy. Mar. Drugs 2017, 15, 309.
• Wu J. J. et al. Validation of an accelerated solvent extraction LC-MS/MS method for Pacific ciguatoxin-1 in fish flesh and comparison with the mouse neuroblastoma assay. Anal. Bioanal. Chem. 2011, 400, 3165–3175.
• Klijnstra M. D., Gerssen A. A Sensitive LC-MS/MS Method for Palytoxin Using Lithium Cationization. Toxins 2018, 10(12), 537.
• Oshiro N. et al. Analytical Studies on Ciguateric Fish in Okinawa, Japan (II): The Grouper Variola albimarginata. J. Mar. Sci. Eng. 2023, 11, 242.