Highly sensitive analysis of the related substance of ciguatoxins by the multiple reaction monitoring and electrospray ionization with LC/MS/MS

Importance of Topic

Ciguatoxins are potent marine toxins responsible for serious seafood poisoning. Their detection at trace levels is critical for food safety monitoring, public health protection, and regulatory compliance. High sensitivity and selectivity in analytical methods help to prevent human exposure and support rapid screening in QA/QC laboratories.

Study Objectives and Overview

This study aimed to develop and optimize an LC–MS/MS method using multiple reaction monitoring (MRM) for the simultaneous quantification of five key ciguatoxin analogues (CTX1B, 52-epi-54-deoxyCTX1B, 51-hydroxyCTX3C, CTX3C, CTX4A). The work included evaluation of chromatographic columns, prediction of MRM transitions based on collision-induced dissociation (CID) product ions, and assessment of method performance at sub-ppb levels.

Methodology and Instrumentation

The analytical workflow combined ultra-high-performance liquid chromatography (UHPLC) with tandem mass spectrometry in positive electrospray ionization mode. Key elements included:

• Chromatographic separation on two high-performance ODS columns (Shim-pack Scepter C18-120 and Shim-pack Velox C18) with a gradient of 5 mmol/L ammonium formate (0.1% formic acid) and methanol.
• Column temperature of 40 °C, 0.4 mL/min flow rate, and 5 µL injection volume.
• MRM transition optimization based on two precursor ions ([M+H]+ and [M+NH4]+) and four characteristic product ions ([M+H]+, [M+H–H2O]+).
• Mass spectrometers: a triple quadrupole LCMS-8060 for quantitation and a Q-TOF LCMS-9030 for CID spectral confirmation.
• Standard solutions quantified by qNMR, diluted to calibration levels between 0.08 and 11.68 ppb.

Main Results and Discussion

The Shim-pack Velox column provided sharper peaks, lower back pressure, and approximately twice the signal intensity compared to the Scepter column. Calibration curves showed excellent linearity (R2>0.997) across all analytes. Limits of quantitation ranged from 0.08 to 0.58 ppb, and inter-day area repeatability (%RSD) remained below 15% for all compounds. The optimized MRM transitions enabled simultaneous detection of five CTXs at concentrations as low as 0.5 ppb.

Benefits and Practical Applications

The developed method offers:

• High sensitivity for trace-level monitoring of multiple ciguatoxins in seafood matrices.
• Robust quantification with low matrix effects due to selective MRM transitions.
• Rapid analysis (18 min run time) suitable for routine testing in research labs and regulatory agencies.
• Flexibility to adapt to additional CTX analogues and related marine toxins.

Future Trends and Possibilities

Advancements may include integration of high-resolution accurate-mass screening for non-targeted toxin surveillance, miniaturized UHPLC-MS platforms for field testing, and automated sample preparation workflows. Expanding the analyte library and improving method throughput will support broader ecological and toxicological studies.

Conclusion

An optimized UHPLC–MS/MS method was established for five ciguatoxins with excellent sensitivity, selectivity, and repeatability. The Shim-pack Velox column combined with tailored MRM transitions enables reliable quantification below 0.5 ppb, facilitating enhanced seafood safety monitoring.

Used Instrumentation

• UHPLC system: Nexera X2 (Shimadzu)
• Chromatographic columns: Shim-pack Scepter C18-120; Shim-pack Velox C18
• Mass spectrometers: LCMS-8060 triple quadrupole; LCMS-9030 Q-TOF

References

• 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.
• Yogi K., Oshiro N., Matsuda S., Sakugawa S., Yasumoto T. Toxin profiles in fish implicated in ciguatera fish poisoning in Amami and Kakeroma islands, Kagoshima prefecture, Japan. Food Hyg. Saf. Sci. 2013, 54, 385–391.