High-resolution mass spectrometry analysis of tetrodotoxin (TTX) and its analogues in puffer fish and shellfish

ABSTRACT

Tetrodotoxin (TTX) is an emerging toxin in the European marine environment. It has various known structural analogues. It acts as a sodium channel blocker; the ability of each analogue to bind to the sodium channel varies with the particular structure of each analogue. Thus, each analogue will vary in its toxic potential. TTX analogues co-occur in food samples at variable concentrations. An LC-MS method was developed for the identification and quantitation of several analogues of TTX using an LTQ-Orbitrap XL mass spectrometer. The LTQ-Orbitrap XL mass spectrometer facilitates high mass accuracy measurement up to 100,000 full width at half maximum (FWHM). Using high resolution at 100,000 FWHM allows for the identification of TTX and its analogues in various matrices, including puffer fish and molluscan shellfish samples (Δ ppm = 0.28–3.38). The confirmation of characteristic fragment ions of TTX and its analogues was achieved by determining their elemental formulae via high mass accuracy. A quantitative method was then developed and optimised using these characteristic fragment ions. The limit of quantitation (LOQ) of the method was 0.136 µg g^–1 (S/N = 10) and the limit of detection (LOD) was 0.041 µg g^–1 (S/N = 3) spiking TTX standard into TTX-free mackerel fish extracts. The method was applied to naturally contaminated puffer fish and molluscan shellfish samples to confirm the presence of TTX and its analogues.

KEYWORDS:

• HRMS
• TTX
• method development
• LTQ Orbitrap-XL
• puffer fish
• molluscan shellfish

Acknowledgments

The authors acknowledge Dr Panagiota Katikou, National Reference Laboratory on Marine Biotoxins, Thessaloniki, Greece, for providing puffer fish (Lagocephalus sceleratus) samples; and Dr Vitor Vasconcelos, Centro Interdiciplinar de Investigacao Marinha e Ambiental (CIIMAR), Rua dos Bragas, Porto, Portugal, for providing molluscan shellfish (Charonia lampas) samples.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The authors acknowledge the funding obtained under the EU/INTERREG IIIB Atlantic Area Programme for projects entitled ATLANTOX ‘Advanced Tests about New Toxins Occurring in the Atlantic Area due to Climate Change’ and PHARMATLANTIC ‘Knowledge Transfer Network for Prevention of Mental Diseases and Cancer in the Atlantic Area’. The Higher Education Authority (Programme for Research in Third-Level Institutions, Cycle 4 (PRTLI IV)) National Collaboration Programme on Environment and Climate Changes: Impacts and Responses is also acknowledged for funding marine research within the Cork Institute of Technology, Cork, Ireland.