Tetrodotoxin (TTX): What do we currently know? The state of the art.

Tetrodotoxin (TTX) is one of the most potent naturally occurring neurotoxins and continues to attract considerable scientific, clinical, forensic, and regulatory attention. Historically associated with pufferfish poisoning in East and Southeast Asia, TTX has recently emerged as a global food safety concern due to its detection in an increasing number of marine and terrestrial organisms and its expanding geographical distribution, including European coastal waters. Despite decades of research, important questions regarding its biosynthesis, ecological origin, toxicokinetics, and environmental dissemination remain unresolved. This review provides a comprehensive overview of current knowledge on TTX. The chemical structure, physicochemical properties, proposed biosynthetic origin, and diversity of naturally occurring TTX analogues are discussed. Attention is devoted to the toxin's mechanism of action, which involves high-affinity binding to voltage-gated sodium channels and subsequent inhibition of sodium ion conductance, leading to neuromuscular paralysis and potentially fatal respiratory failure. Current evidence regarding toxicokinetics, tissue distribution, elimination pathways, and the suitability of different biological matrices for laboratory confirmation of exposure is also summarized. The review further examines the analytical challenges associated with TTX determination, including its high polarity, occurrence at trace concentrations, rapid elimination from blood, and frequent co-occurrence with structurally related analogues. The evolution of analytical methodologies is presented, from early bioassays and chromatographic techniques with fluorescence or UV detection to advanced liquid chromatography-mass spectrometry platforms, high-resolution mass spectrometry, capillary electrophoresis, immunochemical assays, and emerging biosensor-based approaches. Finally, current regulatory perspectives and risk assessment strategies are discussed in the context of the increasing occurrence of TTX in seafood and the growing need for harmonized monitoring programs. Collectively, this review summarizes the current state of the art, identifies key knowledge gaps, and highlights future directions in TTX research.