Ciguatera Fish Poisoning is a worldwide concern caused by the consumption of fish contaminated with ciguatoxins not only in endemic regions in the Pacific Ocean or the Caribbean Sea but also in emerging areas of Macaronesia on the eastern Atlantic. The recent emergence of these toxins in other coastal areas worldwide, prompted the need for the characterization of the risk in these areas. This Ciguatera Fish Poisoning risk has been recently identified as a potential threat in subtropical areas of the Atlantic coast and scientific efforts are being focused in the identification and confirmation of the toxins involved in this potential risk. Neuroblastoma cell assay has been widely used for the evaluation of the toxicity in several marine biotoxin groups, and found to be a very useful tool for toxicity screening. LC-MS/MS has been also used for confirmatory purposes although the main limitation of the advances on LC-MS/MS development is due to commercial unavailability of reference materials and hampers method implementation and validation or even confirmation of the ciguatoxins (CTXs) responsible for the toxic profiles. While neuroblastoma cell assay (N2a) is typically used for toxicity screening as mentioned above, being necessary to confirm this N2a toxicity by LC-MS/MS, this study is designed using N2a as a tool to confirm the toxicity of the fractions obtained corresponding to potential CTXs analogues according to the analysis by LC-MS/MS. With this aim, an amberjack sample (Seriola fasciata) from Selvagen Islads (Portugal) and implicated in Ciguatera Fish Poisoning was analyzed by LC-MS/MS and Caribbean Ciguatoxins were found to be mainly responsible for the toxicity. N2a was used in this work as a tool to help in the confirmation of the toxicity of fractions obtained by HPLC. Caribbean Ciguatoxin-1 was found as the main analogue responsible for the N2a toxicity while three Caribbean Ciguatoxin-1 (C-CTX1) metabolites which contribute to the total toxicity were also identified.

Ciguatoxins (CTXs) are a group of neurotoxins responsible for the syndrome ciguatera fish poisoning (CFP) as a result of the consumption of contaminated fish. The presence of these toxins has been detected around the Pacific, Caribbean and Indian coasts. Recent reports indicate the emergence of CFP in other geographic areas, in particular in European coasts, of the Canary Islands (Spain) and Madeira (Portugal). A neuroblastoma cell line of murine origin (N2a) has been applied to assay different groups of neurotoxins, acting on voltage-gated sodium channel (VGSC) of excitable cells, N2a-MTT. The great potential of N2a-MTT as a sensitive tool for the CTXs screening is clearly recognized, notably because it allows the detection of these toxins at levels below recommended as security levels. However, the complexity of the matrix is a critical point on the application of N2a-MTT, which needs to be evaluated. The aim of this work is to provide recommendations for an implemented N2a-MTT method for CTXs determination in fish that avoids matrix effects, particularly those related to high lipid content.