Development and optimization of lc-ms for the identification and confirmation of ciguatoxins

Resumo

Over recent years, the expansion of the Ciguatera Poisoning (CP) to areas where this contamination had never been reported has been significantly increased and the CP became a potential emerging risk. This expansion had also an impact in certain coastal areas of Europe in particular in the archipelagos located at the central-eastern section of the North Atlantic (Azores, Madeira, Canary Islands, and Cape Verde), known collectively as the Macaronesian islands, in which several cases of CP had been reported after the consumption of certain fish species. This prompted the health authorities to consider that an evaluation of the CP risk should be considered. As an initiative of the Spanish Food Safety Authority. The characterization of the CP risk was considered a priority for the EU Safety Authority (EFSA) which also based on a previous scientific opinion published in 2010, established the recommendation of evaluating this risk. A European project partially supported by EFSA was proposed to carry out the characterization above mentioned, being one of the main objectives to develop the analytical methodology to carry out the confirmation of the CP as well as the characterization of the CP toxins involved in it. The research that supports the overall objective of this doctoral thesis is also one of the main objectives of this EU project in which the development of a confirmatory method through Mass Spectrometry is particularly focused. The limitation of reference materials commercially available for method implementation has been also one of the main limitations to the advance in this analytical field as well as in the advances of the toxicological characterization of the different ciguatoxin analogues. The contribution to the design of an analytical procedure for the preparation of these reference materials has been also considered among the objectives of this EU project also included in the specific objectives of this doctoral thesis. And with this aim, the combination of chromatographic approaches and the different optimizations carried out allowed to prepare reference materials for laboratory used through the efficient isolation and purification of the main CTXs involved in the contamination. The successful development and implementation of Liquid chromatography (LC) coupled to different Mass spectrometry approaches (MS/MS, HRMS) allowed the confirmation of the CP in fish species from the EU areas above mentioned, as well as their characterization being Caribbean Ciguatoxins, and in particular the congener C-CTX1 the main responsible for the toxicity in the areas selected for this study. The access through the EFSA project, to microalgae samples as responsible for the fish contamination, also allowed to evaluate the correlation between precursors in the microalgae and toxin analogues in the contaminated fish. The efforts on method development to evaluate the toxic profile of several microalgae species, in particular, Gambierdiscus and Fukuyoa species allowed to observe a lack of correlation being the toxicity of the microalgae associated to the presence of additional toxins reported in the literature. This lack of correlation suggests that a biotransformation in the fish might also occur.