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Ciguatoxins (CTXs) are naturally occurring neurotoxins that can accumulate in fish and cause Ciguatera Poisoning (CP) in seafood consumers. Ciguatoxic fish have been detected in tropical and subtropical regions of the world including the Pacific and Indian Oceans, the Caribbean Sea, and more recently in the northeast Atlantic Ocean. The biogeographic distribution of ciguatoxic fish appears to be expanding; however, the paucity of CTX standards and reference materials limits the ability of public health authorities to monitor for these toxins in seafood supply chains. Recent reports establish that Caribbean Ciguatoxin-1 (C-CTX1) is the principal toxin responsible for CP cases and outbreaks in the northeast Atlantic Ocean and that C-CTX congener profiles in contaminated fish samples match those from the Caribbean Sea. Therefore, in this work, C-CTX reference materials were prepared from fish obtained from the northeast Atlantic Ocean. The collection of fish specimens (e.g., amberjack, grouper, or snapper) was screened for CTX-like toxicity using the in vitro sodium channel mouse neuroblastoma cytotoxicity assay (N2a cell assay). Muscle and liver tissues from toxic specimens were pooled for extraction and purified products were ultimately profiled and quantified by comparison with authentic C-CTX1 using LC-MS/MS. This work presents a detailed protocol for the preparation of purified C-CTX reference materials to enable continued research and monitoring of the ciguatera public health hazard. To carry out this work, C-CTX1 was isolated and purified from fish muscle and liver tissues obtained from the Canary Islands (Spain) and Madeira archipelago (Portugal). [ABSTRACT FROM AUTHOR]

Ciguatoxins (CTXs) are naturally occurring neurotoxins that can accumulate in fish and cause Ciguatera Poisoning (CP) in seafood consumers. Ciguatoxic fish have been detected in tropical and subtropical regions of the world including the Pacific and Indian Oceans, the Caribbean Sea, and more recently in the northeast Atlantic Ocean. The biogeographic distribution of ciguatoxic fish appears to be expanding; however, the paucity of CTX standards and reference materials limits the ability of public health authorities to monitor for these toxins in seafood supply chains. Recent reports establish that Caribbean Ciguatoxin-1 (C-CTX1) is the principal toxin responsible for CP cases and outbreaks in the northeast Atlantic Ocean and that C-CTX congener profiles in contaminated fish samples match those from the Caribbean Sea. Therefore, in this work, C-CTX reference materials were prepared from fish obtained from the northeast Atlantic Ocean. The collection of fish specimens (e.g., amberjack, grouper, or snapper) was screened for CTX-like toxicity using the in vitro sodium channel mouse neuroblastoma cytotoxicity assay (N2a cell assay). Muscle and liver tissues from toxic specimens were pooled for extraction and purified products were ultimately profiled and quantified by comparison with authentic C-CTX1 using LC-MS/MS. This work presents a detailed protocol for the preparation of purified C-CTX reference materials to enable continued research and monitoring of the ciguatera public health hazard. To carry out this work, C-CTX1 was isolated and purified from fish muscle and liver tissues obtained from the Canary Islands (Spain) and Madeira archipelago (Portugal).

The recent emergence of ciguatera in the eastern Atlantic, particularly in the Canary Islands (Spain) and Madeira (Portugal) prompted the development and implementation of liquid chromatography tandem–mass spectrometry (LC/MS-MS) methods for the detection of ciguatoxins in fish. The complexity of fish tissue matrices, low concentrations of ciguatoxins in hazardous fish, and the scarcity of ciguatoxin standards present challenging issues for successful implementation of routine ciguatoxin analysis. A laboratory reference material of Caribbean Ciguatoxin-1 (C-CTX1), which was previously confirmed in fish responsible for ciguatera outbreaks in the Canary Islands, was used to assess the toxin's stability under strongly acidic conditions and solvent systems commonly used in LC-MS/MS. It was observed that strongly acidic conditions caused the transformation of C-CTX1 to a C56 methoxy congener, C-CTX1-Me. C-CTX1 was structurally characterised by LC-MS/MS and fragmentation pathways are presented showing the same fragmentation pattern as C-CTX1-Me. These results suggest that the use of strongly acidic conditions during sample pretreatment for C-CTX analysis, might produce significant artefacts, and risks failing to detect the presence of C-CTX1. [ABSTRACT FROM AUTHOR]

Ciguatera poisoning (CP) is a common seafood intoxication mainly caused by the consumption of fish contaminated by ciguatoxins. Recent studies showed that Caribbean ciguatoxin-1 (C-CTX1) is the main toxin causing CP through fish caught in the Northeast Atlantic, e.g., Canary Islands (Spain) and Madeira (Portugal). The use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) combined with neuroblastoma cell assay (N2a) allowed the initial confirmation of the presence of C-CTX1 in contaminated fish samples from the abovementioned areas, nevertheless the lack of commercially available reference materials for these particular ciguatoxin (CTX) analogues has been a major limitation to progress research. The EuroCigua project allowed the preparation of C-CTX1 laboratory reference material (LRM) from fish species (Seriola fasciata) from the Madeira archipelago (Portugal). This reference material was used to implement a liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for the detection of C-CTX1, acquisition of full-scan as well as collision-induced mass spectra of this particular analogue. Fragmentation pathways were proposed based on fragments obtained. The optimized LC-HRMS method was then applied to confirm C-CTX1 in fish (Bodianus scrofa) caught in the Selvagens Islands (Portugal).