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Ciguatera poisoning occurs throughout subtropical and tropical regions globally. The Virgin Islands in the Caribbean Sea is a known hyperendemic region for ciguatera and has been associated with Caribbean ciguatoxin (C-CTX) contamination in fish. An algal C-CTX (C-CTX5) was identified in Gambierdiscus silvae and G. caribeaus isolated from benthic algal samples collected in waters south of St. Thomas, US Virgin Islands. The highest CTXproducing isolate, G. silvae 1602 SH-6, was grown at large-scale to isolate sufficient C-CTX5 for structural confirmation by NMR spectroscopy. A series of orthogonal extraction and fractionation procedures resulted in purification of approximately 40 μg of C-CTX5, as estimated by quantitative NMR. A suite of 1D and 2D NMR experiments were acquired that verified the structure originally proposed for C-CTX5. The structural confirmation and successful isolation of C-CTX5 opens the way for work on the stability, toxicology and biotransformation of C-CTXs, as well as for the production of quantitative reference materials for analytical method development and validation. The strategies developed for purification of C-CTX5 may also apply to isolation and purification of CTXs from the Pacific Ocean and other regions. 

Tropical epibenthic dinoflagellate communities produce a plethora of bioactive secondary metabolites, including the toxins ciguatoxins (CTXs) and potentially gambierones, that can contaminate fishes, leading to ciguatera poisoning (CP) when consumed by humans. Many studies have assessed the cellular toxicity of causative dinoflagellate species to better understand the dynamics of CP outbreaks. However, few studies have explored extracellular toxin pools which may also enter the food web, including through alternative and unanticipated routes of exposure. Additionally, the extracellular exhibition of toxins would suggest an ecological function and may prove important to the ecology of the CP-associated dinoflagellate species. In this study, semi-purified extracts obtained from the media of a Coolia palmyrensis strain (DISL57) isolated from the U.S. Virgin Islands were assessed for bioactivity via a sodium channel specific mouse neuroblastoma cell viability assay and associated metabolites evaluated by targeted and non-targeted liquid chromatography tandem and high-resolution mass spectrometry. We found that extracts of C. palmyrensis media exhibit both veratrine enhancing bioactivity and non-specific bioactivity. LC-HR-MS analysis of the same extract fractions identified gambierone and multiple undescribed peaks with mass spectral characteristics suggestive of structural similarities to polyether compounds. These findings implicate C. palmyrensis as a potential contributor to CP and highlight extracellular toxin pools as a potentially significant source of toxins that may enter the food web through multiple exposure pathways. 

Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) or high-resolution MS (LC–HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC–MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard’s reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC–MS/MS and LC–HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1–GRT derivatization strategy mitigates many of the shortcomings of current LC–MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes. 

Ciguatera poisoning is linked to the ingestion of seafood that is contaminated with ciguatoxins (CTXs). The structural variability of these polyether toxins in nature remains poorly understood due to the low concentrations present even in highly toxic fish, which makes isolation and chemical characterization difficult. We studied the mass spectrometric fragmentation of Caribbean CTXs, i.e., the epimers C-CTX-1 and -2 (1 and 2), using a sensitive UHPLC–HRMS/MS approach in order to identify product ions of diagnostic value. We found that the fragmentation of the ladder-frame backbone follows a characteristic pattern and propose a generalized nomenclature for the ions formed. These data were applied to the structural characterization of a pair of so far poorly characterized isomers, C-CTX-3 and -4 (3 and 4), which we found to be reduced at C-56 relative to 1 and 2. Furthermore, we tested and applied reduction and oxidation reactions, monitored by LC–HRMS, in order to confirm the structures of 3 and 4. Reduction of 1 and 2 with NaBH4 afforded 3 and 4, thereby unambiguously confirming the identities of 3 and 4. In summary, this work provides a foundation for mass spectrometry-based characterization of new C-CTXs, including a suite of simple chemical reactions to assist the examination of structural modifications.