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.

Ciguatera poisoning (CP) is a severe seafood-borne disease, caused by the consumption of reef fish contaminated with Caribbean ciguatoxins (C-CTXs) in the Caribbean and tropical Atlantic. However, C-CTXs have not been identified from their presumed algal source, so the relationship to the CTXs in fish causing illness remains unknown. This has hindered the development of detection methods, diagnostics, monitoring programs, and limited fundamental knowledge on the environmental factors that regulate C-CTX production. In this study, in vitro and chemical techniques were applied to unambiguously identify a novel C-CTX analogue, C-CTX5, from Gambierdiscus silvae and Gambierdiscus caribaeus strains from the Caribbean. Metabolism in vitro by fish liver microsomes converted algal C-CTX5 into C-CTX1/2, the dominant CTX in ciguatoxic fish from the Caribbean. Furthermore, C-CTX5 from G. silvae was confirmed to have voltage-gated sodium-channel-specific activity. This finding is crucial for risk assessment, understanding the fate of C-CTXs in food webs, and is a prerequisite for development of effective analytical methods and monitoring programs. The identification of an algal precursor produced by two Gambierdiscus species is a major breakthrough for ciguatera research that will foster major advances in this important seafood safety issue.

The genus Gambierdiscus produces an array of bioactive hydrophilic and lipophilic secondary metabolites that range in mode of action and toxicity. In this study, the metabolite fingerprint was mapped for thirteen Gambierdiscus, five Coolia and two Fukuyoa species (34 isolates) by assessing the production of 56 characterised secondary metabolites. Gambierdiscus polynesiensis was the only species to produce Pacific-ciguatoxin-3B (P-CTX3B), P-CTX3C, iso-P-CTX3B/C, P-CTX4A, P-CTX4B and iso-P-CTX4A/B. G. australes produced maitotoxin-1 (MTX-1) and MTX-5, G. cheloniae produced MTX-6 and G. honu produced MTX-7. Ubiquitous production of 44-methylgambierone was observed amongst all the Gambierdiscus isolates, with nine species also producing gambierone. Additional gambierone analogues, including anhydrogambierone (tentatively described herein), were also detected in all Gambierdiscus species, two Coolia and two Fukuyoa species. Gambieroxide was detected in G. lewisii and G. pacificus and gambieric acid A was detected in ten Gambierdiscus species, with G. australes (CAWD381) being the only isolate to produce gambieric acids A–D. This study has demonstrated that the isolates tested to date produce the known CTXs or MTXs, but not both, and highlighted several species that produced ‘unknown’ compounds displaying characteristics of cyclic polyethers, which will be the focus of future compound discovery efforts.

Identifying compounds responsible for the observed toxicity of the Gambierdiscus species is a critical step to ascertaining whether they contribute to ciguatera poisoning. Macroalgae samples were collected during research expeditions to Rarotonga (Cook Islands) and North Meyer Island (Kermadec Islands), from which two new Gambierdiscus species were characterized, G. cheloniae CAWD232 and G. honu CAWD242. Previous chemical and toxicological investigations of these species demonstrated that they did not produce the routinely monitored Pacific ciguatoxins nor maitotoxin-1 (MTX-1), yet were highly toxic to mice via intraperitoneal (i.p.) injection. Bioassay-guided fractionation of methanolic extracts, incorporating wet chemistry and chromatographic techniques, was used to isolate two new MTX analogs; MTX-6 from G. cheloniae CAWD232 and MTX-7 from G. honu CAWD242. Structural characterization of the new MTX analogs used a combination of analytical chemistry techniques, including LC–MS, LC–MS/MS, HR–MS, oxidative cleavage and reduction, and NMR spectroscopy. A substantial portion of the MTX-7 structure was elucidated, and (to a lesser extent) that of MTX-6. Key differences from MTX-1 included monosulfation, additional hydroxyl groups, an extra double bond, and in the case of MTX-7, an additional methyl group. To date, this is the most extensive structural characterization performed on an MTX analog since the complete structure of MTX-1 was published in 1993. MTX-7 was extremely toxic to mice via i.p. injection (LD₅₀ of 0.235 µg/kg), although no toxicity was observed at the highest dose rate via oral administration (155.8 µg/kg). Future research is required to investigate the bioaccumulation and likely biotransformation of the MTX analogs in the marine food web.

Turmeric is a medicinal herb containing curcuminoids, used as quality markers in dietary supplements. In 2016, an AOAC First Action Official Method^SM was adopted for quantitation of curcuminoids and requires multi-laboratory reproducibility data for Final Action status. To collect reproducibility data for the quantitation of curcuminoids in dietary supplements through the National Institutes of Health Office of Dietary Supplements/National Institute of Standards and Technology Quality Assurance Program (QAP). Laboratories that participated in the QAP by following the Official Methods of Analysis^SM Method 2016.16, submitted data for ten turmeric products. The data were analyzed for mean, repeatability, and reproducibility standard deviations, repeatability, and reproducibility. The initial data collection resulted in insufficient replicates (five) for each test sample to determine reproducibility, therefore laboratories were provided additional materials resulting in an incremental data approach. For homogenous products, reproducibility for curcumin ranged from 3.4 to 10.3%, bisdemethoxycurcumin with reproducibility ranging from 6.4 to 14.8%, and demethoxycurcumin ranging from 5.6 to 9.9%. The method was unsuitable for the quantitation of curcuminoids in complex smoothie products, products containing microbeads, or tinctures based on interlaboratory variances. Recommendations were provided for future multi-laboratory studies performed through QAPs and incremental approaches. Method 2016.16 is suitable for the quantitation of curcuminoids and should be adopted for Final Action status for single and multi-ingredient dietary supplements containing dried roots, dried powders/extracts in bulk material, capsules, and softgels. Reproducibility for Method 2016.16 was collected through a non-traditional incremental data multi-laboratory study. The method is suitable for quantitation of curcuminoids in most common dietary supplements.