Ciguatoxins (CTXs) contaminate fish worldwide and cause the foodborne illness ciguatera. In the Pacific, these toxins are produced by the dinoflagellate Gambierdiscus toxicus, which accumulates in fish through the food chain and undergoes oxidative modification, giving rise to numerous analogs. In this study, we examined the oxidation of CTXs in vitro with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using reference toxins, and found that CTX4A, CTX4B, and CTX3C, which are produced by the alga, are oxidized to the analogs found in fish, namely CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, 2-hydroxyCTX3C, and 2,3-dihydroxyCTX3C. This oxidation was catalyzed by human CYP3A4, fish liver S9 fractions, and microsomal fractions prepared from representative ciguateric fishes (Lutjanus bohar, L. monostigumus, and Oplegnathus punctatus). In addition, fish liver S9 fractions prepared from non-ciguateric fishes (L. gibbus and L. fulviflamma) in Okinawa also converted CTX4A and CTX4B to CTX1B, 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B in vitro. This is the first study to demonstrate the enzymatic oxidation of these toxins, and provides insight into the mechanism underlying the development of species-specific toxin profiles and the fate of these toxins in humans and fish.

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Ciguatera fish poisoning (CFP) is one of the most frequently reported seafood poisoning diseases. It is endemic to the tropical region and occurs most commonly in the regions around the Pacific Ocean, Indian Ocean, and Caribbean Sea. The principal toxins causing CFP are ciguatoxins (CTXs). In the Pacific region, more than 20 analogs of CTXs have been identified to date. Based on their skeletal structures, they are classified into CTX1B-type and CTX3C-type toxins. We have previously reported species-specific and regional-specific toxin profiles. In this study, the levels and profiles of CTXs in fish present in the tropical western Pacific regions were analyzed using the liquid chromatography–tandem mass spectrometry (LC–MS/MS) technique. Forty-two fish specimens, belonging to the categories of snappers, groupers, Spanish mackerel, and moray eel, were purchased from various places such as Fiji, the Philippines, Thailand, and Taiwan. Only the fish captured from Fijian coastal waters contained detectable amounts of CTXs. The toxin levels in the fish species found along the coastal regions of the Viti Levu Island, the main island in Fiji, and the toxin profiles were significantly different from those of the fish species present in other coastal regions. The toxin levels and profiles varied among the different fish samples collected from different coastal areas. Based on the toxin levels and toxin profiles, the coast was demarcated into three zones. In Zone-1, which covers the northern coast of the main island and the regions of the Malake Island and Korovau, CTXs in fish were below the detection level. In Zone-2, CTX3C-type toxins were present in low levels in the fish. CTX1B-type and CTX3C-type toxins co-occurred in the fish present in Zone-3. The toxin profiles may have reflected the variation in Gambierdiscus spp.

Background Ciguatera fish poisoning (CFP) poses serious threat to public health and exploitation of aquatic resources from the various warm-water regions of the world. Hence, a process for the efficient determination of the relevant toxins is required. Objective We sought to develop and validate the first LC-MS/MS method to quantify the major toxins prevalent in fish from the Pacific Ocean. Method Toxins were extracted from fish flesh (2g) using a methanol–water mixture (9:1, v/v). The extract was heated at 80 °C, and low-polarity lipids were eliminated using hexane, initially from the basic solution and later from the acidic solution. Cleanup was performed using solid-phase extraction, Florisil, silica, reversed-phase C18, and primary secondary amine columns. A validation study was conducted by spiking fish flesh with two representative toxins having different skeletal structures and polarities and was calibrated by NMR (qNMR) spectroscopy. Results: The validation parameters for CTX1B and CTX3C at spiked levels of 0.1 μg/kg were as follows: repeatabilities of 2.3-3.5% and 3.2-5.3%; intermediate precisions of 6.3-9.8% and 6.0-7.4%; recoveries of 80-107% and 95-120%. The lowest detection levels were 0.004 μg/kg for CTX1B, 0.005 μg/kg for 51-hydroxyCTX3C, and 0.009 μg/kg for CTX3C. Conclusions: The described method practically clears the international action level of 0.01 μg/kg CTX1B equivalents set by the U.S. Food and Drug Administration and the European Food Safety Authority and satisfies the global standards set by CODEX and AOAC INTERNATIONAL. Highlights A validation study for an LC-MS/MS method for CTXs detection was completed for the first time using calibrated toxin standards.

Ciguatera fish poisoning (CFP) is one of the most frequently encountered seafood poisoning syndromes; it is caused by the consumption of marine finfish contaminated with ciguatoxins (CTXs). The majority of CFP cases result from eating fish flesh, but a traditional belief exists among people that the head and viscera are more toxic and should be avoided. Unlike the viscera, scientific data to support the legendary high toxicity of the head is scarce. We prepared tissue samples from the fillet, head, and eyes taken from five yellow-edged lyretail (Variola louti) individuals sourced from Okinawa, Japan, and analyzed the CTXs by LC-MS/MS. Three CTXs, namely, CTX1B, 52-epi-54-deoxyCTX1B, and 54-deoxyCTX1B, were confirmed in similar proportions. The toxins were distributed nearly evenly in the flesh, prepared separately from the fillet and head. Within the same individual specimen, the flesh in the fillet and the flesh from the head, tested separately, had the same level and composition of toxins. We, therefore, conclude that flesh samples for LC-MS/MS analysis can be taken from any part of the body. However, the tissue surrounding the eyeball displayed CTX levels two to four times higher than those of the flesh. The present study is the first to provide scientific data demonstrating the high toxicity of the eyes.