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Development of an LC-MS/MS method for the determination of Ciguatoxins in fish
Project
Project code: BfR-SiN-08-1322-705
Contract period: 01.07.2018
- 31.12.2019
Purpose of research: Experimental development
Ciguatera fish poisoning (CFP) is a human neuro-intoxication that occurs after consuming certain tropical reef fish which contain ciguatoxins (CTXs) [1]. These toxins are metabolites of CTX precursors produced by benthic dinoflagellates belonging to the genus Gambierdiscus. When cells of Gambierdiscus spp. are ingested by herbivorous fish, the toxins are bioaccumulated and biotransformed into lipid-soluble CTXs. Piscivorous fish accumulate and transformed CTXs by feeding from these herbivorous fish. After the consumption of fish containing CTXs illness is caused involving a complex array of gastrointestinal, cardiovascular, neurological and neuropsychological symptoms. CFP symptoms may last days, weeks or months. Global incidence rates for CFP are estimated to affect between 10,000 to 50,000 individuals annually, presenting the most frequently reported natural marine toxin related illness. The EU does not have regulations setting limits or official methods for CTXs. However, EU regulation requires that no fish products containing CTXs are placed on the market. In contrary, the U.S. Food and Drug Administration (FDA) established guidance levels for CTXs originating in the Caribbean (C-CTXs; 0.1 ng/g C-CTX-1 equivalents) and Pacific (P-CTXs; 0.01 ng/g P-CTX-1 equivalents). CFP cases in Germany (reported each year since 2012) were mainly caused by imported fish from the Indian Ocean. CTXs profiles in those fish are characteristic for that catching area and differ from the Caribbean and Pacific CTXs. Therefore, the toxins can't be identified and quantified unambiguously as no standards are available yet. The occurrence of Gambierdiscus spp in Europe was first reported in 2003 from the island of Crete. The CFP poisoning of fishermen who ate fish from the region around the island of Madeira as well as poisonings from the Canaries show the presence of CTX in European waters [4]. Although most CFP cases in Europe have so far been due to imported fish, it is important to be aware of the possibility that CFP might be an emerging issue of concern in the European Union (E.U.) in terms of risks for the growth of Gambierdiscus in EU waters. Currently conditions in the Mediterranean Sea boarder the zone of habitability for Gambierdiscus, however waters are predicted to warm and become more hospitable for the growth and distribution of Gambierdiscus [2]. Determining the frequency of ciguatera cases and ciguatoxic fish in Europe can be accomplished through the development of reliable methods, tools for diagnosis, sampling efforts, and illness reporting. Methods have been established for detecting CTXs in fish implicated in CFP outbreaks, the results of which are used to support the clinical diagnosis of CFP. The current accepted testing procedure utilizes a two-tiered protocol involving (1) in vitro mouse neuroblastoma (N2a) cell assay as a semi-quantitative screening for toxicity consistent with CTX mode of action; and (2) liquid chromatography tandem-mass spectrometry (LC-MS/MS) for molecular confirmation of CTXs. CTXs from different catching areas vary in their chemical structure. Specific CTX congeners were identified which serve as regional biomarkers. Samples tested positive by N2a cell assay are analyzed for the presence of this CTX congener. In fish from the Gulf of Mexico, Caribbean, and other regions in the Atlantic, the FDA uses the congener C-CTX-1, while P-CTX-1 is used for fish from the Pacific Ocean. Multiple reaction monitoring in positive ion mode is the MS/MS technique employed to confirm the presence of a specific CTX congener. However, the monitored transitions usually include multiple water losses from the precursors and thus are not diagnostic for unambiguous confirmation, unless standards of individual CTX congeners were available. Standards for the specific CTX congeners identified as regional biomarkers for CTXs are difficult to source, limited P-CTX-3C exists to be purchased while all others must currently be purified from fish tissue. When standards are lacking, cytotoxicity profiles of meal remnants or raw fish can be constructed using the N2a assay to test LC-fractionated extracts [3]. The aim of the project is the establishment of an LC-MS / MS method for the detection of C-CTXs and P-CTXs in fish, followed by the optimization of the detection method of Indian CTX and its inhouse validation.
References
[1] Scheuer PJ, et al. 1967. Ciguatoxin: isolation and chemical nature. Science 155:1267-1268.
[2] Shaltout M, Omstedt A. 2014. Recent sea surface temperature trends and future scenarios for the Mediterranean Sea. Oceanologia 56:411-443
[3] Abraham A, et al. 2012. Caribbean ciguatoxin profile in raw and cooked fish implicated in ciguatera. Food Chemistry 131:192-198.
[4] Vlamis, A, Katikou, P. 2015. Human impact in Mediterranean coastal ecosystems and climate change: emerging toxins. Climate change and marine and freshwater toxins. 8: 237-269
Section overview
Subjects
- Process engineering
- Toxicology
