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Background Ciguatera fish poisoning (CFP) poses a serious threat to both public health and the use 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 (2 g) 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. The 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 the ciguatera toxins 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%, respectively. 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 ciguatoxin detection was completed for the first time using calibrated toxin standards.

Development and Validation of an LC-MS/MS Method for the Ultra-Trace Analysis of Pacific Ciguatoxins in Fish