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

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.