Japan Food Research Laboratories, Tama Laboratory, 6-11-10 Nagayama, Tama, Tokyo 206-0025, Japan.
National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki, Kanagawa 210-9501, Japan.
J AOAC Int. 2021 Sep 27;104(5):1272-1281. doi: 10.1093/jaoacint/qsab052.
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.
We sought to develop and validate the first LC-MS/MS method to quantify the major toxins prevalent in fish from the Pacific Ocean.
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.
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.
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.
A validation study for an LC-MS/MS method for ciguatoxin detection was completed for the first time using calibrated toxin standards.
赤潮鱼中毒（CFP）对来自世界各温暖水域的公共健康和水生资源的利用构成了严重威胁。因此，需要开发和验证一种用于有效测定相关毒素的方法。
我们旨在开发并验证首个可定量测定来自太平洋海域鱼类中主要毒素的 LC-MS/MS 方法。
使用甲醇-水混合物（9:1，v/v）从鱼肉（2g）中提取毒素。将提取物在 80°C 下加热，并使用正己烷首先从碱性溶液，然后从酸性溶液中去除低极性脂质。使用固相萃取、Florisil、硅胶、反相 C18 和仲胺柱进行净化。通过向鱼肉中添加两种具有不同骨架结构和极性的代表性毒素进行验证研究，并通过 NMR（qNMR）光谱进行校准。
在以 0.1μg/kg 浓度添加赤潮毒素 CTX1B 和 CTX3C 时，验证参数如下：重复性为 2.3-3.5%和 3.2-5.3%；中间精密度为 6.3-9.8%和 6.0-7.4%；回收率为 80-107%和 95-120%。CTX1B 的最低检测水平为 0.004μg/kg，51-羟基 CTX3C 为 0.005μg/kg，CTX3C 为 0.009μg/kg。
该方法实质上符合美国食品和药物管理局以及欧洲食品安全局设定的赤潮毒素 0.01μg/kg CTX1B 等效物的国际行动水平，并满足了 Codex 和 AOAC INTERNATIONAL 设定的全球标准。
首次使用校准毒素标准完成了用于赤潮毒素检测的 LC-MS/MS 方法的验证研究。
