Universal Method for Determination of Ciguatoxins by LC-MS/MS

Abstract

Ciguatera poisoning (CP) is one of the most frequent seafood poisonings across the globe. CP results from the consumption of fish flesh that has accumulated principal toxins known as ciguatoxins (CTXs), and it mainly occurs in tropical and subtropical regions. In Japan, incidents of CP have been reported primarily from Okinawa and Amami Islands in the subtropical area. Meanwhile, there have also been reports from Mainland sporadically. Since the amount of CTXs contained in fish flesh is extremely low, a highly sensitive detection method by LC-MS/MS is required. But the currently reported detection method is applicable only to specific equipment, and many laboratories have difficulty to respond CP. In this study, to prepare for the risk of nationwide CP, we researched a universal analytical method for CTXs based on LC-MS/MS. Using a water/acetonitrile mobile phase supplemented with lithium hydroxide and formic acid gave rise to prominent peaks of the stable [M+Li]+ions. As the [M+Li]+ions did not produce valid product ions even with high collision energy, the [M+Li]+ions of each analog were set for both precursor and product ions ([M+Li]+>[M+Li]+) and monitored under the multiple reaction monitoring (MRM) mode. With the method described above, analyses of nine CTX congeners were carried out. The limit of detection (LOD, S/N>5) and quantitation (LOQ, S/N>10) were estimated as 0.005-0.030 ng/mL and 0.010-0.061 ng/mL, respectively. When the 1 mL of extract solution is prepared from 5 g of the fish tissue, the LOD and LOQ will be at 0.001-0.006 μg/kg and 0.002-0.012 μg/kg, respectively. This result indicates that we could detect the required level of 0.175 μg/kg CTX1B equivalent in fish flesh which is recommended for safe consumption in Japan. This method is considered to be a universal analytical method without depending on the specific equipment. Thus it could contribute to improving the CP investigations in nationwide laboratories.