Analytical Method Optimization of Tetrodotoxin and Its Contamination in Gastropods

Tetrodotoxin (TTX) is an extremely potent marine biotoxin. An analytical method was developed for both trace contamination and extremely high levels of TTX in gastropods by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with clean-up of cation exchange solid phase extraction (SPE) in this study. The limit of detection (LOD) in the sample matrix was 0.5 μg/kg. With the calibration of a screened internal standard (validamycin, IS), the linear range was 0.1-100 ng/mL (1.5-1500 μg/kg in sample matrix) with a correlation coefficient of r² > 0.999. The average recoveries at three spiking levels (1.5 μg/kg, 44 μg/kg, and 1500 μg/kg) were 82.6-94.4% with relative standard deviations (RSDs) less than 8.4%. TTX levels in seven gastropods (741 samples) were studied. The contamination and analogues in Neverita didyma (N. didyma, 565 samples collected in Zhejiang province, China, from 2016 to 2022) were first reported. The detection rate of TTX in N. didyma was 34.2%. The average concentration was 23.1 μg/kg, and the maximum value was 2327 μg/kg. The time distribution study indicated that high contaminations of TTX occurred from May to August for N. didyma.