4.4. Extraction and Clean-Up of Fish Samples for LC/MS

The lyophilized fish tissues were hydrated overnight. The tissue was extracted with 150 mL of acetone, and the acetone extract was evaporated. The remaining residue was taken up in 40 mL of water and partitioned with 40 mL of diethyl ether. The diethyl ether layer, which contained the toxin, was collected and evaporated, and the residue was partitioned between MeOH/water (9:1, v/v) and hexane [60].

Sample clean-up was carried out according to Yogi et al. [32]. An extract equivalent to 5 g of flesh was dissolved in 2 mL of ethyl acetate/MeOH (9:1). The solution was passed through a Florisil cartridge (InertSep FL-PR, 500 mg), eluted with 2 mL of the same solvent, and then dried under nitrogen at 40 °C. The residue was dissolved in 3 mL of MeCN, applied to a PSA cartridge (InertSep PSA, 200 mg), and washed with 3 mL of MeCN, and the target toxins were eluted with 3 mL of MeOH. The eluate was dried and dissolved in 200 μL of MeOH for LC/MS analysis.