Jouan mr23i

A triple extraction with methanol was performed on the fully homogenized tissues (1 g) according to the extraction process proposed by Gersen et al. [42 (link)], which was also validated by our group [43 (link)] based on the EU-Harmonized Standard Operating Procedure for the Determination of Lipophilic marine biotoxins in mollusks via LC-MS/MS (Version 5, January 2015). In summary, 1 g of the homogenized tissue of each sample was weighed in a 15-mL centrifuge tube with a Sartotius 1702 analytical balance, and 3 mL of methanol was added, proceeding to homogenization with a MS2 Minishaker vortex mixer (IKA Labortechnik, Staufen, Germany) for 1 min at 2500 rpm. Subsequently, the samples were centrifuged using a Jouan MR 23i centrifuge (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 5 min at 2000 G and room temperature, and the supernatant was transferred to a 10-mL volumetric matrix. Then, 3 mL of methanol was added to the solid residue in the tubes, and the process was repeated two more times, adding the supernatant to the 10-mL volumetric flask, and a final volume of 10 mL in methanol was prepared (extraction volume ratio 1:10, v/v). The extracts were filtered through single-use plastic syringes with 0.2-µm PTFE filters into 15-mL centrifuge tubes. For chromatographic analysis, approximately 1 mL of extract was transferred to an amber vial.