Tetrodontoxin Quantification in Tissue

The tissue from one individual was homogenized, and approximately 0.2 g of the homogenate was used for extraction of TTXs according to the method of Suo et al. [11 (link)]. The extract was filtered through a 0.45 μm Supra Pure Syringe Filter (Recenttec, Taipei, Taiwan), and LC-MS/MS analysis was performed on a LC-20AD solvent delivery system (Shimadzu, Kyoto, Japan) connected to a X500R Q-TOF Mass Spectrometer (SCIEX, Framingham, MA, USA) with an ESI ion source. Following the method of Suo et al. [11 (link)], the sample solutions were analyzed by multiple reaction monitoring (MRM) mode at a flow rate of 0.3 mL/min on an Atlantis HILIC Silica column (Waters, Milford, MA, USA). The optimized transitions for qualification and quantification were m/z 320.1 > 302.1 and m/z 320.1 > 162.06 for TTX, respectively, while they were m/z 272.1 > 254.1 and m/z 272.1 > 162.1 for 5,6,11-trideoxyTTX, respectively. The calibration curve was created using 1–50 ng/mL of standard for TTX (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 5,6,11-trideoxyTTX [39 (link)], and showed good linearity and precision (TTX: y = 445.36012x, r² = 0.99; 5,6,11-trideoxyTTX: y = 168.20873x, r² = 0.99).