Ttx standard

TTX extraction and LC-MS/MS analysis were done following the method used in a previous study [29 (link)]. The calibration curve was generated with 1–100 ng/mL of TTX standard (FUJIFILM Wako Pure Chemicals, Osaka, Osaka, Japan), which showed good linearity and precision (y = 28.7128x + 21.1116, r² = 0.9911). The limit of detection value was calculated at 4.4 ng/mL for TTX.

LC-MS/MS was conducted according to a previously reported method [39 (link)]. LC was performed on an Alliance 2690 Separations Module (Waters). A Mightysil RP-18 GP column (2.0 × 250 mm, particle size 5 µm, Kanto Chemical Co., Inc.) was used with the mobile phase of 30 mM heptafluorobutyric acid in 1 mM ammonium acetate buffer (pH 5.0). The flow rate was set at 0.2 mL/min. The eluate was introduced into a Quattro micro^TM API detector (Waters). TTX was ionized by positive-mode electrospray ionization with a desolvation temperature of 400 °C, source block temperature of 150 °C, and cone voltage of 50 V, and monitored at m/z 162 (quantitative) and m/z 302 (qualitative) as product ions (collision voltage 38 V) with m/z 320 as a precursor ion through a MassLynx^TM NT operating system (Waters). TTX standard (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was used as an external standard. The LOD of TTX was 0.009 nmol/g tissue (S/N = 3) and the LOQ of TTX was 0.03 nmol/g tissue; (S/N = 10).