LC-MS/MS was conducted according to a previously reported method [26 (link)]. LC was performed on an Alliance 2690 Separations Module (Waters Corp., Milford, MA, USA). A Mightysil RP-18 GP column (2.0 mm i.d × 250 mm, particle size 5 µm, Kanto Chemical Co., Inc.) was used with a mobile phase of 30 mM heptafluorobutyric acid in 1 mM ammonium acetate buffer (pH 5.0). The flow rate was set to 0.2 mL/min. The eluate was introduced into a Quattro micro API detector (Waters). TTX was ionized by positive-mode electrospray ionization with a desolvation temperature of 400 °C, a source block temperature of 150 °C, and a 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 NT operating system (Waters). TTX standard (purified from pufferfish ovary) was used as an external standard. The limit of detection (LOD) of TTX was 0.01 μg/g tissue (S/N = 3) and the limit of quantification (LOQ) of TTX was 0.03 μg/g tissue (S/N = 10). The other analogs were detected at m/z 336 > 162 for 11-oxoTTX, m/z 304 > 162 for deoxyTTXs, m/z 302 > 162 for 4,9-anhydroTTX, m/z 330 > 162 for 4-epiTTX and m/z 290 > 272 for 11-norTTX-6(R/S)-ol.
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