PSTs were analyzed using HPLC (High-performance liquid chromatography) with postcolumn derivatization. [31 (link),32 (link)]. HPLC analysis was performed using an Alliance 2690 Separations Module (Waters) fitted with porous graphitic carbon Hypercarb® column (4.6 mm i.d. × 100 mm length; 5 µm Thermo Fisher Scientific) at 25 °C. The separation of toxins was performed using two mobile phases—(A) 0.075% (v/v) TFA (Trifluoroacetic Acid) in water; (B) 0.025% (v/v) TFA in 50% (v/v) acetonitrile: water. The flow rate was set at 0.8 mL/min. Initial conditions were 4% B, followed by a linear gradient from 4% B to 25% B over 30 min, then returned to 4% B at 30.01 min, and held and re-equilibrated for over 7 min until the next injection. The eluate from the column was mixed continuously with 50 mM periodic acid and 0.2 M KOH containing 1 M ammonium formate and 50% formamide, and heated at 65 °C. The formation of fluorophores was monitored at 392 nm using 336 nm excitation. Reference materials of C1, C2, C3, GTX1-4, and dcGTX2,3 were provided by the Japan Fisheries Research and Education Agency, and neoSTX, dcSTX, and STX purified from the toxic crab Zosimus aeneus [33 (link)] were used as external standards to identify/quantify individual analogues (Figure 6 ). The toxicity of each PST was calculated with the recommended toxicity equivalency factors (TEFs) for the STX group [34 ].
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