PSTs analysis by HPLC-FLD was also conducted as previous methods [56 (link)]. An Alliance 2690 Separations Module (Waters) connected with a carbon Hypercarb® column (4.6 mm i.d. × 100 mm length; 5 µm; Thermo Fisher Scientific, Waltham, MA, USA) was used. The column temperature was set at 25 °C and the separation of total 12 STX components was performed using two mobile phases—(A) 0.075% (v/v) TFA (Trifluoroacetic Acid) in water and (B) 0.025% (v/v) TFA in 50% (v/v) acetonitrile: water. The flow rate was set at 0.8 mL/min. Linear gradients were 96% A and 4% B to 75% A and 25% B over 30 min, then returned to 96% A and 4% B at 30.01 min and re-equilibrated for 8 min until the next injection. The eluate from the column was continuously mixed with 0.2 M KOH containing 1 M ammonium formate and 50% formamide with 50 mM periodic acid at a flow rate of 0.4 mL/min each and heated at 65 °C. The intensity of the fluorescence was measured at 392 nm with 336 nm excitation. PSTs mixed standard containing GTX1-4 and dcGTX2,3 were provided by the Japan Fisheries Research and Education Agency, and dcneoSTX, neoSTX, hyneoSTX, hySTX, dcSTX, and STX purified from the toxic crab Zosimus aeneus were used to identify. The LOD of STXs was 0.02–0.04 μg/g tissue (S/N = 3) and the LOQ was 0.06–0.12 μg/g tissue (S/N = 10).
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