Alliance 2690 separations module

HPLC analysis was performed with an Alliance 2690 Separations Module with a Waters 996 Photodiode Array Detector and Millennium 32 Chromatography Manager Version 3.2 (Waters, MA, USA). For preparative HPLC, a Nucleosil C18 column was used (5 μm, 4.0 mm × 250 mm I.D.; Macherey-Nagel, Germany). Acetonitrile, methanol, and water (J.T. Baker, USA) were used for separation. For simultaneous analysis of fustin, Fisetin, and sulfuretin, the mobile phase consisted of 2% acetic acid (A) and methanol (B). The flow rate was fixed at 1.0 ml/min and the wavelength was set at 254 nm. The gradient elution was as follows: 5% B for 0 min, 20% B for 10 min, 60% B for 40 min, and 80% B for 50 min. For analysis of the sample, 10 ml of methanol was added to 100 mg of the extract, and the resulting mixture was sonicated for 30 min and then filtered through a 0.45 μm membrane filter. Fisetin used as a reference standard was purchased from Sigma (MO, USA), and fustin and sulfuretin were purchased from the company (Indofine Chemical Co Inc, NJ, USA).

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 ].

The mass spectrometric analysis was conducted using the Alliance 2690 Separations Module, (Waters Alliance, USA) with 10 mL of the sample injected into the C18 column (3.5 mm, 2.1 x 100 mm), and a guard column of the same phase. The extracts were distilled at a rate of 0.2 mL/min. The initial conditions were water-acetonitrile (75:25), for 16 min, followed by water-acetonitrile (10:90) for 24 min. The column was pre-conditioned with 25% acetonitrile. The HPLC system was linked to a MicroMass Quattro Micro triple-quadrupole mass spectrometer (Micromass Ltd., Manchester, UK) with a positive-mode electrospray ionization probe. The compounds were identified and quantified using the multiple reaction monitoring mode [15 ].

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).