4.5.2. Q1 Scanning and MS/MS Analysis of the Unknown DST-Like Compound

Initial LC-MS experiments on the semi-purified extract from the 26–27 min fraction obtained from the PPIA bioactivity-guided fractionation of Sample 2 (from Figure 1 and Table 1) were performed using an Acquity Ultra-Performance Liquid Chromatography system (Waters Corporation, Manchester, UK) coupled to a Sciex QTrap 5500 mass spectrometer equipped with a Turbo V ionization source (SCIEX, Framingham, MA, USA). The column used for separations was a Waters BEH C18 (1.7 µm, 1.0 mm × 150 mm) (Waters Corp., Milford, MA). The aqueous mobile phase (A) consisted of 2 mM ammonium formate and 50 mM formic acid in water. The organic mobile phase (B) consisted of 2 mM ammonium formate and 50 mM formic acid in 95% acetonitrile/5% water. For initial method development studies, gradient conditions started at 50% B, were maintained for two min at 50% B, and were then linearly increased to 70% B in four min, followed by 100% B in two min, held at 100% B for five min, then decreased to 50% B in 0.5 min and lastly, were held at 50% B for 4.5 min. The total run time was 18 min at a flow rate of 0.12 mL/min. The column temperature was maintained at 40 ◦C while the autosampler temperature was 10 ◦C. The injection volume was 5 µL.

The electrospray ionization (ESI) source parameters were as follows: source temperature 550 ◦C, ion spray voltage −4500 V, curtain gas 25 psi, gas 1 and 2 both 40 psi. Full scan, negative ionization mode data were collected using a mass range from 500 to 900 Da and a scan rate of 200 Da s−<sup>1</sup> . Product ion scans for *m*/*z* 819.50 were collected using a mass range from 100 to 900 Da and a scan rate of 1000 Da s−<sup>1</sup> . The declustering and entrance potentials were −110 V and −10 V, respectively, and for product ion scans the collision energy was −70 V and collision cell exit potential was −15 V. Analyst®chromatography software (ver. 1.6.2, SCIEX, Framingham, MA, USA) was used for data visualization and analysis.

#### 4.5.3. LC-HRMS Analysis of Dihydrodinophysistoxin-1

LC-HRMS measurements of the semi-purified extract from the 26–27 min fraction of Sample 2 (from Figure 1 and Table 1) was performed using a Nexera LC system (Shimadzu, Columbia, MD, USA) coupled with a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA). The column and mobile phases were the same as described in Section 4.5.2., with the LC run time shortened to 15 min. Gradient conditions starting at 50% B were maintained for two min, then linearly increased to 70% B in four min, followed by 99% B in two min, held at 99% B for two min, then decreased to 50% B in 0.5 min and held at this level for 4.5 min to re-equilibrate. All other chromatography and autosampler settings were the same as described in Section 4.5.2.

Analytes were ionized using ESI in negative mode with source conditions as follows: spray voltage −3000 V, capillary temperature 320 ◦C, sheath gas 5 arbitrary units (au), and aux gas 0 au. A targeted-single ion monitoring (SIM)/data dependent (dd)-MS<sup>2</sup> experiment was performed on the sample. The instrument was set to monitor and perform MS/MS on *m*/*z* 819.49002. The parameters for SIM were a mass resolution setting of 70,000, automatic gain control (AGC) of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> , maximum injection time of 200 ms, and an isolation window of 2 *m*/*z*. The parameters for dd-MS<sup>2</sup> were as follows: mass resolution setting of 35,000, AGC 2 <sup>×</sup> <sup>10</sup><sup>5</sup> , maximum injection time 100 ms, normalized collision energy 35. The dd settings to initiate MS/MS was a minimum AGC of 8 <sup>×</sup> <sup>10</sup><sup>3</sup> .
