*5.3. Quantitive Analysis of Gambierone and 44-Methylgambierone Production* 5.3.1. Microalgal Culturing and Sample Extraction

The microalgal isolates studied (20 in total) consisted of 14 species from three genera, *Gambierdiscus*, *Coolia* and *Fukuyoa*. All isolates were grown in f2/seawater (1:3) except for the *G. carpenteri* isolate, which was also grown in K media. The growth chamber was set at 25 ◦C (±2 ◦C), 40–70 µmol m−<sup>2</sup> s <sup>−</sup><sup>1</sup> photon irradiance (12:12 h light:dark cycle). Isolates were either sourced from the CICCM; or donated by researchers from Hong Kong and Australia. Cultures were harvested in the late exponential or stationary phase and contained at least 5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells. The cells were harvested by centrifugation (3200× *g*, 10 ◦C, 10 min), the growth medium was decanted and the resulting cell pellets were frozen (−20 ◦C).

Each cell pellet was extracted twice with 90% aq. MeOH, at a ratio of 1 mL per <sup>2</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> cells, and ultrasonication (10 min at 59 kHz). Cellular debris was pelleted by centrifugation (3200× *g*, 4 ◦C, 5 min) between extractions and the supernatant transferred to another vial. The resulting supernatants were pooled to give a final extract concentration equivalent to 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL. The combined extracts were stored at −20 ◦C for 24–48 h to precipitate insoluble matrix co-extractives, which were removed using centrifugation (3200× *g*, 4 ◦C, 5 min) prior to analysis. An aliquot of the clarified extract was transferred into a 2 mL glass autosampler vial and analysed using a modification of the LC–MS/MS method described in Murray et al., 2018 [48].

#### 5.3.2. Liquid Chromatography–Tandem Mass Spectrometry Analysis

Quantitative analysis of gambierone and 44-methylgambierone (monoisotopic masses 1024.5 and 1038.5 g/mol, respectively) was performed on a Waters Xevo TQ-S triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC i-Class with a flowthrough needle sample manager. Chromatographic separation used a Waters Acquity UPLC BEH phenyl column (1.7 µm, 100 × 2.1 mm) held at 50 ◦C. The column was eluted at 0.55 mL/min with (A) Milli-Q water and (B) MeCN mobile phases, each containing 0.2% (*v*/*v*) of a 25% NH4OH solution. Fresh mobile phases were prepared daily to ensure optimal sensitivity and stable retention times. The initial solvent composition was 5% B with a linear gradient to 50% B from 0 to 2.5 min, ramped up to 95% B by 3 min and held at 95% B until 3.2 min, followed by a linear gradient back to 5% B at 3.5 min. The column was then re-equilibrated with 5% B until 4 min. The autosampler chamber was maintained at 10 ◦C and the injection volume was 1 µL. The mass spectrometer used an electrospray ionization source operated in negative ion mode. Other settings were capillary voltage 3.0 kV, cone voltage 40 V, source temperature 150 ◦C, nitrogen gas desolvation flow rate 1000 L/h at 600 ◦C, cone gas 150 L/h and the collision cell was operated with 0.15 mL/min argon. Multiple reaction monitoring (MRM) transitions for gambierone were *m*/*z* 1023.3 > 96.8 (Channel 1) and *m*/*z* 899.6 > 96.8 (Channel 2), with collision energies of 50 eV. 44-Methylgambierone was monitored using the *m*/*z* 1037.6 > 96.8 (Channel 1) and 899.6 > 96.8 (Channel 2), with collision energies of 70 and 48 eV, respectively. All transitions had a dwell time of 30 ms. Channels 1 and 2 were used for quantitation and confirmation, respectively.

Data acquisition and processing were performed with TargetLynx software (Waters, Milford, MA, USA). Gambierone and 44-methylgambierone were identified in sample extracts based on the retention time (2.54 and 2.58 min, respectively) and a fragment ion ratio of 8:1 (Channel 1/Channel 2; as determined using reference material). Quantitative analysis was performed using a five-point linear regression calibration (0–1000 ng/mL) prepared in 90% aq. MeOH and a relative response factor of 1 to 44-methylgambierone. The LoQ of

the analytical method was 1 ng/mL, which equates to 0.01 pg/cell in an extract generated from a cell pellet of 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL.

5.3.3. Quantitation of Gambierone Using Liquid Chromatography–Tandem Mass Spectrometry

The purified gambierone material (Section 5.1.2) was quantified against a qNMRcalibrated 44-methylgambierone reference standard [28] using LC–MS/MS and a relative response factor of 1. The instrument parameters and chromatographic conditions outlined above (Section 5.3.2) were used. Triplicate injections of a 100 ng/mL standard were compared, followed by calibration of the gambierone material using a five-point linear regression calibration (0–1000 ng/mL) of 44-methylgambierone.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/toxins13050333/s1. Table S1. Comparison of the <sup>13</sup>C (125 MHz) and <sup>1</sup>H (500 MHz) NMR chemical shifts (ppm), multiplicity and coupling constants (Hz) for gambierone purified from *Gambierdiscus cheloniae* CAWD232, generated in d4-MeOH, and those published by Rodriguez et al., 2015 [38]. Figure S1. Comparison of the mass spectra of gambierone in positive electrospray ionization mode (**A**) purified from *Gambierdiscus cheloniae* CAWD232 and (**B**) published by Rodriguez et al., 2015 [38]. Figure S2. <sup>1</sup>H NMR spectrum of gambierone purified from *Gambierdiscus cheloniae* CAWD232 acquired on a Bruker Advance III 500 MHz instrument in CD3OH (≥99.8% atom D). Figure S3. Expansion of the <sup>1</sup>H NMR spectrum (0.6–2.7 ppm) of gambierone in CD3OD (≥99.8% atom D) at 500 MHz. Figure S4. Expansion of the <sup>1</sup>H NMR spectrum (2.8–5.2 ppm) of gambierone in CD3OD (≥99.8% atom D) at 500 MHz. Figure S5. Expansion of the <sup>1</sup>H NMR spectrum (5.3–7.3 ppm) of gambierone in CD3OD (≥99.8% atom D) at 500 MHz. Figure S6. Comparison of the <sup>1</sup>H NMR spectrum of (**A**) gambierone purified from *Gambierdiscus cheloniae* CAWD232 acquired on a Bruker Advance III 500 MHz instrument and (**B**) the published spectrum from Rodriguez et al., acquired on a Varian Inova 750 MHz instrument [38]. Figure S7. COSY NMR spectrum of gambierone purified from *Gambierdiscus cheloniae* CAWD232 in CD3OD (≥99.8% atom D) at 500 MHz. Figure S8. HSQC NMR spectrum of gambierone purified from *Gambierdiscus cheloniae* CAWD232 in CD3OD (≥99.8% atom D) at 500 MHz. Figure S9. Comparison of the COSY NMR spectrum (1.0–6.5 ppm) of gambierone purified from *Gambierdiscus cheloniae* CAWD232 (black) and that published by Rodriguez et al., 2015 (red) [38]. Long range (~2 Hz) couplings not displayed. Figure S10. Expansion (1H: 1.0–2.7 ppm; <sup>13</sup>C: 10–55 ppm) of the HSQC spectrum of gambierone purified from *Gambierdiscus cheloniae* CAWD232 (black), and the published spectrum from Rodriguez et al., 2015 (red) [38]. Figure S11. Expansion (1H: 2.5–4.7 ppm; <sup>13</sup>C: 62–85 ppm) of the HSQC spectrum of gambierone purified from *Gambierdiscus cheloniae* CAWD232 (black), and the published spectrum from Rodriguez *et al*., 2015 (red) [38]. Figure S12. Expansion (1H: 4.7–6.5 ppm; <sup>13</sup>C: 110–140 ppm) of the HSQC spectrum of gambierone purified from *Gambierdiscus cheloniae* CAWD232 (black), and the published spectrum from Rodriguez et al., 2015 (red) [38]. Figure S13. Purification scheme for the isolation of gambierone from *Gambierdiscus cheloniae* CAWD232.

**Author Contributions:** Experimental design was performed by J.S.M., J.P., D.T.H. and M.R.P.; the research was carried out by J.S.M.; isolations and microalgal culturing was performed by L.L.R.; compound purification was conducted by J.S.M. and R.v.G.; toxicity assessment was performed by S.C.F.; funding was acquired by J.S.M. and D.T.H.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was conducted with funding from a doctoral scholarship from the New Zealand Food Safety Science and Research Centre awarded to J.S.M, the Cawthron Institute Capability Investment Fund and the Seafood Safety research programme (contract CAWX1801).

**Acknowledgments:** The authors would like to acknowledge Kirsty Smith from Cawthron for collecting the microalgal samples in the Cook Islands, Lucy Thompson from Cawthron for her technical assistance, Tom Trnski from the Auckland War Memorial Museum for collecting microalgal samples from the Kermadec Islands, Meng Yan and Priscilla Leung for the *Fukuyoa* isolates from Hong Kong, Michaela Larsson for the *Coolia* isolates from Australia, and Andrew Lewis from Callaghan Innovation for the NMR analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
