Acute Toxicity of Gambierone and Quantitative Analysis of Gambierones Produced by Cohabitating Benthic Dinoflagellates

Understanding the toxicity and production rates of the various secondary metabolites produced by Gambierdiscus and cohabitating benthic dinoflagellates is essential to unravelling the complexities associated with ciguatera poisoning. In the present study, a sulphated cyclic polyether, gambierone, was purified from Gambierdiscus cheloniae CAWD232 and its acute toxicity was determined using intraperitoneal injection into mice. It was shown to be of low toxicity with an LD50 of 2.4 mg/kg, 9600 times less toxic than the commonly implicated Pacific ciguatoxin-1B, indicating it is unlikely to play a role in ciguatera poisoning. In addition, the production of gambierone and 44-methylgambierone was assessed from 20 isolates of ten Gambierdiscus, two Coolia and two Fukuyoa species using quantitative liquid chromatography–tandem mass spectrometry. Gambierone was produced by seven Gambierdiscus species, ranging from 1 to 87 pg/cell, and one species from each of the genera Coolia and Fukuyoa, ranging from 2 to 17 pg/cell. The production of 44-methylgambierone ranged from 5 to 270 pg/cell and was ubiquitous to all Gambierdiscus species tested, as well as both species of Coolia and Fukuyoa. The relative production ratio of these two secondary metabolites revealed that only two species produced more gambierone, G. carpenteri CAWD237 and G. cheloniae CAWD232. This represents the first report of gambierone acute toxicity and production by these cohabitating benthic dinoflagellate species. While these results demonstrate that gambierones are unlikely to pose a risk to human health, further research is required to understand if they bioaccumulate in the marine food web.


Introduction
Ciguatera fish poisoning (CFP) is the most common non-microbial foodborne illness related to finfish consumption in the world [1,2]. It is generally associated with the bioaccumulation of ciguatoxins (CTXs) in the flesh and viscera of fish from all trophic levels. While published estimates of CFP vectors range between 60 [3,4] and 400 species [1], it is Determining the toxicity of these secondary metabolites and their abundance in Gambierdiscus and cohabitating genera is a critical step in understanding whether they have the potential to cause human illness. Our recent study assessed one of these secondary metabolites, 44-methylgambierone, and determined it had low acute toxicity via intraperitoneal (i.p) injection to mice (LD 50 between 20 and 38 mg/kg). Its production was also qualitatively assessed in commonly found cohabitating genera of toxin-producing benthic dinoflagellates. The results demonstrated that three genera produced this secondary metabolite: Gambierdiscus, Coolia and Fukuyoa [28].
The work presented in this manuscript expands on this knowledge, where another structurally related secondary metabolite, gambierone, was purified in sufficient quantities to allow for the first determination of its acute toxicity and ascertain if it could play a role in CP events. In addition, quantitative analysis of both gambierone and 44-methylgambierone production by 20 isolates of cohabitating dinoflagellates from the genera Gambierdisucs, Coolia and Fukuyoa was performed, as reference material had been generated.

Purification and Identification of Gambierone from Gambierdiscus cheloniae CAWD232
Gambierone was purified from G. cheloniae CAWD232 monoclonal cultures using a combination of liquid-liquid partitioning, solid-phase clean-up, flash-chromatography and preparative high-performance liquid chromatography.
Analysis of the purified gambierone material by electrospray ionization (ESI) mass spectrometry (    In addition to the 1 H NMR spectrum, COSY (Supplementary Figure S7) and HSQC spectra (Supplementary Figure S8) were acquired. The 1 H-1 H and 1 H-13 C correlations were also closely aligned to those published by Rodriguez et al. [38] (Supplementary Figures S9-S12). Collectively, the MS and NMR data provided confirmation that the isolated compound was gambierone.
Quantification of the gambierone purified from G. cheloniae CAWD232 against a qNMRcalibrated 44-methlygambierone standard using LC-MS/MS, and a relative response factor of 1, determined the yield to be 1.84 mg.

Acute Toxicity of Gambierone by Intraperitoneal Injection
On the basis of the low i.p toxicity previously determined for 44-methylgambierone (between 20 and 38 mg/kg) [28], it was anticipated that gambierone would have a similarly low toxicity. Single mice were dosed at 10, 5, 3.2 and 2.54 mg/kg, all of which died within 4−9 h post-dosing ( Table 2). Symptoms of toxicity included abdominal stretching, which was evident 10 min post-dosing. Mice also showed classic signs of discomfort such as pulling their ears back and orbital tightening [39]. As the toxicity progressed the mice became reluctant to move and any movement was jerky. Further deterioration resulted in laboured breathing, and at this point, mice were euthanised to prevent long-term suffering in accordance with the requirements of the Organisation for Economic Cooperation and Development (OECD) Human Endpoints Guidance Document [40]. A single mouse dosed at 2.04 mg/kg gambierone survived, and having established an appropriate dosing range within the OECD guideline 425 [41], the up-and-down method was used to determine the LD 50 of gambierone as 2.4 mg/kg with 95% confidence intervals of 2.04 and 2.54 mg/kg. All mice dosed at 2.54 mg/kg died in an analogous manner to that described above (Table 2). At necropsy, the small intestine and caecum of each mouse that was administered a lethal dose of gambierone, were found to contain a pale green fluid. All mice dosed at less than 2.04 mg/kg survived. These mice showed abdominal stretching as well as the signs of discomfort mentioned above. All mice appeared normal within 3.5 and 7 h post-dosing. A low food intake and subsequent weight loss over the first 24-h period post-dosing was observed in mice dosed at 2.04 and 1.58 mg/kg. However, for the remainder of the 14-day observation period, food intake and weight gain were normal. This effect on food intake was not noted in mice dosed at rates lower than 1.58 mg/kg. At necropsy, none of the surviving mice showed any abnormalities and the organ weights, as expressed as % of bodyweight, were all within the normal range (data not provided).

Gambierone and 44-Methylgambierone Production by Cohabitating Benthic Dinoflagellates
Dinoflagellate isolates (n = 20) were analysed for the production of gambierone and 44-methylgambierone by LC-MS/MS to determine pg/cell quotas. Quantitative analysis was now possible due to the generation of well-characterised reference material for both secondary metabolites. 44-Methylgambierone production was ubiquitous in the ten Gambierdiscus species tested, with levels ranging from 26 to 270 pg/cell ( Table 3). The highest levels were produced by G. lapillus CAWD338 (270 pg/cell) and G. australes CAWD149 (259 pg/cell), while the G. lapillus CAWD336 isolate produced considerably less (46 pg/cell). Compared to the Gambierdiscus species, 44-methylgambierone cell quotas from the Coolia and Fukuyoa isolates were generally lower (5-65 pg/cell).
The production of gambierone was more varied, with only seven of the Gambierdiscus species and one each of the Coolia and Fukuyoa species producing detectable levels (LoQ = 0.01 pg/cell). The gambierone cell quotas for the Gambierdiscus isolates ranged from 1 to 87 pg/cell (Table 3), with the highest levels observed in G. carpenteri CAWD237 (87 pg/cell). When G. carpenteri CAWD237 was grown in K media instead of f/2 media, a higher gambierone quota was observed (87 compared to 65 pg/cell). Similar to what was observed for 44-methylgambierone production, the range and level of gambierone cell quotas by the Coolia and Fukuyoa isolates was lower (2-17 pg/cell). The largest variation was observed between the two C. malayensis isolates CAWD154 and CAWD175 (2 and 17 pg/cell, respectively). Gambierone was not detected in the two C. tropicalis or the two F. paulensis isolates.
The relative production ratio, calculated as pmol/cell, between gambierone and 44methylgambierone was varied and ranged from 0.01 to 2.15. Of the 14 microalgal species tested, only two produced more gambierone than 44-methylgambierone; G. carpenteri CAWD237 (1.19 in K media and 1.46 in f/2 media) and G. cheloniae (2.15). Of the isolates where gambierone was detected, the lowest levels of relative production were observed for G. lewisii CAWD369 and G. pacificus CAWD337, both of which had a ratio of 0.01.

Discussion
In the present study, G. cheloniae CAWD232 was grown (100 L) to purify enough gambierone to determine its acute toxicity and ascertain if it could play a role in CP events. Fractionation of a methanolic extract and subsequent purification was tracked using LC-MS/MS. The compound was identified as gambierone through its mass spectral properties (the deprotonated and protonated molecular ions; the observed sulphate-/waterloss ions characteristic of cyclic polyethers) and comparison of NMR spectroscopic data with those previously published by Rodriguez et al. in 2015 [38]. The chemical shifts, coupling constants and overlaying of the 1 H, COSY and HSQC NMR spectra provided unequivocal evidence that the isolated compound was gambierone. As the HSQC spectrum was used to assign the 13 C chemical shifts, quaternary carbons were unable to be assigned using the current dataset. Small differences in the peak resolution, chemical shifts and coupling constants between the two studies were observed and are a result of the 1 H NMR data in the current study being acquired at 500 MHz, and the Rodriguez et al., 2015 [38] data being acquired at 750 MHz.
Due to the structural similarities between gambierone and 44-methylgambierone (i.e., the presence of a monosulphate, terminal diol and 1,3-diene) the quantity of gambierone generated was determined by LC-MS/MS against a qNMR reference standard of 44-methylgambierone [28,36]. A relative response factor of 1 was used as the only structural difference between these two compounds is an additional methyl group on C-44, which is unlikely to grossly affect its ionisation properties. Analysis was performed in -ESI mode for two reasons: (1) it afforded a clean spectrum with good sensitivity and (2) the charge is on the sulphate group, which is at the opposite end of the molecule to the additional methyl group, hence, it would not yield a noticeable effect on the ionisation efficiency of the two compounds.
Based on the low acute i.p toxicity of 44-methylgambierone (between 20 and 38 mg/kg) [28], it was anticipated that gambierone would also have a low acute toxicity. The LD 50 of gambierone was found to be 2.4 mg/kg, indicating that it is 10-15 times more toxic than 44-methylgambierone. Given that the only structural difference between these two compounds is the addition of a methyl group on C-44, this difference in toxicity is surprising. It also highlights that toxicity cannot be predicted on the basis of structure, and that small structural changes have the potential to greatly affect toxicity. This may be due to the structural change affecting the three-dimensional shape of the molecule such that its access to the active site responsible for the toxic effect is altered. While the i.p toxicity data of gambierone is new and important information, oral dosing is the most relevant administration route to assess the risk posed to humans. Previous studies on other shellfish toxins have shown that, although toxicity is reduced when given orally rather than by i.p, there is no correlation between the two. For example, among the paralytic shellfish toxins, the difference between oral and i.p toxicity ranges from 30-fold for decarbamoyl neosaxitoxin to 400-fold for gonyautoxin 5 [42]. These differences are likely due to differences in metabolism or absorption of the metabolites, parameters which are bypassed when the compounds are administered by i.p. Unfortunately, it was not possible to isolate the quantities of gambierone that would be required to determine an oral LD 50 .
Although the i.p LD 50 of gambierone showed it to be more toxic than 44-methylgambierone, an LD 50 of 2.4 mg/kg is still indicative of low toxicity. This is consistent with results published by Rodriguez et al. [38], who showed that gambierone had low activity in sodium channels and little impact on cytosolic Ca 2+ levels in the neuroblastoma SH-SY5Y cell line. In comparison, other toxins implicated in CP are of far greater toxicity. Algal CTXs have i.p LD 50 values of 2−10 µg/kg and fish metabolite CTXs have i.p LD 50 values of 0.25−0.45 µg/kg, thereby ranging from being 240 (for 10 µg/kg) to 9600 (for 0.25 µg/kg) times more toxic than gambierone [43]. Although further work is required to assess the bioaccumulation of this secondary metabolite in the marine food web, the hydrophilic nature of gambierone along with the vast difference in toxicity compared to CTXs mean that it is highly unlikely that gambierone contributes to CP. The symptoms of gambierone intoxication are also significantly different to those observed for the CTXs. CTX toxicity is characterised by diarrhoea and hypersalivation, neither of which were observed in this study. A comparison of the symptoms induced by gambierone and 44-methylgambierone is limited, since only one mouse dosed with 44-methylgambierone (38 mg/kg, a lethal dose) showed toxicity. However, it is noted that this mouse did not show the abdominal stretching that was consistently seen in mice dosed with gambierone, although both toxins affected food intake. In addition, this effect was limited to 1−2 days for gambierone, whereas a much more pronounced anorexic effect was seen in the one mouse given a lethal dose of 44-methylgambierone [28]. Although there are some apparent differences in symptomology between gambierone and 44-methylgambierone, additional toxicity work with the latter compound is required for confirmation.
Previous research on 44-methylgambierone production showed that it was ubiquitous to all Gambierdiscus species tested [28]. The current work has expanded on this by demonstrating that G. holmesii CAWD368 and G. lewisii CAWD369 also produce 44methylgambierone, and by assessing the gambierone production in a range of Gambierdiscus, Coolia and Fukuyoa isolates (n = 20). These isolates were sourced from the Cawthron Institute Culture Collection of Microalgae (CICCM) or were donated from research collaborators in Hong Kong and Australia. The Gambierdiscus isolates were collected in Australia, the Cook Islands, French Polynesia, the Kermadec Islands and the Federated States of Micronesia. Representative isolates from the ten species held in culture-G. australes, G. caribaeus, G. carpenteri, G. cheloniae, G. holmesii, G. honu, G. lapillus, G. lewisii, G. pacificus and G. polynesiensis-were selected. Two isolates were selected for both G. lapillus and G. polynesiensis, and for G. carpenteri the selected isolate was grown in two different growth media to investigate if differences in the production of secondary metabolites would be observed. The four Coolia isolates from two species, C. malayensis and C. tropicalis, and four Fukuyoa isolates from two species, F. paulensis and F. ruetzeri, were selected as they had previously been shown to produce 44-methylgambierone [28].
A quantitative analysis of 44-methylgambierone and gambierone was also undertaken in the current study to evaluate cell quotas. Production of 44-methylgambierone varied between the dinoflagellate isolates, with G. lapillus CAWD338 producing the most. Interestingly, the second highest producer was G. australes CAWD149, which is the only known MTX-1 producer [36]. Our previous work with G. carpenteri isolates from around the Pacific region reported this species to produce 44-methylgambierone, except in the case of G. carpenteri CAWD237 collected in Australia [28]. In contrast, the present study demonstrated that the isolate does produce this secondary metabolite. However, because the original analysis was performed on a cell pellet grown at a different laboratory, it is possible that the different seawater/growth medium source, along with different growth cabinets (light intensity, temperature controls), affected the production of this secondary metabolite. This suggests that culturing conditions play a critical role in the production of these compounds, but further investigations are required to properly understand this.
The production of gambierone by Gambierdiscus species was varied, with only seven species producing detectable levels (>0.01 pg/cell; G. carpenteri, G. cheloniae, G. holmesii, G. honu, G. lewisii, G. pacificus and G. polynesiensis). G. carpenteri CAWD237 was the highest producer followed by G. cheloniae CAWD232, which was the isolate used for the purification of gambierone in the present study. To date, G. belizeanus has been the only species reported to produce gambierone, thereby making this the first report of gambierone production by an additional seven Gambierdiscus species.
An interesting observation was made with the G. carpenteri CAWD237 isolate, in that it produced more gambierone and 44-methylgambierone when grown in K media compared to f/2 media. This is unusual, as it is well known that the preferred growth medium for Gambierdiscus is f/2 media [44]. K media has been specifically designed for growing marine dinoflagellates that cannot survive in higher levels of trace metals. One potential explanation is that this culture had reduced metal availability resulting in a stressed state [45], which in turn increased the production rates of these secondary metabolites. Further research with the remaining isolates is required to test this hypothesis, as well as the effects of the presence/absence of inorganic phosphates on growth and toxin production [45].
Both strains of the two Coolia species, C. malayensis CAWD154 and CAWD175 and C. tropicalis UTS2 and UTS3, produced 44-methylgambierone, whilst only the C. malayensis isolates produced gambierone (2 and 17 pg/cell). A similar result was observed with both strains of the two Fukuyoa species, F. paulensis CAWD238 and CAWD306 and F. ruetzeri S044 and S051, producing 44-methylgambierone, while only the F. ruetzeri isolates produced gambierone (6 and 8 pg/cell). This is the first report of gambierone production by the genera Coolia and Fukuyoa.
Investigation of the production ratio between gambierone and 44-methylgambierone across all species revealed that only two species produced more gambierone than 44methylgambierone. The first was G. cheloniae CAWD232, which produced more than twice the amount of gambierone compared to 44-methylgambierone (ratio of 2.12), followed by G. carpenteri CAWD237 (ratio of 1.17 and 1. 44 for K and f/2 media, respectively). The largest inverse variation was observed for G. lewisii CAWD369 and G. pacificus CAWD337 (both with a ratio of 0.01).
While this is the first study to accurately quantify gambierone and 44-methylgambierone production by Gambierdiscus species, little research has been conducted to accurately quantify cells quotas using LC-MS/MS for the additional cyclic polyethers produced by this genus. The few that have been reported show that G. australes isolates can produce MTX-1 at 2−9 pg/cell, and G. polynesiensis CAWD212 produces total CTXs (sum of P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) at 0.44 pg/cell [46]. One research group demonstrated that G. polynesiensis TB92 produced 11.9 ± 0.4 pg P-CTX-3C equivalence/cell, as determined using the receptor-binding assay. Subsequent analysis using LC-MS/MS revealed that five CTX analogues were present (P-CTX-3B, P-CTX-3C, P-CTX-4A, P-CTX-4B and M-seco-CTX-3C); however, they were not quantified [13].

Conclusions
Gambierdiscus produces a complex array of bioactive ladder-shaped polyether secondary metabolites. Understanding the toxicity of these compounds is essential in determining if they contribute to CP. One of these compounds, gambierone, was purified from G. cheloniae CAWD232 and found to have low acute toxicity by i.p injection in mice (LD 50 2.4 mg/kg). The hydrophilic nature of this secondary metabolite and the low acute toxicity compared to CTXs, 9600 times less toxic than P-CTX-1B, indicate that gambierone is unlikely to play a role in CP. However, to confirm this prediction, further research is required to assess the bioaccumulation of this secondary metabolite in the marine food web. In addition, cell quotas were determined and seven of the ten Gambierdiscus species tested produced gambierone, along with one species from the genera Coolia and Fukuyoa. The cell quotas of another structurally related analogue, 44-methylgambierone, were also determined and ubiquitous production by all Gambierdiscus species, as well as both the Coolia and Fukuyoa species tested, was demonstrated. This is the first report of the acute toxicity of gambierone and the quantitative analysis of gambierones produced by these genera of cohabitating benthic dinoflagellates.
Fractionation was then performed on a Reveleris Flash Chromatography system (Büchi, Flawil, Switzerland) fitted with an Agilent Superflash C 18 SF 25-75 g column (four injections; Santa Clara, CA, USA). The column was eluted at 20 mL/min with (A) Milli-Q water and (B) acetonitrile (MeCN; Fisher-Optima) mobile phases. The initial solvent composition was 20% B for 5 min before a linear gradient to 95% B from 5-30 min, and then held at 95% B for 10 min. Fractions were collected every 30 s (10 mL). A final fractionation was performed on a Shimadzu preparative HPLC system (Kyoto, Japan) using isocratic elution, 35% aq. MeCN with 0.2% (v/v) of a 25% ammonium hydroxide (NH 4 OH) solution, on a Phenomenex Gemini C18 column (5 µm, 150 × 21 mm; five injections) with a flow rate of 25 mL/min. The fractions containing gambierone were combined, dried down using rotary-evaporation (50 mBar and 50 • C) and resuspended in 100% MeOH (1 mL; for a schematic of the purification scheme refer Supplementary Figure S13).

Nuclear Magnetic Resonance Spectroscopy Evaluation of Gambierone
1-and 2-D NMR spectroscopy experiments were conducted on a Bruker Avance III 500 MHz instrument with a 5 mm BBOF smart probe (Billerica, MA, USA). The instrument was operated at 500 MHz for 1 H and 125 MHz for 13 C, with the chemical shifts being determined at 303 K and referenced to the MeOH signal at 3.31 ppm. The gambierone material was taken to dryness under a stream of N 2 gas at 40 • C, resuspended in d4-MeOH (600 µL, >99.8% deuterium; Sigma-Aldrich, St. Louis, MO, USA) and transferred to a Wilmad ® (Vineland, NJ, USA) 5 mm high-precision NMR tube prior to analysis.

Liquid Chromatography-Mass Spectrometry Evaluation of Gambierone
The purified gambierone (from Section 5.1.2) was assessed using a Waters Xevo TQ-S triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC i-Class with a flow-through needle sample manager (Waters; Milford, MA, USA). The mass spectrometer utilized electrospray ionization (positive and negative ion modes) with a scan range of m/z 850-1150. 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 using mobile phases containing 0.2% (v/v) of a 25% NH 4 OH solution in (A) Milli-Q water and (B) MeCN. Initial solvent conditions were 5% B for 1 min with a linear gradient to 95% B from 1.0 to 7.5 min, held at 95% B for 1 min, followed by a linear gradient back to 5% B from 8.5 to 9 min. The column was re-equilibrated with 5% B for 1 min before the next injection. The autosampler chamber was maintained at 10 • C and the injection volume was 1 µL.

Toxicity Assessment
Acute toxicity was determined using the principles of OECD guideline 425 [41]. This guideline employs an up-and-down procedure, whereby one animal is dosed and if it survives, the next mouse receives an increased dose, whereas if it dies, the next mouse is dosed with a reduced amount of the test material. To determine the LD 50 , dosing is continued until four live-death reversals have been achieved.
Toxicity was determined by i.p injection. Each mouse was weighed prior to dosing and the required amount of gambierone calculated to yield the chosen dose on a mg/kg basis. The dose was prepared by taking the appropriate volume of stock solution (pure gambierone in 90% aq. MeOH), drying it down under nitrogen and immediately redissolving in 1% Tween 60 in normal saline (1 mL). This freshly prepared solution was then immediately injected into mice. All dosing was conducted between 8 and 9.30 a.m. to avoid any diurnal variations in response. Mice were monitored closely during the day of dosing and those that survived were monitored for a 14 day observation period, which included a daily measurement of food consumption and bodyweight. After 14 days, the mice were euthanised by carbon dioxide inhalation and necropsied. The weights of the liver, kidneys, spleen, heart, lungs, stomach (full and empty) and the whole gut were measured and calculated as a percentage of bodyweight (data not provided).

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 −2 s −1 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 × 10 5 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 2 × 10 5 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 × 10 5 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].

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% NH 4 OH 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 × 10 5 cells/mL.

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.