1/-2 using sodium borohydride (NaBH₄) and borodeuteride (NaBD₄) generated pairs of isomers with protonated molecules at *m/z* 1143.6463 and 1144.6535, respectively [120]. This led to the structural characterisation of two new analogues, C-CTX-3 and C-CTX-4, containing an open N-ring, and thus corresponding to C-CTX-1 and -2 reduced at C-56 [120]. Caribbean CTX congeners with a protonated molecule at *m/z* 1143.6 were identified earlier in several fish from the Caribbean [23,38,41,42,108], and more recently from the Eastern Atlantic Ocean [131]. However, these are an open (unstable) form of C-CTX-1 that are generated under acidic conditions and lack stereochemistry, making the assignment of the two isomers controversial. Kryuchkov et al. also showed that a pentafluorophenyl-propyl UHPLC column (100×2.1 mm, 1.7 µm) significantly improved the shape of C-CTX-1 and -2 peaks and provided better chromatographic separation between C-CTX-1/-2 and C-CTX-3/-4. However, the octadecylsilane-based LC column (100 × 2.1 mm, 1.5 µm) allowed for the best anomer separation regardless of chromatographic conditions [120]. A comparison of the peak shapes of C-CTX-1 and -2 during LC-HRMS in acidic and neutral mobile phases on the C-18 column were indicative of a rapid on-column interchange between C-CTX-1 and -2 that was accelerated under acidic conditions, whereas the peak shapes of 3 and 4 were unaffected [120].

To improve the HRMS sensitivity compared to that of targeted LC-MS/MS, Kryuchkov et al. also proposed a promising chemical derivatization approach employing a fast and simple one-pot derivatization with Girard's reagent T (GRT) [205]. The sensitivity of their LC-MS/MS method using a triple quadrupole (6495B, Agilent Technologies, Santa Clara, CA, USA) and the LC-HRMS method on a Q-Orbitrap (Q-Exactive, Thermo Fisher Scientific, Waltham, MA, USA) was improved for C-CTX-1 by ~40- and 17-fold, respectively [205]. Derivatization with GRT improved method selectivity and ionization efficiency by inserting a quaternary ammonium ion onto C-CTX-1. However, the use of GRT derivatization for routine applications still requires validation [205].

UHPLC-HRMS methods in negative and positive ionisation modes on a Q-Orbitrap (Q-Exactive, Thermo Fisher Scientific) were also adapted for CYP and UGT metabolism experiments and chemical reduction by Gwinn et al. They reported the production of phase II GlcA conjugates of C-CTX-1/-2 in vitro by liver microsomes of five fish species from the northern Gulf of Mexico and Atlantic salmon, which was the first time it was observed [165]. Borohydride reduction of C-CTX-1/2-GlcA metabolites confirmed that the metabolites were comprised of four distinct glucuronide products, with the sugar attached at two separate sites on C-CTX-1/-2, excluding the C56 hydroxyl group as the conjugation site [165].

### 5.3.2. Contribution of HRMS to Study *Gambierdiscus* spp. Toxins

The HRMS technique has also been applied to study algal toxin profiles [43,73,86,109,206,207]. Combining LC-MS/MS for rapid screening with a quantitative estimation and LC-HRMS for confirmation and characterisation, Estevez et al. showed that the toxin profiles of *Gambierdiscus* spp. dinoflagellates from the Mediterranean Sea and Northeast Atlantic were similar to those detected in CP endemic regions, except for the presence of CTXs, with the detection of 44-methylgambierone, gambieric acid C and D, gambierone analogue, and gambieroxide in *Gambierdiscus australes* strains (Balearic Islands, Spain) [207]. This study highlighted species and regional specificity of *Gambierdiscus* spp., with a 44-methylgambierone and gambierone-producing *Gambierdiscus* sp. strain from Crete (Greece) and an MTX-4 producing *G. excentricus* strain from the Canary Islands (Spain) [207]. HRMS did not detect maitotoxin-1 (MTX1), desulfo-MTX1, and didehydro-desulfo-MTX1 above the detection limit in Mediterranean strains of *G. australes* [207]. Sulfo-gambierone and dihydrosulfo-gambierone, two new analogues of gambierone, were also characterised in two *G. excentricus* strains initially isolated from the Bahamas and the Florida Keys [208], however, no algal ciguatoxins were detected in these two strains, which was consistent with the low ciguatoxin-like content evaluated using the CBA-N2a assay [208]. An m-aminophenylboronic acid agarose gel was used to confirm the
presence of gambierone and identify a novel isomer of 44-methylgambierone in an extract of *Gambierdiscus silvae* from the U.S. Virgin Islands [206].

Recently, a major breakthrough was achieved with the identification of an algal toxin C-CTX-5 that was extracted from a *Gambierdiscus silvae* strain and two *Gambierdiscus caribaeus* strains. The toxin was isolated from macroalgae collected from reefs surrounding St. Thomas (U. S. Virgin Islands) [43]. These *Gambierdiscus* strains showed C-CTX-like activity on voltage-gated sodium channel using CBA-N2a, with a higher toxicity for *G. silvae* compared to the two *G. caribaeus* strains tested [43]. The toxins were screened for known CTXs using reference standards (C-CTX-1, gambierone and 44-methylgambierone) and unknown putative CTXs using LC-HRMS on a Q-Orbitrap (Q Exactive, Thermo Fischer Scientific) with a heated electrospray ionization probe (HESI-II) [43]. With a full-scan acquisition (m/z 1000–1250), product-ion spectra using targeted parallel reaction monitoring (PRM) scan mode, and chemical transformations, Mudge et al. identified a novel C-CTX analogue, C-CTX-5, for the first time from Caribbean *Gambierdiscus* strains [43]. An initial assessment of CTX-like activity was made using in vitro CBA-N2a, which confirmed that C-CTX-5 was ciguatoxic, with an IC₅₀~½ IC₅₀ of C-CTX-1/-2. However, this remains to be quantitatively evaluated using quantified purified materials [43]. Moreover, C-CTX-1/-2 were also detected compared with a reference standard, and it was estimated at ~7% of the level of C-CTX-5 in *G. silvae*. Therefore, it could also contribute directly to toxicity of fish that ingest *Gambierdiscus* cells [43]. Gambierone was detected in all algal extracts, and 44-methylgambierone was detected in *G. silvae* and one of the two *G. caribaeus* [43]. Interestingly, the transformation of C-CTX-5 into C-CTX-1/-2 occurred via a reduction step [43] rather than oxidation, as observed for Pacific toxins, CTX-4A, CTX-4B, and CTX-3C, which are oxidized to the corresponding CTXs found in fish from the Pacific Ocean [90].

# 6. Discussion and Perspectives

## 6.1. *Bioassays*

The cytotoxicity assay CBA-N2a reflects the overall toxicity attributed to sodium-channel blocking toxins and is currently used as an initial screening method for toxic fish. It provides an alternative to the mouse bioassay and the use of radiolabeled molecules. However, in most studies, higher values for composite toxicities were obtained compared with C-CTX-1 concentrations in fish flesh samples [37,109,110,112,155,175,176]. Discrepancies between composite cytotoxicity evaluated using CBA-N2a and concentrations of C-CTX-1 evaluated using LC-MS/MS could be due to the presence of other C-CTX congeners. Therefore, C-CTX-1 was not the principal toxin. These results emphasize the challenges of combining biological and/or functional and/or pharmacological assays with analytical methods, considering their different specificities and sensitivities. The possibility that the CBA-N2a assay can overestimate ciguatera risk needs to be investigated. Radiolabeled ligand binding assays can also be used to control fish toxicity and monitor toxic LC fractions of slow- and fast-acting C-CTXs [41,172,194]. However, the CBA-N2a assay was 12-fold more sensitive to detecting C-CTX-1 than the radiolabeled RBA [194]. It was also shown to be more sensitive than LC-MS/MS and fRBA using fluorescent brevetoxin (BODIPY®-PbTx-2) in a comparative study on P-CTXs from *Gambierdiscus polynesiensis* [83]. P-CTX-1 was 5–10-fold more potent than C-CTX-1 using RBA [172]; in agreement with the fact that P-CTX-1 was 14 times more toxic to mice than C-CTX-1 [38]. Thus, although it is no longer recommended by the European Food Safety Authority, the mouse bioassay could allow for the estimation of the relative toxicity of new analogues compared with C-CTX-1 or P-CTX-1 which are more accessible. This is important considering that differences in ADME processes may influence in vivo toxicity. To accelerate screening and address species differences in CTX sensitivity, Lewis et al. established a new FLIPR-based screen using human SH-SY5Y cells sensitised using ouabain and veratridine [81]. This assay demonstrated comparable sensitivity to the CBA-N2a assay, and could differentially detect MTX and CTX in extracts of *Gambierdiscus* spp. [81].
presence of gambierone and identify a novel isomer of 44-methylgambierone in an extract of *Gambierdiscus silvae* from the U.S. Virgin Islands [206].

Recently, a major breakthrough was achieved with the identification of an algal toxin C-CTX-5 that was extracted from a *Gambierdiscus silvae* strain and two *Gambierdiscus caribaeus* strains. The toxin was isolated from macroalgae collected from reefs surrounding St. Thomas (U. S. Virgin Islands) [43]. These *Gambierdiscus* strains showed C-CTX-like activity on voltage-gated sodium channel using CBA-N2a, with a higher toxicity for *G. silvae* compared to the two *G. caribaeus* strains tested [43]. The toxins were screened for known CTXs using reference standards (C-CTX-1, gambierone and 44-methylgambierone) and unknown putative CTXs using LC-HRMS on a Q-Orbitrap (Q Exactive, Thermo Fischer Scientific) with a heated electrospray ionization probe (HESI-II) [43]. With a full-scan acquisition (m/z 1000–1250), product-ion spectra using targeted parallel reaction monitoring (PRM) scan mode, and chemical transformations, Mudge et al. identified a novel C-CTX analogue, C-CTX-5, for the first time from Caribbean *Gambierdiscus* strains [43]. An initial assessment of CTX-like activity was made using in vitro CBA-N2a, which confirmed that C-CTX-5 was ciguatoxic, with an IC₅₀~½ IC₅₀ of C-CTX-1/-2. However, this remains to be quantitatively evaluated using quantified purified materials [43]. Moreover, C-CTX-1/-2 were also detected compared with a reference standard, and it was estimated at ~7% of the level of C-CTX-5 in *G. silvae*. Therefore, it could also contribute directly to toxicity of fish that ingest *Gambierdiscus* cells [43]. Gambierone was detected in all algal extracts, and 44-methylgambierone was detected in *G. silvae* and one of the two *G. caribaeus* [43]. Interestingly, the transformation of C-CTX-5 into C-CTX-1/-2 occurred via a reduction step [43] rather than oxidation, as observed for Pacific toxins, CTX-4A, CTX-4B, and CTX-3C, which are oxidized to the corresponding CTXs found in fish from the Pacific Ocean [90].

# 6. Discussion and Perspectives

## 6.1. *Bioassays*

The cytotoxicity assay CBA-N2a reflects the overall toxicity attributed to sodium-channel blocking toxins and is currently used as an initial screening method for toxic fish. It provides an alternative to the mouse bioassay and the use of radiolabeled molecules. However, in most studies, higher values for composite toxicities were obtained compared with C-CTX-1 concentrations in fish flesh samples [37,109,110,112,155,175,176]. Discrepancies between composite cytotoxicity evaluated using CBA-N2a and concentrations of C-CTX-1 evaluated using LC-MS/MS could be due to the presence of other C-CTX congeners. Therefore, C-CTX-1 was not the principal toxin. These results emphasize the challenges of combining biological and/or functional and/or pharmacological assays with analytical methods, considering their different specificities and sensitivities. The possibility that the CBA-N2a assay can overestimate ciguatera risk needs to be investigated. Radiolabeled ligand binding assays can also be used to control fish toxicity and monitor toxic LC fractions of slow- and fast-acting C-CTXs [41,172,194]. However, the CBA-N2a assay was 12-fold more sensitive to detecting C-CTX-1 than the radiolabeled RBA [194]. It was also shown to be more sensitive than LC-MS/MS and fRBA using fluorescent brevetoxin (BODIPY®-PbTx-2) in a comparative study on P-CTXs from *Gambierdiscus polynesiensis* [83]. P-CTX-1 was 5–10-fold more potent than C-CTX-1 using RBA [172]; in agreement with the fact that P-CTX-1 was 14 times more toxic to mice than C-CTX-1 [38]. Thus, although it is no longer recommended by the European Food Safety Authority, the mouse bioassay could allow for the estimation of the relative toxicity of new analogues compared with C-CTX-1 or P-CTX-1 which are more accessible. This is important considering that differences in ADME processes may influence in vivo toxicity. To accelerate screening and address species differences in CTX sensitivity, Lewis et al. established a new FLIPR-based screen using human SH-SY5Y cells sensitised using ouabain and veratridine [81]. This assay demonstrated comparable sensitivity to the CBA-N2a assay, and could differentially detect MTX and CTX in extracts of *Gambierdiscus* spp. [81].
## 6.2. *Considerations for Analytical Approaches for C-CTX Detection and Quantification*

Significant impediments to the study of ciguatera include the scarcity of pure ciguatoxins standards [129,130] and the number of structural analogues contributing to the toxicity of hazardous fish [5, 113, 116, 129]. The lack of sufficient quantities of pure CTX standards impedes the development and harmonization of methods for their detection and public health protection. These difficulties are compounded by the commensurately low toxin thresholds for human poisoning and a sufficient sample pretreatment to reduce matrix interferences. Typically, ultra-trace analysis of lipophilic analytes (<1 ppb) is heavily influenced by the matrix involved, with biological matrices such as fish tissues often the most challenging. Although it is time-consuming and expensive, the only way to ensure potential biological activity of new C-CTX congeners is to carry out accurate chromatographic fractionation and to test for biological activity using adapted bioassays [118, 170, 195, 196, 198].

The selection of extraction and purification procedures depends on the specific objectives of the study. Given these diverse needs, a wide range of accelerated and comprehensive protocols have been developed, which are tailored to address specific matrix issues (e.g., fish tissue, microalgae, or blood). The desired outcomes include isolating reference materials, investigating fish toxin profiles, ensuring fish safety, or facilitating clinical diagnosis. Currently, no extraction protocol from biological matrix has been validated for CTXs, regardless of their origin. Various extraction and purification methodologies (see Section 4) are used for C-CTXs [118]. Currently, extraction procedures have been adapted from the method of Lewis [195] and the FDA method developed by Dickey [209]. Studies comparing the extraction of cooked and raw samples of flesh of C-CTX-containing fish have revealed only slight differences in toxin profiles and contents [23, 91, 104, 108, 131]. Therefore, within the fish flesh matrix, both slow and fast-acting ciguatoxins appear to be heat stable. However, particular attention should be paid during sample purification of certain fish species, such as moray eel and barracuda, due to the possible presence of fast-acting toxins (FATs). Some FATs have been characterised by specific symptoms in mice, and generally include more polar compounds than C-CTX-1 [38, 41, 104]. Potential FATs with molecular masses <900 Da, which are more comparable to the brevetoxins (850–900 Da) than known ciguatoxins (1100–1200 Da), were suspected to contribute to the total toxicity of a highly ciguatoxic barracuda [40, 41]. For this particular fish species, purification using Florisil® could be deleterious [104]. Additionally, losses of toxicity in the mouse bioassay observed during TSK gel filtration suggested that these losses were due to the disappearance of FATs [41]. The chemistry of the various chromatographic resins or solvents used is crucial for FAT stability. A consensus method should be established for the extraction and purification of C-CTXs from fish samples, considering the evaluation of pre-analytic efficiency and fish species-dependent matrix effects in mass spectrometry analyses. This will enable the comparison of studies on toxin profiles in ciguatoxic fish. Currently, several scientific groups in Europe and the USA are working on C-CTXs. Thus, an interlaboratory study could be useful in validating a unique protocol, or at least for the main stages of extraction and purification (e.g., the order of extraction and defatting or the type of SPE sorbents).

The chromatographic separation stage must be optimized to minimize the co-elution of the molecules of interest. The use of UHPLC systems and the availability of numerous stationary phases contributes to improving the resolution between analytes peaks, detection sensitivity, shorter analytical run times, and reduced solvent consumption [83]. Moreover, sub-2-µm solid-core particles could enable large increases in efficiency. However, it is crucial to implement an efficient preanalytical step to eliminate interfering substances present in fish flesh, such as lipids, which can affect sample loading, MS sensitivity, and column life. The most commonly used stationary phase is reversed-phase C18 (octadecylsilane), but some authors have also used a pentafluorophenyl-propyl stationary phase to significantly improve the separation of C-CTX-1/-2 and C-CTX-3/-4 peaks [120].

Since the 2010s, three methodological approaches in low-resolution LC-MS/MS using single (SRM) or multiple reaction monitoring (MRM) modes have been distinguished (Table 6). These approaches depend on the type and sensitivity of the mass spectrome-
## 6.2. *Considerations for Analytical Approaches for C-CTX Detection and Quantification*

Significant impediments to the study of ciguatera include the scarcity of pure ciguatoxins standards [129,130] and the number of structural analogues contributing to the toxicity of hazardous fish [5, 113, 116, 129]. The lack of sufficient quantities of pure CTX standards impedes the development and harmonization of methods for their detection and public health protection. These difficulties are compounded by the commensurately low toxin thresholds for human poisoning and a sufficient sample pretreatment to reduce matrix interferences. Typically, ultra-trace analysis of lipophilic analytes (<1 ppb) is heavily influenced by the matrix involved, with biological matrices such as fish tissues often the most challenging. Although it is time-consuming and expensive, the only way to ensure potential biological activity of new C-CTX congeners is to carry out accurate chromatographic fractionation and to test for biological activity using adapted bioassays [118, 170, 195, 196, 198].

The selection of extraction and purification procedures depends on the specific objectives of the study. Given these diverse needs, a wide range of accelerated and comprehensive protocols have been developed, which are tailored to address specific matrix issues (e.g., fish tissue, microalgae, or blood). The desired outcomes include isolating reference materials, investigating fish toxin profiles, ensuring fish safety, or facilitating clinical diagnosis. Currently, no extraction protocol from biological matrix has been validated for CTXs, regardless of their origin. Various extraction and purification methodologies (see Section 4) are used for C-CTXs [118]. Currently, extraction procedures have been adapted from the method of Lewis [195] and the FDA method developed by Dickey [209]. Studies comparing the extraction of cooked and raw samples of flesh of C-CTX-containing fish have revealed only slight differences in toxin profiles and contents [23, 91, 104, 108, 131]. Therefore, within the fish flesh matrix, both slow and fast-acting ciguatoxins appear to be heat stable. However, particular attention should be paid during sample purification of certain fish species, such as moray eel and barracuda, due to the possible presence of fast-acting toxins (FATs). Some FATs have been characterised by specific symptoms in mice, and generally include more polar compounds than C-CTX-1 [38, 41, 104]. Potential FATs with molecular masses <900 Da, which are more comparable to the brevetoxins (850–900 Da) than known ciguatoxins (1100–1200 Da), were suspected to contribute to the total toxicity of a highly ciguatoxic barracuda [40, 41]. For this particular fish species, purification using Florisil® could be deleterious [104]. Additionally, losses of toxicity in the mouse bioassay observed during TSK gel filtration suggested that these losses were due to the disappearance of FATs [41]. The chemistry of the various chromatographic resins or solvents used is crucial for FAT stability. A consensus method should be established for the extraction and purification of C-CTXs from fish samples, considering the evaluation of pre-analytic efficiency and fish species-dependent matrix effects in mass spectrometry analyses. This will enable the comparison of studies on toxin profiles in ciguatoxic fish. Currently, several scientific groups in Europe and the USA are working on C-CTXs. Thus, an interlaboratory study could be useful in validating a unique protocol, or at least for the main stages of extraction and purification (e.g., the order of extraction and defatting or the type of SPE sorbents).

The chromatographic separation stage must be optimized to minimize the co-elution of the molecules of interest. The use of UHPLC systems and the availability of numerous stationary phases contributes to improving the resolution between analytes peaks, detection sensitivity, shorter analytical run times, and reduced solvent consumption [83]. Moreover, sub-2-µm solid-core particles could enable large increases in efficiency. However, it is crucial to implement an efficient preanalytical step to eliminate interfering substances present in fish flesh, such as lipids, which can affect sample loading, MS sensitivity, and column life. The most commonly used stationary phase is reversed-phase C18 (octadecylsilane), but some authors have also used a pentafluorophenyl-propyl stationary phase to significantly improve the separation of C-CTX-1/-2 and C-CTX-3/-4 peaks [120].

Since the 2010s, three methodological approaches in low-resolution LC-MS/MS using single (SRM) or multiple reaction monitoring (MRM) modes have been distinguished (Table 6). These approaches depend on the type and sensitivity of the mass spectrome-
ter, as well as the intended purpose, such as quantifying C-CTX-1 with a purified standard or identifying C-CTXs based on their MS characteristic pattern of ion formation. Currently, for sensitive C-CTX-1 quantification, a water/methanol gradient is combined with a SRM of the prominent adduct [M+Na]$^+$ as precursor and product ions, using an ESI-triple quadrupole [109,110,112,155,175,176], which has been previously used for P-CTXs [30,210]. Confirmation of C-CTX-1 and search for C-CTX analogues are performed using a water/acetonitrile gradient and MRM using the most prominent peak of each analogue [M+Na]$^+$, [M+H–H₂O], [M+H]$^+$, or [M+H–CH₃–H₂O]$^+$, and serially dehydrated ions [110,175,176]. However, a comparison with the approach based on SRM of sodium adduct as precursor and product ions [30] showed that using an ESI-triple quadrupole/linear ion trap (API 4000, Sciex) and a methanol-based mobile phase, better selectivity through confirmatory transitions and better sensitivity with less matrix effects allowed for the detection of P-CTX-1 and P-CTX-3C by choosing [M+NH₄]$^+$ and [M+H]$^+$ ion precursors, respectively, and [M+H–2H₂O]$^+$ as product ions [211].

<table><caption>**Table 6.** Summary of the main analytical conditions used for the confirmation and quantification of C-CTX-1 and the screening of new/putative CTX analogues using (U)HPLC-MS/MS in multiple reaction monitoring, as detailed in Part 5.</caption><thead><tr><th>Review Section</th><th>Organic Eluent</th><th>Eluent Modifier</th><th>Goal (MS Mode)</th><th>Precursor Ions</th><th>References</th></tr></thead><tbody><tr><td>5.2.2</td><td>Methanol</td><td>Formic acid and ammonium formate</td><td>C-CTX-1 quantification (SRM)</td><td>[M+Na]$^+$</td><td>[109,110,112,155, 175,176]</td></tr><tr><td rowspan="2">5.2.3</td><td rowspan="2">Acetonitrile</td><td rowspan="2">Formic acid or ammonium formate or ammonium acetate</td><td>C-CTX-1 confirmation (MRM)</td><td>[M+H–H₂O]$^+$</td><td>[37,39,108,174]</td></tr><tr><td>C-CTXs analogues identification (MRM)</td><td>[M+Na]$^+$; [M+H–H₂O]; [M+H]$^+$; [M+H–CH₃–H₂O]$^+$</td><td>[110,175,176]</td></tr><tr><td rowspan="2">5.2.4</td><td rowspan="2">Methanol and acetonitrile (3:1)</td><td rowspan="2">Formic acid and ammonium acetate</td><td>4 CTX groups identification (MRM)</td><td>[M+Na]$^+$</td><td rowspan="2">[34,180]</td></tr><tr><td>CTXs confirmation (MRM)</td><td>[M+NH₄]$^+$</td></tr></tbody></table>

A third approach was developed for multi-analytes screening of CTXs sorted into four groups: CTX-4A and CTX3C groups (for P-CTXs), a C-CTX group, and an I-CTX, by monitoring sodium adducts separated with a water/methanol/acetonitrile gradient [180]. MS confirmation was achieved by monitoring four predicted MRM transitions per CTX and choosing the ammonium adduct [M+NH₄]$^+$ as the precursor ion and the corresponding [M+H]$^+$ and [M+H–nH₂O]$^+$ with *n* = 1–3 as product ions [180]. This approach allowed for the relative contribution of the identified ciguatoxins from the CTX3C group to be compared using LC-MS/MS in 52 tissue samples of *Lutjanus bohar* [34]. This approach could be extended to species-specific CTX metabolism and inter-oceanic CTX toxin profile analyses. Indeed, analytical validation performed on three different fish genera (*Lutjanus*, *Scarus*, and *Epinephelus*) also highlighted quantitative species-specific differences in MS responses, suggesting fish-specific matrix effects, which is a major problem for quantification from different matrices [180]. Thus, another promising perspective is the synthesis of the ¹⁸O-labeled C-CTX-1/2, C-CTX-3/4, gambierone, and 44-methylgambierone quantified by Mudge et al. [132]. Importantly, isotope-labeled standards allow for isotope dilution mass spectrometry and gold-standard correction for matrix effects and analyte losses during sample preparation.

In addition to the targeted mass spectrometry approach using (U)HPLC-MS/MS, high-resolution mass spectrometry (HRMS) represents a promising advance for the structural identification of marine biotoxins by improving the accuracy of assigning chemical formulas [212]. However, HRMS instruments such as the Quadrupole Time-of-Flight (QTOF) and Q-Orbitrap are expensive and can generate large amounts of complex data, which is not always convenient for routine analysis. Moreover, HRMS is usually less sensitive than targeted LC-MS/MS and is insufficient to quantify CTX levels in the sub-ppb range. In
ter, as well as the intended purpose, such as quantifying C-CTX-1 with a purified standard or identifying C-CTXs based on their MS characteristic pattern of ion formation. Currently, for sensitive C-CTX-1 quantification, a water/methanol gradient is combined with a SRM of the prominent adduct [M+Na]$^+$ as precursor and product ions, using an ESI-triple quadrupole [109,110,112,155,175,176], which has been previously used for P-CTXs [30,210]. Confirmation of C-CTX-1 and search for C-CTX analogues are performed using a water/acetonitrile gradient and MRM using the most prominent peak of each analogue [M+Na]$^+$, [M+H–H₂O], [M+H]$^+$, or [M+H–CH₃–H₂O]$^+$, and serially dehydrated ions [110,175,176]. However, a comparison with the approach based on SRM of sodium adduct as precursor and product ions [30] showed that using an ESI-triple quadrupole/linear ion trap (API 4000, Sciex) and a methanol-based mobile phase, better selectivity through confirmatory transitions and better sensitivity with less matrix effects allowed for the detection of P-CTX-1 and P-CTX-3C by choosing [M+NH₄]$^+$ and [M+H]$^+$ ion precursors, respectively, and [M+H–2H₂O]$^+$ as product ions [211].

<table><caption>**Table 6.** Summary of the main analytical conditions used for the confirmation and quantification of C-CTX-1 and the screening of new/putative CTX analogues using (U)HPLC-MS/MS in multiple reaction monitoring, as detailed in Part 5.</caption><thead><tr><th>Review Section</th><th>Organic Eluent</th><th>Eluent Modifier</th><th>Goal (MS Mode)</th><th>Precursor Ions</th><th>References</th></tr></thead><tbody><tr><td>5.2.2</td><td>Methanol</td><td>Formic acid and ammonium formate</td><td>C-CTX-1 quantification (SRM)</td><td>[M+Na]$^+$</td><td>[109,110,112,155, 175,176]</td></tr><tr><td rowspan="2">5.2.3</td><td rowspan="2">Acetonitrile</td><td rowspan="2">Formic acid or ammonium formate or ammonium acetate</td><td>C-CTX-1 confirmation (MRM)</td><td>[M+H–H₂O]$^+$</td><td>[37,39,108,174]</td></tr><tr><td>C-CTXs analogues identification (MRM)</td><td>[M+Na]$^+$; [M+H–H₂O]; [M+H]$^+$; [M+H–CH₃–H₂O]$^+$</td><td>[110,175,176]</td></tr><tr><td rowspan="2">5.2.4</td><td rowspan="2">Methanol and acetonitrile (3:1)</td><td rowspan="2">Formic acid and ammonium acetate</td><td>4 CTX groups identification (MRM)</td><td>[M+Na]$^+$</td><td rowspan="2">[34,180]</td></tr><tr><td>CTXs confirmation (MRM)</td><td>[M+NH₄]$^+$</td></tr></tbody></table>

A third approach was developed for multi-analytes screening of CTXs sorted into four groups: CTX-4A and CTX3C groups (for P-CTXs), a C-CTX group, and an I-CTX, by monitoring sodium adducts separated with a water/methanol/acetonitrile gradient [180]. MS confirmation was achieved by monitoring four predicted MRM transitions per CTX and choosing the ammonium adduct [M+NH₄]$^+$ as the precursor ion and the corresponding [M+H]$^+$ and [M+H–nH₂O]$^+$ with *n* = 1–3 as product ions [180]. This approach allowed for the relative contribution of the identified ciguatoxins from the CTX3C group to be compared using LC-MS/MS in 52 tissue samples of *Lutjanus bohar* [34]. This approach could be extended to species-specific CTX metabolism and inter-oceanic CTX toxin profile analyses. Indeed, analytical validation performed on three different fish genera (*Lutjanus*, *Scarus*, and *Epinephelus*) also highlighted quantitative species-specific differences in MS responses, suggesting fish-specific matrix effects, which is a major problem for quantification from different matrices [180]. Thus, another promising perspective is the synthesis of the ¹⁸O-labeled C-CTX-1/2, C-CTX-3/4, gambierone, and 44-methylgambierone quantified by Mudge et al. [132]. Importantly, isotope-labeled standards allow for isotope dilution mass spectrometry and gold-standard correction for matrix effects and analyte losses during sample preparation.

In addition to the targeted mass spectrometry approach using (U)HPLC-MS/MS, high-resolution mass spectrometry (HRMS) represents a promising advance for the structural identification of marine biotoxins by improving the accuracy of assigning chemical formulas [212]. However, HRMS instruments such as the Quadrupole Time-of-Flight (QTOF) and Q-Orbitrap are expensive and can generate large amounts of complex data, which is not always convenient for routine analysis. Moreover, HRMS is usually less sensitive than targeted LC-MS/MS and is insufficient to quantify CTX levels in the sub-ppb range. In
the study of Sibat et al., HRMS sensitivity was 2- to 12-fold lower for P-CTXs, but with an increased mass accuracy, the presence of more concentrated P-CTX analogues was confirmed, and interferences determined by co-eluting compounds was also identified [211]. Some LC-HRMS studies have confirmed the presence of targeted C-CTX-1 when reference materials are available [113,120,131,204,205] and have provided structural information on new C-CTX analogues [113,120,131,205]. LC-HRMS remains a complementary technique. Due to its cost and complexity, utilizing this method requires significant investments and technical skills, making it inaccessible to many Caribbean countries where the control of ciguateric fishes is necessary.

### 6.3. Multiplicity of Caribbean Ciguatoxins

A key point emerging from the literature is the presence of multiple Caribbean ciguatoxins in fish from the Atlantic Ocean and Caribbean Sea. This multiplicity, reminiscent of P-CTX multiplicity [33,119], could be due to strain-dependent variations in toxin content of dinoflagellates belonging to the genus *Gambierdiscus*, as well as differences in the metabolic activities of fish, especially in their microsomal detoxication capacities including glucuronidation [165]. For decades, research has focused on ciguatera fish poisoning in the Lesser and Greater Antilles, as well as Southern Florida. Since 2004, fish caught in the Northeast Atlantic (Canary Islands and Archipelago of Madeira) have been associated with ciguatera-type poisoning. So far, no P-CTXs have been identified in fish from this new ciguatera area. To date, C-CTX-1 (C<sub>62</sub>H<sub>92</sub>O<sub>19</sub>, 1140.6 Da) and occasionally its isomer C-CTX-2 have been found in fish from the Caribbean Sea ([22,23,35,37-39,41,42,108,172,174,200], and now from the Canaries Islands and Madeira Archipelago [109,110,112,130,131,155,175,176].

Only C-CTX-1 (1140.6 Da) and one of its isomers, C-CTX-2, have been structurally characterized using NMR. Their in vivo toxicity in mice is known, with lethal doses of 3.6 and 1 µg·kg<sup>-1</sup>, respectively, and their mode of action has been demonstrated. They target receptor site 5 of voltage-gated sodium channels. Few studies relating to the toxin concentrations of C-CTX-1 have been carried out with a calibration, using a standard of C-CTX-, due to limited amounts of C-CTX-1-purified material [23,41,42,131,200]. Lewis at al. estimated that ~50% of the toxicity determined using the MBA was explained by the presence of C-CTX-1 in 30 fish from the Caribbean [200]. Thus, C-CTX-1 may not be the only biomarker that can confirm the presence of C-CTXs in fish flesh, as C-CTX-1 and other C-CTX analogues have been detected in fish from the Lesser Antilles [23,37-39,41,42,108] or the Northeast Atlantic ocean [109,110,131,175]. By using HRMS combined with chemical reactions, some C-CTX congeners have been tentatively characterised (see Section 5.3.1). The congeners C-CTX-1159, C-CTX-3/-4, C-CTX-1157, and C-CTX-1127, were identified in fractions that showed toxic activity either using CBA-N2a or RBAs (Table 7). However, due to their similar structures, they are difficult to separate in reverse-phase LC, posing a challenge in confirming their toxicity with certainty.

Finally, among the analogues, two groups of C-CTX congeners have been recurrently reported in fish from the Atlantic zone:

* Compounds with [M+H]<sup>+</sup> at *m/z* 1143.6: isomers previously known as C-CTX-1143 showed no significant activity in radio-ligand binding assays [23,41]. However, RBA may not be sensitive enough to detect low amounts in fish, and another study identified an analogue, C-CTX-1143, using LC-MS/MS in a positive fraction by CBA-N2a [108]. Two anomers, C-CTX-3 and C-CTX-4 (C<sub>62</sub>H<sub>94</sub>O<sub>19</sub>), have been characterised using HRMS as the reduced forms of C-CTX-1 [120,131]. Their toxic potential and their role in fish total toxicity are yet to be clearly demonstrated due to their unstable hemiketal structure.

* Compounds with [M+H]<sup>+</sup> at *m/z* 1157.6: two compounds with [M+H]<sup>+</sup> at *m/z* 1157.6 were recently identified using LC-HRMS, 17-hydroxy-C-CTX-1 ([M+H]<sup>+</sup> *m/z* 1157.6255) with a possible location of the -OH group in the E-ring, and putative 17-hydroxy-50,51-didehydro-C-CTX-3 ([M+H]<sup>+</sup> *m/z* 1157.6255) [131]. An analogue C-CTX-1157 was also
the study of Sibat et al., HRMS sensitivity was 2- to 12-fold lower for P-CTXs, but with an increased mass accuracy, the presence of more concentrated P-CTX analogues was confirmed, and interferences determined by co-eluting compounds was also identified [211]. Some LC-HRMS studies have confirmed the presence of targeted C-CTX-1 when reference materials are available [113,120,131,204,205] and have provided structural information on new C-CTX analogues [113,120,131,205]. LC-HRMS remains a complementary technique. Due to its cost and complexity, utilizing this method requires significant investments and technical skills, making it inaccessible to many Caribbean countries where the control of ciguateric fishes is necessary.

### 6.3. Multiplicity of Caribbean Ciguatoxins

A key point emerging from the literature is the presence of multiple Caribbean ciguatoxins in fish from the Atlantic Ocean and Caribbean Sea. This multiplicity, reminiscent of P-CTX multiplicity [33,119], could be due to strain-dependent variations in toxin content of dinoflagellates belonging to the genus *Gambierdiscus*, as well as differences in the metabolic activities of fish, especially in their microsomal detoxication capacities including glucuronidation [165]. For decades, research has focused on ciguatera fish poisoning in the Lesser and Greater Antilles, as well as Southern Florida. Since 2004, fish caught in the Northeast Atlantic (Canary Islands and Archipelago of Madeira) have been associated with ciguatera-type poisoning. So far, no P-CTXs have been identified in fish from this new ciguatera area. To date, C-CTX-1 ($C_{62}H_{92}O_{19}$, 1140.6 Da) and occasionally its isomer C-CTX-2 have been found in fish from the Caribbean Sea ([22,23,35,37–39,41,42,108,172,174,200], and now from the Canaries Islands and Madeira Archipelago [109,110,112,130,131,155,175,176]).

Only C-CTX-1 (1140.6 Da) and one of its isomers, C-CTX-2, have been structurally characterized using NMR. Their in vivo toxicity in mice is known, with lethal doses of 3.6 and 1 µg·kg⁻¹, respectively, and their mode of action has been demonstrated. They target receptor site 5 of voltage-gated sodium channels. Few studies relating to the toxin concentrations of C-CTX-1 have been carried out with a calibration, using a standard of C-CTX-, due to limited amounts of C-CTX-1-purified material [23,41,42,131,200]. Lewis at al. estimated that ~50% of the toxicity determined using the MBA was explained by the presence of C-CTX-1 in 30 fish from the Caribbean [200]. Thus, C-CTX-1 may not be the only biomarker that can confirm the presence of C-CTXs in fish flesh, as C-CTX-1 and other C-CTX analogues have been detected in fish from the Lesser Antilles [23,37–39,41,42,108] or the Northeast Atlantic ocean [109,110,131,175]. By using HRMS combined with chemical reactions, some C-CTX congeners have been tentatively characterised (see Section 5.3.1). The congeners C-CTX-1159, C-CTX-3/-4, C-CTX-1157, and C-CTX-1127, were identified in fractions that showed toxic activity either using CBA-N2a or RBAs (Table 7). However, due to their similar structures, they are difficult to separate in reverse-phase LC, posing a challenge in confirming their toxicity with certainty.

Finally, among the analogues, two groups of C-CTX congeners have been recurrently reported in fish from the Atlantic zone:

* Compounds with $[M+H]⁺$ at *m/z* 1143.6: isomers previously known as C-CTX-1143 showed no significant activity in radio-ligand binding assays [23,41]. However, RBA may not be sensitive enough to detect low amounts in fish, and another study identified an analogue, C-CTX-1143, using LC-MS/MS in a positive fraction by CBA-N2a [108]. Two anomers, C-CTX-3 and C-CTX-4 ($C_{62}H_{94}O_{19}$), have been characterised using HRMS as the reduced forms of C-CTX-1 [120,131]. Their toxic potential and their role in fish total toxicity are yet to be clearly demonstrated due to their unstable hemiketal structure.

* Compounds with $[M+H]⁺$ at *m/z* 1157.6: two compounds with $[M+H]⁺$ at *m/z* 1157.6 were recently identified using LC-HRMS, 17-hydroxy-C-CTX-1 ($[M+H]⁺$ *m/z* 1157.6255) with a possible location of the -OH group in the E-ring, and putative 17-hydroxy-50,51-didehydro-C-CTX-3 ($[M+H]⁺$ *m/z* 1157.6255) [131]. An analogue C-CTX-1157 was also
reported in a CTX-positive fraction analysed using CBA-N2a. [108] and in a positive fraction analysed using RBA (unpublished data from [40]).

<table><caption>**Table 7.** Caribbean ciguatoxins analogues (C-CTXs) identified in fish from the Caribbean Sea and Macaronesia. Published pseudomolecular ion $[M+H]^{+} m/z$, polarity, potential toxicity using radio-ligand binding assays, or neuroblastoma cell-based assay of the fraction containing C-CTX and putative structure.</caption><thead><tr><th>Name</th><th>C-CTX-1159</th><th>C-CTX-3/-4 [120]</th><th>C-CTX-1157</th><th>C-CTX-1127</th></tr></thead><tbody><tr><td>[M+H]<sup>+</sup> m/z</td><td>1159.6</td><td>1143.6</td><td>1157.6</td><td>1127.6</td></tr><tr><td>Polarity *</td><td>+</td><td>—</td><td>+</td><td>—</td></tr><tr><td>Reported in Caribbean fish</td><td>[42,108]</td><td>[23,38,41,42,108,120]</td><td>[23,38,41,42,108]</td><td>[23,41,42]</td></tr><tr><td>Reported in fish from Macaronesia</td><td>[110]</td><td>[131]</td><td>[175,176]</td><td>[113,175]</td></tr><tr><td>Potential toxicity</td><td>[108,110]</td><td>[108]</td><td>[40,108]</td><td>[175]</td></tr><tr><td>Putative structure</td><td>Hydroxylated C-CTX-1 or oxidised C-CTX-1143</td><td>reduced C-CTX-1/-2 hemiketals [120]</td><td>Oxidised C-CTX-1</td><td>Loss of CH<sub>2</sub> or demethylated C-CTX-1</td></tr></tbody></table>

C-CTX profiles may vary depending on the fish species and their regional distribution. A notable observation from studying toxin profiles [23,41,42,108,131] is that in the most toxic fish, at the limit of edibility or inedible, C-CTX-1 appears to be predominant. Among the 12 samples of *Caranx latus*, the highest relative levels of C-CTX-1 (~75–90%) were observed in low toxic specimens. Their slight losses of activity during the purification stage indicated that unstable CTXs were not present [42]. In contrast, highly toxic horse-eye jacks had relatively low levels of C-CTX-1 (~50%) and were subject to larger toxicity losses upon purification, indicating that relatively high levels of unstable CTXs were present [42]. In a highly ciguotoxic barracuda from Guadeloupe, two distinct fractions were purified depending on symptomatology in mice. C-CTX-1 accounted for ~90% of the total toxicity (49 ppb in first fraction). However, despite important losses in toxicity (>60%) during purification of the second fraction, peak of sodium channel activity was identified, and two pseudo-molecular ions at *m/z* 809.43 and *m/z* 857.42 were detected using LC-MS. In a barracuda associated with a CP, C-CTX-1 contributed to ~60% of the toxicity found using CBA-N2a [108]. In fish from Macaronesia, C-CTX-1 was detected in the CTX-like active fractions exhibiting the highest toxicity [130,175], which represented more than 60% of the total toxin content (0.16 to 0.56 ppb), followed by 17-hydroxy-C-CTX-1 (0.11 to 0.23 ppb) in four fish [131]. However, these results must be analysed with caution, as these studies mostly evaluated variable percentages of congeners (or groups of isomers with the same molecular weight) depending on the fish and their total toxicity [42]. Thus, the relative percentages considered all the putative C-CTXs identified through LC-MS or LC-MS/MS analyses, whereas the data on their in vitro and/or in vivo toxicity compared to that of C-CTX-1 are still lacking.

Finally, the recent discovery by Mudge et al. of C-CTX-5 (1138.6 Da) is a significant step towards an understanding of chemical ciguatera biogenesis [43]. This new algal toxin related to C-CTX-1 was found in isolates of dinoflagellates collected in St. Thomas in the U. S. Virgin Islands, a geographical area at risk in the Caribbean Sea [43]. C-CTX-5 is likely to be half as potent as C-CTX-1 based on in vitro CBA-N2a. It was demonstrated that metabolization by liver microsomes of an omnivorous fish (sheepshead *Archosargus probatocephalus*) at the C3 position produces C-CTX-1/-2 [43]. In addition, *G. silvae* appears to be capable of producing low levels of C-CTX-1 (~7–8% of the level of C-CTX-5) [43]. Thus, *Gambierdiscus silvae* and *Gambierdiscus caribaeus* can be implicitly considered as algal producers of the Caribbean ciguatera food chain. To our knowledge, no analogue with $[M+H]^{+}$ at *m/z* 1139.6 has been reported in Atlantic fish. This research work [43] offers

<footnote>* in terms of polarity, C-CTX-1 is before C-CTX-1127.</footnote>
reported in a CTX-positive fraction analysed using CBA-N2a. [108] and in a positive fraction analysed using RBA (unpublished data from [40]).

<table><caption>**Table 7.** Caribbean ciguatoxins analogues (C-CTXs) identified in fish from the Caribbean Sea and Macaronesia. Published pseudomolecular ion $[M+H]^{+} m/z$, polarity, potential toxicity using radio-ligand binding assays, or neuroblastoma cell-based assay of the fraction containing C-CTX and putative structure.</caption><thead><tr><th>Name</th><th>C-CTX-1159</th><th>C-CTX-3/-4 [120]</th><th>C-CTX-1157</th><th>C-CTX-1127</th></tr></thead><tbody><tr><td>[M+H]<sup>+</sup> m/z</td><td>1159.6</td><td>1143.6</td><td>1157.6</td><td>1127.6</td></tr><tr><td>Polarity *</td><td>+</td><td>—</td><td>+</td><td>—</td></tr><tr><td>Reported in Caribbean fish</td><td>[42,108]</td><td>[23,38,41,42,108,120]</td><td>[23,38,41,42,108]</td><td>[23,41,42]</td></tr><tr><td>Reported in fish from Macaronesia</td><td>[110]</td><td>[131]</td><td>[175,176]</td><td>[113,175]</td></tr><tr><td>Potential toxicity</td><td>[108,110]</td><td>[108]</td><td>[40,108]</td><td>[175]</td></tr><tr><td>Putative structure</td><td>Hydroxylated C-CTX-1 or oxidised C-CTX-1143</td><td>reduced C-CTX-1/-2 hemiketals [120]</td><td>Oxidised C-CTX-1</td><td>Loss of CH<sub>2</sub> or demethylated C-CTX-1</td></tr></tbody></table>

C-CTX profiles may vary depending on the fish species and their regional distribution. A notable observation from studying toxin profiles [23,41,42,108,131] is that in the most toxic fish, at the limit of edibility or inedible, C-CTX-1 appears to be predominant. Among the 12 samples of *Caranx latus*, the highest relative levels of C-CTX-1 (~75–90%) were observed in low toxic specimens. Their slight losses of activity during the purification stage indicated that unstable CTXs were not present [42]. In contrast, highly toxic horse-eye jacks had relatively low levels of C-CTX-1 (~50%) and were subject to larger toxicity losses upon purification, indicating that relatively high levels of unstable CTXs were present [42]. In a highly ciguotoxic barracuda from Guadeloupe, two distinct fractions were purified depending on symptomatology in mice. C-CTX-1 accounted for ~90% of the total toxicity (49 ppb in first fraction). However, despite important losses in toxicity (>60%) during purification of the second fraction, peak of sodium channel activity was identified, and two pseudo-molecular ions at *m/z* 809.43 and *m/z* 857.42 were detected using LC-MS. In a barracuda associated with a CP, C-CTX-1 contributed to ~60% of the toxicity found using CBA-N2a [108]. In fish from Macaronesia, C-CTX-1 was detected in the CTX-like active fractions exhibiting the highest toxicity [130,175], which represented more than 60% of the total toxin content (0.16 to 0.56 ppb), followed by 17-hydroxy-C-CTX-1 (0.11 to 0.23 ppb) in four fish [131]. However, these results must be analysed with caution, as these studies mostly evaluated variable percentages of congeners (or groups of isomers with the same molecular weight) depending on the fish and their total toxicity [42]. Thus, the relative percentages considered all the putative C-CTXs identified through LC-MS or LC-MS/MS analyses, whereas the data on their in vitro and/or in vivo toxicity compared to that of C-CTX-1 are still lacking.

Finally, the recent discovery by Mudge et al. of C-CTX-5 (1138.6 Da) is a significant step towards an understanding of chemical ciguatera biogenesis [43]. This new algal toxin related to C-CTX-1 was found in isolates of dinoflagellates collected in St. Thomas in the U. S. Virgin Islands, a geographical area at risk in the Caribbean Sea [43]. C-CTX-5 is likely to be half as potent as C-CTX-1 based on in vitro CBA-N2a. It was demonstrated that metabolization by liver microsomes of an omnivorous fish (sheepshead *Archosargus probatocephalus*) at the C3 position produces C-CTX-1/-2 [43]. In addition, *G. silvae* appears to be capable of producing low levels of C-CTX-1 (~7–8% of the level of C-CTX-5) [43]. Thus, *Gambierdiscus silvae* and *Gambierdiscus caribaeus* can be implicitly considered as algal producers of the Caribbean ciguatera food chain. To our knowledge, no analogue with $[M+H]^{+}$ at *m/z* 1139.6 has been reported in Atlantic fish. This research work [43] offers

<footnote>* in terms of polarity, C-CTX-1 is before C-CTX-1127.</footnote>
new perspectives for studying the dynamics of benthic *Gambierdiscus* CTX-producers and their conditions of production of C-CTX-5 and small amounts of C-CTX-1, as well as for the production of analytical standards based on C-CTX-5 using chemical or enzymatic reactions. It also enables the study of the C-CTXs dynamics during the Caribbean food chain transmission.

## 7. Conclusions

The recent finding that C-CTX-5, produced by *G. silvae* and *G. caribeus*, is the likely algal precursor of fish C-CTX and undergoes biotransformation to C-CTX-1/-2 in fish liver microsomes fills a significant knowledge gap in understanding the origins of Caribbean C-CTXs. This finding provides a coherent link to C-CTX-1, which typically dominates ciguatoxic fish from the Caribbean. Importantly, C-CTX-5 from *G. silvae* was confirmed to be a voltage-gated sodium channel activator, indicating its potential to contribute to ciguatera.

Despite these advances, the relative potencies of new Caribbean ciguatoxins still remains to be determined using pure and/or calibrated standards. However, until these become available, an estimation of total CTX activity based on voltage-gated sodium channel activity (ideally human sodium channels) should be used to quantify ciguatera risk when multiple contaminating congeners are present. Recent developments in LC-MS/MS allow for confirmation of C-CTXs in fish from the Caribbean Sea and Northeast Atlantic. Quantitation of C-CTX-1 is dependent on the availability of reference materials, which continue to limit analytical approaches, including the development of new extraction and purification procedures. The recent use of high-resolution mass spectrometry also provides an important contribution to chemical and toxin profile analysis in fish and dinoflagellates. However, it is not appropriate for public health controls, especially in small, under-resourced islands where ciguatera is a major public health problem. The lack of commercially available certified C-CTX reference materials is currently the most critical concern. This constitutes the main difficulty in developing an official LC-MS monitoring program for Caribbean CTXs.

Finally, since the first studies in the 1950s, ciguatera researchers initially investigated the origins, distribution, chemistry and detection of P-CTXs. Despite significant, more recent progress, Caribbean ciguatoxins remain less understood, in part due to the multiplicity and variability of the congeners involved. This is reinforced by the diversity of associated vectors, dinoflagellates, and fish species at risk, and by the variability of ciguatoxic effects and responses in humans. This complexity likely reflects differences in food chain-associated detoxification mechanisms, which are only now being studied. However, despite this complexity, the clinical features of Caribbean ciguatera have remained constant over the last 50 years, supporting the view that appropriate toxicity measures can accurately reflect ciguatera risk.

To conclude, the methodological approach used for monitoring ciguatoxins usually includes two-tiers, with a first tier using a biological assay screen and the second tier using analytical methods. Protocols that have been developed for the identification and/or quantification of CTXs were compared in this review, including basic (HPLC-MS) to more sophisticated (UHPLC-MS/MS or HRMS) approaches. Each approach has advantages, disadvantages, and contributions to the field discussed. However, to control fish toxicity within a public health framework, alternative bioassays can more simply account for the multiplicity of similar toxins that often contribute to human poisoning. This includes, for example, the use of the mouse bioassay to control the shellfish toxins (50 lipophilic toxins with a common unique backbone) responsible for paralytic shellfish poisoning (PSP), which was the reference biological method validated by the AOAC (Association of Official Analytical Chemists) [213] and used in Europe until January 2019 [214–217]. Recently, the MBA was replaced by the HPLC-fluorescence method developed by Lawrence et al., which is now the official reference method for PSP detection in the European Union [217]. Thus, presently, liquid chromatography (LC) coupled with mass spectrometry (MS) appears
new perspectives for studying the dynamics of benthic *Gambierdiscus* CTX-producers and their conditions of production of C-CTX-5 and small amounts of C-CTX-1, as well as for the production of analytical standards based on C-CTX-5 using chemical or enzymatic reactions. It also enables the study of the C-CTXs dynamics during the Caribbean food chain transmission.

## 7. Conclusions

The recent finding that C-CTX-5, produced by *G. silvae* and *G. caribeus*, is the likely algal precursor of fish C-CTX and undergoes biotransformation to C-CTX-1/-2 in fish liver microsomes fills a significant knowledge gap in understanding the origins of Caribbean C-CTXs. This finding provides a coherent link to C-CTX-1, which typically dominates ciguatoxic fish from the Caribbean. Importantly, C-CTX-5 from *G. silvae* was confirmed to be a voltage-gated sodium channel activator, indicating its potential to contribute to ciguatera.

Despite these advances, the relative potencies of new Caribbean ciguatoxins still remains to be determined using pure and/or calibrated standards. However, until these become available, an estimation of total CTX activity based on voltage-gated sodium channel activity (ideally human sodium channels) should be used to quantify ciguatera risk when multiple contaminating congeners are present. Recent developments in LC-MS/MS allow for confirmation of C-CTXs in fish from the Caribbean Sea and Northeast Atlantic. Quantitation of C-CTX-1 is dependent on the availability of reference materials, which continue to limit analytical approaches, including the development of new extraction and purification procedures. The recent use of high-resolution mass spectrometry also provides an important contribution to chemical and toxin profile analysis in fish and dinoflagellates. However, it is not appropriate for public health controls, especially in small, under-resourced islands where ciguatera is a major public health problem. The lack of commercially available certified C-CTX reference materials is currently the most critical concern. This constitutes the main difficulty in developing an official LC-MS monitoring program for Caribbean CTXs.

Finally, since the first studies in the 1950s, ciguatera researchers initially investigated the origins, distribution, chemistry and detection of P-CTXs. Despite significant, more recent progress, Caribbean ciguatoxins remain less understood, in part due to the multiplicity and variability of the congeners involved. This is reinforced by the diversity of associated vectors, dinoflagellates, and fish species at risk, and by the variability of ciguatoxic effects and responses in humans. This complexity likely reflects differences in food chain-associated detoxification mechanisms, which are only now being studied. However, despite this complexity, the clinical features of Caribbean ciguatera have remained constant over the last 50 years, supporting the view that appropriate toxicity measures can accurately reflect ciguatera risk.

To conclude, the methodological approach used for monitoring ciguatoxins usually includes two-tiers, with a first tier using a biological assay screen and the second tier using analytical methods. Protocols that have been developed for the identification and/or quantification of CTXs were compared in this review, including basic (HPLC-MS) to more sophisticated (UHPLC-MS/MS or HRMS) approaches. Each approach has advantages, disadvantages, and contributions to the field discussed. However, to control fish toxicity within a public health framework, alternative bioassays can more simply account for the multiplicity of similar toxins that often contribute to human poisoning. This includes, for example, the use of the mouse bioassay to control the shellfish toxins (50 lipophilic toxins with a common unique backbone) responsible for paralytic shellfish poisoning (PSP), which was the reference biological method validated by the AOAC (Association of Official Analytical Chemists) [213] and used in Europe until January 2019 [214–217]. Recently, the MBA was replaced by the HPLC-fluorescence method developed by Lawrence et al., which is now the official reference method for PSP detection in the European Union [217]. Thus, presently, liquid chromatography (LC) coupled with mass spectrometry (MS) appears
to be the preferred method of obtaining accurate toxin profile data for ciguatoxins, as it combines a qualitative structural approach and a quantitative analysis when standards are available. Analytical approaches also have merit in distinguishing between the three structural families of ciguatoxins, depending on the ocean. However, until standards are available for all significant forms, bioassays, including CBA-N2a and MBA, continue to play a key role in screening for ciguateric fishes.

**Author Contributions:** Conceptualization, J.-P.V.; writing—original draft preparation, I.P., R.J.L. and J.-P.V.; writing—review and editing, I.P., R.J.L. and J.-P.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

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8. Darius, H.T.; Roué, M.; Sibat, M.; Viallon, J.; Gatti, C.M.I.; Vandersea, M.W.; Tester, P.A.; Litaker, R.W.; Amzil, Z.; Hess, P.; et al. Toxicological Investigations on the Sea Urchin *Tripneustes Gratilla* (Toxopneustidae, Echinoid) from Anaho Bay (Nuku Hiva, French Polynesia): Evidence for the Presence of Pacific Ciguatoxins. *Mar. Drugs* **2018**, *16*, 122. [CrossRef]

9. Mak, Y.L.; Wai, T.-C.; Murphy, M.B.; Chan, W.H.; Wu, J.J.; Lam, J.C.W.; Chan, L.L.; Lam, P.K.S. Pacific Ciguatoxins in Food Web Components of Coral Reef Systems in the Republic of Kiribati. *Environ. Sci. Technol.* **2013**, *47*, 14070–14079. [CrossRef]

10. Rongo, T.; van Woesik, R. Ciguatera Poisoning in Rarotonga, Southern Cook Islands. *Harmful Algae* **2011**, *10*, 345–355. [CrossRef]

11. Roué, M.; Darius, H.T.; Picot, S.; Ung, A.; Viallon, J.; Gaertner-Mazouni, N.; Sibat, M.; Amzil, Z.; Chinain, M. Evidence of the Bioaccumulation of Ciguatoxins in Giant Clams (*Tridacna Maxima*) Exposed to *Gambierdiscus* spp. Cells. *Harmful Algae* **2016**, *57*, 78–87. [CrossRef] [PubMed]

12. Silva, M.; Rodriguez, I.; Barreiro, A.; Kaufmann, M.; Neto, A.I.; Hassouani, M.; Sabour, B.; Alfonso, A.; Botana, L.M.; Vasconcelos, V. First Report of Ciguatoxins in Two Starfish Species: *Ophidiaster Ophidianus* and *Marthasterias Glacialis*. *Toxins* **2015**, *7*, 3740–3757. [CrossRef]

13. Bagnis, R.; Mazelier, P.; Bennett, J.; Christian, E. *Poissons de Polynésie*, 3rd ed.; Les Editions du Pacifique: Papeete, Tahiti, 1973.

14. Randall, J.E. *Caribbean Reef Fishes*, 3rd ed.; T.F.H. Publications, Inc. Ltd.: Hong Kong, China, 1996.

15. Vernoux, J.P.; Magras, M.; Magras, P. *Poissons Coralliens Des Antilles*, 1st ed.; Faune & flore des Tropiques; Les Edition du Latanier: Gustavia, Saint Barthelémy, 1988; ISBN 2-9502284-2-9. Available online: https://www.researchgate.net/publication/301295899_Poissons_coralliens_des_AntillesCoral_fishes_of_the_West_Indies_128_pages (accessed on 22 June 2023).

16. Lehane, L.; Lewis, R.J. Ciguatera: Recent Advances but the Risk Remains. *Int. J. Food Microbiol.* **2000**, *61*, 91–125. [CrossRef]

17. Mattei, C.; Vetter, I.; Eisenblätter, A.; Krock, B.; Ebbecke, M.; Desel, H.; Zimmermann, K. Ciguatera Fish Poisoning: A First Epidemic in Germany Highlights an Increasing Risk for European Countries. *Toxicon* **2014**, *91*, 76–83. [CrossRef] [PubMed]

18. Louis Malardé Institute French Polynesia Ciguawatch Initiative. Available online: https://ciguawatch.ilm.pf/fr/pacific-ciguatera-risk-map-and-declaration-system/ (accessed on 27 June 2023).

19. FAO and WHO. *Report of the Expert Meeting on Ciguatera Poisoning Rome, 19–23 November 2018; Food Safety and Quality, FAO and WHO: Rome, Italy, 2020; pp. 1–134.*
to be the preferred method of obtaining accurate toxin profile data for ciguatoxins, as it combines a qualitative structural approach and a quantitative analysis when standards are available. Analytical approaches also have merit in distinguishing between the three structural families of ciguatoxins, depending on the ocean. However, until standards are available for all significant forms, bioassays, including CBA-N2a and MBA, continue to play a key role in screening for ciguateric fishes.

**Author Contributions:** Conceptualization, J.-P.V.; writing—original draft preparation, I.P., R.J.L. and J.-P.V.; writing—review and editing, I.P., R.J.L. and J.-P.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

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52. Banner, A.H.; Helfrich, P. *The Distribution of Ciguatera in the Tropical Pacific*; Hawaii Marine Laboratory, University of Hawaii: Honolulu, HI, USA, 1964; pp. 1–48.

53. Hashimoto, Y.; Konosu, S.; Yasumoto, T.; Kamiya, H. Ciguatera in the Ryukyu and Amami Islands. *Nippon. Suisan Gakkaishi* **1969**, *35*, 316–326. [CrossRef]

54. Yasumoto, T.; Scheuer, P.J. Marine Toxins of the Pacific. 8. Ciguatoxin from Moray Eel Livers. *Toxicon Off. J. Int. Soc. Toxinol.* **1969**, *7*, 273–276. [CrossRef] [PubMed]

55. Randall, J.E. A Review of Ciguatera, Tropical Fish Poisoning, with a Tentative Explanation of Its Cause. *Bull. Mar. Sci.* **1958**, *8*, 236–267.

56. Randall, J.E. Food Habits of Reef Fishes of the West Indies. In *Proceedings of the Studies in Tropical Oceanography, Miami Beach, FL, USA*, 17–24 November 1965; University of Miami, Institute of Marine Sciences: Coral Gables, FL, USA, 1967; Volume 5, pp. 665–847.

57. Yasumoto, T.; Hashimoto, Y.; Bagnis, R.; Randall, J.E.; Banner, A.H. Toxicity of the Surgeonfishes. *Bull. Jpn. Soc. Sci. Fish.* **1971**, *37*, 724–734. [CrossRef]

58. Bagnis, R.; Vernoux, J.P. Ciguatoxin and edible reef fishes. *Bull. Soc. Pathol. Exot. Filiales* **1975**, *68*, 320–325.

59. Vernoux, J.P.; Bagnis, R. Fractionnement d'extraits lipidiques ciguatoxiques en milieu alcalin. *Biochimie* **1976**, *58*, 479–484. [CrossRef]

60. Chanteau, S.; Bagnis, R.; Yasumoto, T. Purification de la ciguatoxine de la loche *Epinephelus microdon* (Bleecker). *Biochimie* **1976**, *58*, 1149–1151. [CrossRef]

61. Chungue, E.; Bagnis, R.; Fusetani, N.; Hashimoto, Y. Isolation of Two Toxins from a Parrotfish *Scarus Gibbus*. *Toxicon* **1977**, *15*, 89–93. [CrossRef]

62. Vernoux, J.P. L'ichtyosarcotoxisme de Type Ciguatera Aux Antilles et En Polynésie Française: Test de Ciguatoxicité et Chaîne Trophique Ciguatérigène. Ph.D. Thesis, Univeristé Bordeaux I, Bordeaux, France, 1981.

63. Bagnis, R.; Chanteau, S.; Yasumoto, T. Significance of the various toxins present on dead coral substrata in the origin of ciguatera. *Bull. Soc. Pathol. Exot. Filiales* **1977**, *70*, 320–324.

64. Yasumoto, T.; Nakajima, I.; Bagnis, R.; Adachi, R. Finding of a Dinoflagellate as a Likely Culprit of Ciguatera. *Bull. Jpn. Soc. Sci. Fish.* **1977**, *43*, 1021–1026. [CrossRef]

65. Adachi, R.; Fukuyo, Y. The Thecal Structure of a Marine Toxic Dinoflagellate *Gambierdiscus Toxicus* Gen. et Sp. Nov. Collected in a Ciguatera-Endemic Area. *Bull. Jpn. Soc. Sci. Fish.* **1979**, *45*, 67–71. [CrossRef]

66. Bagnis, R.; Chanteau, S.; Chungue, E.; Hurtel, J.M.; Yasumoto, T.; Inoue, A. Origins of Ciguatera Fish Poisoning: A New Dinoflagellate, *Gambierdiscus Toxicus* Adachi and Fukuyo, Definitively Involved as a Causal Agent. *Toxicon* **1980**, *18*, 199–208. [CrossRef] [PubMed]

67. Yasumoto, T.; Bagnis, R.; P. Vernoux, J. Toxicity of the Surgeonfishes-II. *Bull. Jpn. Soc. Sci. Fish.* **1976**, *42*, 359–365. [CrossRef]

68. Vernoux, J.-P.; Lejeune, J. Ciguatera in the French West Indies. In *Memoirs of the Queensland Museum*; Queensland Museum: Brisbane, Australia, 1994; Volume 34, pp. 631–638.

69. Murata, M.; Naoki, H.; Iwashita, T.; Matsunaga, S.; Sasaki, M.; Yokoyama, A.; Yasumoto, T. Structure of Maitotoxin. *J. Am. Chem. Soc.* **1993**, *115*, 2060–2062. [CrossRef]

70. Zheng, W.; DeMattei, J.A.; Wu, J.-P.; Duan, J.J.-W.; Cook, L.R.; Oinuma, H.; Kishi, Y. Complete Relative Stereochemistry of Maitotoxin. *J. Am. Chem. Soc.* **1996**, *118*, 7946–7968. [CrossRef]

71. Litaker, R.W.; Vandersea, M.W.; Faust, M.A.; Kibler, S.R.; Nau, A.W.; Holland, W.C.; Chinain, M.; Holmes, M.J.; Tester, P.A. Global Distribution of Ciguatera Causing Dinoflagellates in the Genus *Gambierdiscus*. *Toxicon* **2010**, *56*, 711–730. [CrossRef]

72. Holmes, M.J.; Lewis, R.J. Purification and Characterisation of Large and Small Maitotoxins from Cultured *Gambierdiscus Toxicus*. *Nat. Toxins* **1994**, *2*, 64–72. [CrossRef]

73. Murray, J.S.; Finch, S.C.; Mudge, E.M.; Wilkins, A.L.; Puddick, J.; Harwood, D.T.; Rhodes, L.L.; van Ginkel, R.; Rise, F.; Prinsep, M.R. Structural Characterization of Maitotoxins Produced by Toxic *Gambierdiscus* Species. *Mar. Drugs* **2022**, *20*, 453. [CrossRef]

74. Wang, D.-Z.; Xin, Y.-H.; Wang, M.-H. *Gambierdiscus* and Its Associated Toxins: A Minireview. *Toxins* **2022**, *14*, 485. [CrossRef]
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49. Wheeler, J.F.G.; Ommanney, F.D. *Report on the Mauritius-Seychelles Fisheries Survey, 1948–1949, Etc*; Colonial Office, H.M. Stationery Office: London, UK, 1953.

50. Lewis, R.J. Ciguatera: Australian Perspectives on a Global Problem. *Toxicon* **2006**, *48*, 799–809. [CrossRef]

51. Banner, A.H.; Scheuer, P.J.; Sasaki, S.; Helfrich, P.; Alender, C.B. Observations on Ciguatera-Type Toxin in Fish. *Ann. N. Y. Acad. Sci.* **1960**, *90*, 770–787. [CrossRef] [PubMed]

52. Banner, A.H.; Helfrich, P. *The Distribution of Ciguatera in the Tropical Pacific*; Hawaii Marine Laboratory, University of Hawaii: Honolulu, HI, USA, 1964; pp. 1–48.

53. Hashimoto, Y.; Konosu, S.; Yasumoto, T.; Kamiya, H. Ciguatera in the Ryukyu and Amami Islands. *Nippon. Suisan Gakkaishi* **1969**, *35*, 316–326. [CrossRef]

54. Yasumoto, T.; Scheuer, P.J. Marine Toxins of the Pacific. 8. Ciguatoxin from Moray Eel Livers. *Toxicon Off. J. Int. Soc. Toxinol.* **1969**, *7*, 273–276. [CrossRef] [PubMed]

55. Randall, J.E. A Review of Ciguatera, Tropical Fish Poisoning, with a Tentative Explanation of Its Cause. *Bull. Mar. Sci.* **1958**, *8*, 236–267.

56. Randall, J.E. Food Habits of Reef Fishes of the West Indies. In *Proceedings of the Studies in Tropical Oceanography, Miami Beach, FL, USA*, 17–24 November 1965; University of Miami, Institute of Marine Sciences: Coral Gables, FL, USA, 1967; Volume 5, pp. 665–847.

57. Yasumoto, T.; Hashimoto, Y.; Bagnis, R.; Randall, J.E.; Banner, A.H. Toxicity of the Surgeonfishes. *Bull. Jpn. Soc. Sci. Fish.* **1971**, *37*, 724–734. [CrossRef]

58. Bagnis, R.; Vernoux, J.P. Ciguatoxin and edible reef fishes. *Bull. Soc. Pathol. Exot. Filiales* **1975**, *68*, 320–325.

59. Vernoux, J.P.; Bagnis, R. Fractionnement d'extraits lipidiques ciguatoxiques en milieu alcalin. *Biochimie* **1976**, *58*, 479–484. [CrossRef]

60. Chanteau, S.; Bagnis, R.; Yasumoto, T. Purification de la ciguatoxine de la loche *Epinephelus microdon* (Bleecker). *Biochimie* **1976**, *58*, 1149–1151. [CrossRef]

61. Chungue, E.; Bagnis, R.; Fusetani, N.; Hashimoto, Y. Isolation of Two Toxins from a Parrotfish *Scarus Gibbus*. *Toxicon* **1977**, *15*, 89–93. [CrossRef]

62. Vernoux, J.P. L'ichtyosarcotoxisme de Type Ciguatera Aux Antilles et En Polynésie Française: Test de Ciguatoxicité et Chaîne Trophique Ciguatérigène. Ph.D. Thesis, Univeristé Bordeaux I, Bordeaux, France, 1981.

63. Bagnis, R.; Chanteau, S.; Yasumoto, T. Significance of the various toxins present on dead coral substrata in the origin of ciguatera. *Bull. Soc. Pathol. Exot. Filiales* **1977**, *70*, 320–324.

64. Yasumoto, T.; Nakajima, I.; Bagnis, R.; Adachi, R. Finding of a Dinoflagellate as a Likely Culprit of Ciguatera. *Bull. Jpn. Soc. Sci. Fish.* **1977**, *43*, 1021–1026. [CrossRef]

65. Adachi, R.; Fukuyo, Y. The Thecal Structure of a Marine Toxic Dinoflagellate *Gambierdiscus Toxicus* Gen. et Sp. Nov. Collected in a Ciguatera-Endemic Area. *Bull. Jpn. Soc. Sci. Fish.* **1979**, *45*, 67–71. [CrossRef]

66. Bagnis, R.; Chanteau, S.; Chungue, E.; Hurtel, J.M.; Yasumoto, T.; Inoue, A. Origins of Ciguatera Fish Poisoning: A New Dinoflagellate, *Gambierdiscus Toxicus* Adachi and Fukuyo, Definitively Involved as a Causal Agent. *Toxicon* **1980**, *18*, 199–208. [CrossRef] [PubMed]

67. Yasumoto, T.; Bagnis, R.; P. Vernoux, J. Toxicity of the Surgeonfishes-II. *Bull. Jpn. Soc. Sci. Fish.* **1976**, *42*, 359–365. [CrossRef]

68. Vernoux, J.-P.; Lejeune, J. Ciguatera in the French West Indies. In *Memoirs of the Queensland Museum*; Queensland Museum: Brisbane, Australia, 1994; Volume 34, pp. 631–638.

69. Murata, M.; Naoki, H.; Iwashita, T.; Matsunaga, S.; Sasaki, M.; Yokoyama, A.; Yasumoto, T. Structure of Maitotoxin. *J. Am. Chem. Soc.* **1993**, *115*, 2060–2062. [CrossRef]

70. Zheng, W.; DeMattei, J.A.; Wu, J.-P.; Duan, J.J.-W.; Cook, L.R.; Oinuma, H.; Kishi, Y. Complete Relative Stereochemistry of Maitotoxin. *J. Am. Chem. Soc.* **1996**, *118*, 7946–7968. [CrossRef]

71. Litaker, R.W.; Vandersea, M.W.; Faust, M.A.; Kibler, S.R.; Nau, A.W.; Holland, W.C.; Chinain, M.; Holmes, M.J.; Tester, P.A. Global Distribution of Ciguatera Causing Dinoflagellates in the Genus *Gambierdiscus*. *Toxicon* **2010**, *56*, 711–730. [CrossRef]

72. Holmes, M.J.; Lewis, R.J. Purification and Characterisation of Large and Small Maitotoxins from Cultured *Gambierdiscus Toxicus*. *Nat. Toxins* **1994**, *2*, 64–72. [CrossRef]

73. Murray, J.S.; Finch, S.C.; Mudge, E.M.; Wilkins, A.L.; Puddick, J.; Harwood, D.T.; Rhodes, L.L.; van Ginkel, R.; Rise, F.; Prinsep, M.R. Structural Characterization of Maitotoxins Produced by Toxic *Gambierdiscus* Species. *Mar. Drugs* **2022**, *20*, 453. [CrossRef]

74. Wang, D.-Z.; Xin, Y.-H.; Wang, M.-H. *Gambierdiscus* and Its Associated Toxins: A Minireview. *Toxins* **2022**, *14*, 485. [CrossRef]
75. Holmes, M.J.; Lewis, R.J. Origin of Ciguateric Fish: Quantitative Modelling of the Flow of Ciguatoxin through a Marine Food Chain. *Toxins* **2022**, *14*, 534. [CrossRef]

76. Holmes, M.J.; Lewis, R.J.; Gillespie, N.C. Toxicity of Australian and French Polynesian Strains of *Gambierdiscus Toxicus* (Dinophyceae) Grown in Culture: Characterization of a New Type of Maitotoxin. *Toxicon* Off. J. Int. Soc. Toxinol. **1990**, *28*, 1159–1172. [CrossRef] [PubMed]

77. Holmes, M.J.; Lewis, R.J.; Poli, M.A.; Gillespie, N.C. Strain Dependent Production of Ciguatoxin Precursors (Gambiertoxins) by *Gambierdiscus Toxicus* (Dinophyceae) in Culture. *Toxicon* Off. J. Int. Soc. Toxinol. **1991**, *29*, 761–775. [CrossRef] [PubMed]

78. Sakami, T.; Nakahara, H.; Chinain, M.; Ishida, Y. Effects of Epiphytic Bacteria on the Growth of the Toxic Dinoflagellate *Gambierdiscus Toxicus* (Dinophyceae). *J. Exp. Mar. Biol. Ecol.* **1999**, *233*, 231–246. [CrossRef]

79. Chinain, M.; Darius, H.T.; Ung, A.; Cruchet, P.; Wang, Z.; Ponton, D.; Laurent, D.; Pauillac, S. Growth and Toxin Production in the Ciguatera-Causing Dinoflagellate *Gambierdiscus Polynesiensis* (Dinophyceae) in Culture. *Toxicon* **2010**, *56*, 739–750. [CrossRef]

80. Gillespie, N.C.; Lewis, R.J.; Burke, J.; Holmes, M.J. The Significance of the Absence of Ciguatoxin in a Wild Population of a *Gambierdiscus Toxicus*. In Proceedings of the Fifth International Coral Reef Congress, Tahiti, French Polynesia, 27 May–1 June 1985; Delsalle, B., Galzin, R., Salvat, B., Eds.; Antenne Museum-Ephe: Tahiti, French Polynesia, 1985; Volume 4, pp. 437–442.

81. Lewis, R.J.; Inserra, M.; Vetter, I.; Holland, W.C.; Hardison, D.R.; Tester, P.A.; Litaker, R.W. Rapid Extraction and Identification of Maitotoxin and Ciguatoxin-Like Toxins from Caribbean and Pacific *Gambierdiscus* Using a New Functional Bioassay. *PLoS ONE* **2016**, *11*, e0160006. [CrossRef]

82. Longo, S.; Sibat, M.; Viallon, J.; Darius, H.T.; Hess, P.; Chinain, M. Intraspecific Variability in the Toxin Production and Toxin Profiles of In Vitro Cultures of *Gambierdiscus Polynesiensis* (Dinophyceae) from French Polynesia. *Toxins* **2019**, *11*, 735. [CrossRef]

83. Darius, H.T.; Revel, T.; Viallon, J.; Sibat, M.; Cruchet, P.; Longo, S.; Hardison, D.R.; Holland, W.C.; Tester, P.A.; Litaker, R.W.; et al. Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of *Gambierdiscus Polynesiensis*. *Mar. Drugs* **2022**, *20*, 348. [CrossRef]

84. Stuart, J.; Smith, K.F.; Rhodes, L.; Murray, J.S.; Viallon, J.; Henry, K.; Darius, H.T.; Murray, S.A.; De Azevedo, C.D.; Argyle, P.; et al. Geographical Distribution, Molecular and Toxin Diversity of the Dinoflagellate Species *Gambierdiscus Honu* in the Pacific Region. *Harmful Algae* **2022**, *118*, 102308. [CrossRef]

85. Chinain, M.; Faust, M.A.; Pauillac, S. Morphology and Molecular Analyses of Three Toxic Species of *Gambierdiscus* (Dinophyceae): *G. Pacificus*, *Sp. Nov.*, *G. Australes*, *Sp. Nov.*, and *G. Polynesiensis*, *Sp. Nov.*. *J. Phycol.* **1999**, *35*, 1282–1296. [CrossRef]

86. Yon, T.; Sibat, M.; Réveillon, D.; Bertrand, S.; Chinain, M.; Hess, P. Deeper Insight into *Gambierdiscus Polynesiensis* Toxin Production Relies on Specific Optimization of High-Performance Liquid Chromatography-High Resolution Mass Spectrometry. *Talanta* **2021**, *232*, 122400. [CrossRef] [PubMed]

87. Kibler, S.R.; Davenport, E.D.; Tester, P.A.; Hardison, D.R.; Holland, W.C.; Litaker, R.W. *Gambierdiscus* and *Fukuyoa* Species in the Greater Caribbean: Regional Growth Projections for Ciguatera-Associated Dinoflagellates. *Ecol. Model.* **2017**, *360*, 204–218. [CrossRef]

88. Laza-Martínez, A.; David, H.; Riobó, P.; Miguel, I.; Orive, E. Characterization of a Strain of *Fukuyoa Paulensis* (Dinophyceae) from the Western Mediterranean Sea. *J. Eukaryot. Microbiol.* **2016**, *63*, 481–497. [CrossRef] [PubMed]

89. Holmes, M.J.; Venables, B.; Lewis, R.J. Critical Review and Conceptual and Quantitative Models for the Transfer and Depuration of Ciguatoxins in Fishes. *Toxins* **2021**, *13*, 515. [CrossRef]

90. Ikehara, T.; Kuniyoshi, K.; Oshiro, N.; Yasumoto, T. Biooxidation of Ciguatoxins Leads to Species-Specific Toxin Profiles. *Toxins* **2017**, *9*, 205. [CrossRef] [PubMed]

91. Vernoux, J.P.; Lahlou, N.; Abbad el Andaloussi, S.; Riyeche, N.; Magras, L.P. A Study of the Distribution of Ciguatoxin in Individual Caribbean Fish. *Acta Trop.* **1985**, *42*, 225–233.

92. Gillespie, N.C.; Lewis, R.J.; Pearn, J.H.; Bourke, A.T.; Holmes, M.J.; Bourke, J.B.; Shields, W.J. Ciguatera in Australia. Occurrence, Clinical Features, Pathophysiology and Management. *Med. J. Aust.* **1986**, *145*, 584–590. [CrossRef]

93. Crump, J.A.; McLay, C.L.; Chambers, S.T. Ciguatera Fish Poisoning. *Postgrad. Med. J.* **1999**, *75*, 678–679. [CrossRef]

94. Vernoux, J.-P. La Ciguatera Dans l'île de Saint-Barthélémy: Aspects Épidémiologiques, Toxicologiques et Préventifs. *Oceanol. Acta* **1988**, *11*, 37–46.

95. Clausing, R.J.; Losen, B.; Oberhaensli, F.R.; Darius, H.T.; Sibat, M.; Hess, P.; Swarzenski, P.W.; Chinain, M.; Dechraoui Bottein, M.-Y. Experimental Evidence of Dietary Ciguatoxin Accumulation in an Herbivorous Coral Reef Fish. *Aquat. Toxicol.* **2018**, *200*, 257–265. [CrossRef]

96. Inserra, M.C.; Israel, M.R.; Caldwell, A.; Castro, J.; Deuis, J.R.; Harrington, A.M.; Keramidas, A.; Garcia-Caraballo, S.; Maddern, J.; Erickson, A.; et al. Multiple Sodium Channel Isoforms Mediate the Pathological Effects of Pacific Ciguatoxin-1. *Sci. Rep.* **2017**, *7*, 42810. [CrossRef]

97. Inserra, M.; Lavrukhina, Y.; Jones, A.; Lewis, R.J.; Vetter, I. Ciguatoxin Detection Methods and High-Throughput Assays. In *Analysis of Food Toxins and Toxicants*; Wong, Y., Lewis, R.J., Eds.; John Wiley & Sons, Ltd.: Hoboken, NJ, USA, 2017; pp. 469–488, ISBN 978-1-118-99268-5.

98. Murata, M.; Legrand, A.M.; Ishibashi, Y.; Fukui, M.; Yasumoto, T. Structures and Configurations of Ciguatoxin from the Moray Fèl Gymnothorax Javanicus and Its Likely Precursor from the Dinoflagellate *Gambierdiscus Toxicus*. *J. Am. Chem. Soc.* **1990**, *112*, 4380–4386. [CrossRef]
75. Holmes, M.J.; Lewis, R.J. Origin of Ciguateric Fish: Quantitative Modelling of the Flow of Ciguatoxin through a Marine Food Chain. *Toxins* **2022**, *14*, 534. [CrossRef]

76. Holmes, M.J.; Lewis, R.J.; Gillespie, N.C. Toxicity of Australian and French Polynesian Strains of *Gambierdiscus Toxicus* (Dinophyceae) Grown in Culture: Characterization of a New Type of Maitotoxin. *Toxicon* Off. J. Int. Soc. Toxinol. **1990**, *28*, 1159–1172. [CrossRef] [PubMed]

77. Holmes, M.J.; Lewis, R.J.; Poli, M.A.; Gillespie, N.C. Strain Dependent Production of Ciguatoxin Precursors (Gambiertoxins) by *Gambierdiscus Toxicus* (Dinophyceae) in Culture. *Toxicon* Off. J. Int. Soc. Toxinol. **1991**, *29*, 761–775. [CrossRef] [PubMed]

78. Sakami, T.; Nakahara, H.; Chinain, M.; Ishida, Y. Effects of Epiphytic Bacteria on the Growth of the Toxic Dinoflagellate *Gambierdiscus Toxicus* (Dinophyceae). *J. Exp. Mar. Biol. Ecol.* **1999**, *233*, 231–246. [CrossRef]

79. Chinain, M.; Darius, H.T.; Ung, A.; Cruchet, P.; Wang, Z.; Ponton, D.; Laurent, D.; Pauillac, S. Growth and Toxin Production in the Ciguatera-Causing Dinoflagellate *Gambierdiscus Polynesiensis* (Dinophyceae) in Culture. *Toxicon* **2010**, *56*, 739–750. [CrossRef]

80. Gillespie, N.C.; Lewis, R.J.; Burke, J.; Holmes, M.J. The Significance of the Absence of Ciguatoxin in a Wild Population of a *Gambierdiscus Toxicus*. In Proceedings of the Fifth International Coral Reef Congress, Tahiti, French Polynesia, 27 May–1 June 1985; Delsalle, B., Galzin, R., Salvat, B., Eds.; Antenne Museum-Ephe: Tahiti, French Polynesia, 1985; Volume 4, pp. 437–442.

81. Lewis, R.J.; Inserra, M.; Vetter, I.; Holland, W.C.; Hardison, D.R.; Tester, P.A.; Litaker, R.W. Rapid Extraction and Identification of Maitotoxin and Ciguatoxin-Like Toxins from Caribbean and Pacific *Gambierdiscus* Using a New Functional Bioassay. *PLoS ONE* **2016**, *11*, e0160006. [CrossRef]

82. Longo, S.; Sibat, M.; Viallon, J.; Darius, H.T.; Hess, P.; Chinain, M. Intraspecific Variability in the Toxin Production and Toxin Profiles of In Vitro Cultures of *Gambierdiscus Polynesiensis* (Dinophyceae) from French Polynesia. *Toxins* **2019**, *11*, 735. [CrossRef]

83. Darius, H.T.; Revel, T.; Viallon, J.; Sibat, M.; Cruchet, P.; Longo, S.; Hardison, D.R.; Holland, W.C.; Tester, P.A.; Litaker, R.W.; et al. Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of *Gambierdiscus Polynesiensis*. *Mar. Drugs* **2022**, *20*, 348. [CrossRef]

84. Stuart, J.; Smith, K.F.; Rhodes, L.; Murray, J.S.; Viallon, J.; Henry, K.; Darius, H.T.; Murray, S.A.; De Azevedo, C.D.; Argyle, P.; et al. Geographical Distribution, Molecular and Toxin Diversity of the Dinoflagellate Species *Gambierdiscus Honu* in the Pacific Region. *Harmful Algae* **2022**, *118*, 102308. [CrossRef]

85. Chinain, M.; Faust, M.A.; Pauillac, S. Morphology and Molecular Analyses of Three Toxic Species of *Gambierdiscus* (Dinophyceae): *G. Pacificus*, *Sp. Nov.*, *G. Australdes*, *Sp. Nov.*, and *G. Polynesiensis*, *Sp. Nov.*. *J. Phycol.* **1999**, *35*, 1282–1296. [CrossRef]

86. Yon, T.; Sibat, M.; Réveillon, D.; Bertrand, S.; Chinain, M.; Hess, P. Deeper Insight into *Gambierdiscus Polynesiensis* Toxin Production Relies on Specific Optimization of High-Performance Liquid Chromatography-High Resolution Mass Spectrometry. *Talanta* **2021**, *232*, 122400. [CrossRef] [PubMed]

87. Kibler, S.R.; Davenport, E.D.; Tester, P.A.; Hardison, D.R.; Holland, W.C.; Litaker, R.W. *Gambierdiscus* and *Fukuyoa* Species in the Greater Caribbean: Regional Growth Projections for Ciguatera-Associated Dinoflagellates. *Ecol. Model.* **2017**, *360*, 204–218. [CrossRef]

88. Laza-Martínez, A.; David, H.; Riobó, P.; Miguel, I.; Orive, E. Characterization of a Strain of *Fukuyoa Paulensis* (Dinophyceae) from the Western Mediterranean Sea. *J. Eukaryot. Microbiol.* **2016**, *63*, 481–497. [CrossRef] [PubMed]

89. Holmes, M.J.; Venables, B.; Lewis, R.J. Critical Review and Conceptual and Quantitative Models for the Transfer and Depuration of Ciguatoxins in Fishes. *Toxins* **2021**, *13*, 515. [CrossRef]

90. Ikehara, T.; Kuniyoshi, K.; Oshiro, N.; Yasumoto, T. Biooxidation of Ciguatoxins Leads to Species-Specific Toxin Profiles. *Toxins* **2017**, *9*, 205. [CrossRef] [PubMed]

91. Vernoux, J.P.; Lahlou, N.; Abbad el Andaloussi, S.; Riyeche, N.; Magras, L.P. A Study of the Distribution of Ciguatoxin in Individual Caribbean Fish. *Acta Trop.* **1985**, *42*, 225–233.

92. Gillespie, N.C.; Lewis, R.J.; Pearn, J.H.; Bourke, A.T.; Holmes, M.J.; Bourke, J.B.; Shields, W.J. Ciguatera in Australia. Occurrence, Clinical Features, Pathophysiology and Management. *Med. J. Aust.* **1986**, *145*, 584–590. [CrossRef]

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116. Tester, P.A.; Feldman, R.L.; Nau, A.W.; Kibler, S.R.; Wayne Litaker, R. Ciguatera Fish Poisoning and Sea Surface Temperatures in the Caribbean Sea and the West Indies. *Toxicon* **2010**, *56*, 698–710. [CrossRef]

117. Tester, P.A.; Litaker, R.W.; Berdalet, E. Climate Change and Harmful Benthic Microalgae. *Harmful Algae* **2020**, *91*, 101655. [CrossRef]

118. Pasinszki, T.; Lako, J.; Dennis, T.E. Advances in Detecting Ciguatoxins in Fish. *Toxins* **2020**, *12*, 494. [CrossRef] [PubMed]

119. Yasumoto, T.; Igarashi, T.; Legrand, A.-M.; Cruchet, P.; Chinain, M.; Fujita, T.; Naoki, H. Structural Elucidation of Ciguatoxin Congeners by Fast-Atom Bombardment Tandem Mass Spectroscopy. *J. Am. Chem. Soc.* **2000**, *122*, 4988–4989. [CrossRef]

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121. Estevez, P.; Leao, J.M.; Yasumoto, T.; Dickey, R.W.; Gago-Martinez, A. Caribbean Ciguatoxin-1 Stability under Strongly Acidic Conditions: Characterisation of a New C-CTX1 Methoxy Congener. *Food Addit. Contam. Part A* **2020**, *37*, 519–529. [CrossRef] [PubMed]

122. Lewis, R.J.; Sellin, M.; Poli, M.A.; Norton, R.S.; MacLeod, J.K.; Sheil, M.M. Purification and Characterization of Ciguatoxins from Moray Eel (*Lycodontis Javanicus*, Muraenidae). *Toxicon* **1991**, *29*, 1115–1127. [CrossRef]

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100. Satake, M.; Morohashi, A.; Oguri, H.; Oishi, T.; Hirama, M.; Harada, N.; Yasumoto, T. The Absolute Configuration of Ciguatoxin. *J. Am. Chem. Soc.* **1997**, *119*, 11325–11326. [CrossRef]

101. Dickey, R.W.; Granade, H.R.; Bencsath, F.A.; Martin, G.E. Characterization of Polyether Biotoxins from Caribbean Barracuda (*Sphyraena Barracuda*) and Horse Eye Jack (*Caranx Latus*). In Proceedings of the International Symposium on Ciguatera and Marine Natural Product, Honolulu, HI, USA, 8–10 August 1994; Hokama, Y., Scheuer, P.J., Yasumoto, T., Eds.; Asian Pacific Research Foundation: Honolulu, HI, USA, 1995; p. 292.

102. Crouch, R.C.; Martin, G.E.; Musser, S.M.; Ray Grenade, H.; Dickey, R.W. Improvements in the Sensitivity of Inverse-Detected Heteronuclear Correlation Spectra Using Micro Inverse Probes and Micro Cells: HMQC and HMBC Spectra of Caribbean Ciguatoxin — Preliminary Structural Inferences. *Tetrahedron Lett.* **1995**, *36*, 6827–6830. [CrossRef]

103. Vernoux, J.-P.; Abbad el Andaloussi, S. Hétérogénéité des ciguatoxines extraites de poissons péchés aux Antilles françaises. *Biochimie* **1986**, *68*, 287–291. [CrossRef] [PubMed]

104. Vernoux, J.P.; Talha, F. Fractionation and Purification of Some Muscular and Visceral Ciguatoxins Extracted from Caribbean Fish. *Comp. Biochem. Physiol. B* **1989**, *94*, 499–504. [CrossRef]

105. Pérez-Arellano, J.-L.; Luzardo, O.P.; Brito, A.P.; Cabrera, M.H.; Zumbado, M.; Carranza, C.; Angel-Moreno, A.; Dickey, R.W.; Boada, L.D. Ciguatera Fish Poisoning, Canary Islands. *Emerg. Infect. Dis. J.* -CDC **2005**, *11*, 1981–1982. [CrossRef]

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108. Abraham, A.; Jester, E.L.E.; Granade, H.R.; Plakas, S.M.; Dickey, R.W. Caribbean Ciguatoxin Profile in Raw and Cooked Fish Implicated in Ciguatera. *Food Chem.* **2012**, *131*, 192–198. [CrossRef]

109. Costa, P.R.; Estevez, P.; Castro, D.; Soliño, L.; Gouveia, N.; Santos, C.; Rodrigues, S.M.; Leao, J.M.; Gago-Martínez, A. New Insights into the Occurrence and Toxin Profile of Ciguatoxins in Selvagens Islands (Madeira, Portugal). *Toxins* **2018**, *10*, 524. [CrossRef] [PubMed]

110. Estevez, P.; Castro, D.; Manuel Leao, J.; Yasumoto, T.; Dickey, R.; Gago-Martinez, A. Implementation of Liquid Chromatography Tandem Mass Spectrometry for the Analysis of Ciguatera Fish Poisoning in Contaminated Fish Samples from Atlantic Coasts. *Food Chem.* **2019**, *280*, 8–14. [CrossRef] [PubMed]

111. Sanchez-Henao, J.A.; García-Álvarez, N.; Fernández, A.; Saavedra, P.; Silva Sergent, F.; Padilla, D.; Acosta-Hernández, B.; Martel Suárez, M.; Diogène, J.; Real, F. Predictive Score and Probability of CTX-like Toxicity in Fish Samples from the Official Control of Ciguatera in the Canary Islands. *Sci. Total Environ.* **2019**, *673*, 576–584. [CrossRef]

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114. Otero, P.; Silva, M. Emerging Marine Biotoxins in European Waters: Potential Risks and Analytical Challenges. *Mar. Drugs* **2022**, *20*, 199. [CrossRef]

115. Estevez, P.; Castro, D.; Pequeño-Valtierra, A.; Giraldez, J.; Gago-Martinez, A. Emerging Marine Biotoxins in Seafood from European Coasts: Incidence and Analytical Challenges. *Foods* **2019**, *8*, 149. [CrossRef]

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117. Tester, P.A.; Litaker, R.W.; Berdalet, E. Climate Change and Harmful Benthic Microalgae. *Harmful Algae* **2020**, *91*, 101655. [CrossRef]

118. Pasinszki, T.; Lako, J.; Dennis, T.E. Advances in Detecting Ciguatoxins in Fish. *Toxins* **2020**, *12*, 494. [CrossRef] [PubMed]

119. Yasumoto, T.; Igarashi, T.; Legrand, A.-M.; Cruchet, P.; Chinain, M.; Fujita, T.; Naoki, H. Structural Elucidation of Ciguatoxin Congeners by Fast-Atom Bombardment Tandem Mass Spectroscopy. *J. Am. Chem. Soc.* **2000**, *122*, 4988–4989. [CrossRef]

120. Kryuchkov, F.; Robertson, A.; Miles, C.O.; Mudge, E.M.; Uhlig, S. LC-HRMS and Chemical Derivatization Strategies for the Structure Elucidation of Caribbean Ciguatoxins: Identification of C-CTX-3 and -4. *Mar. Drugs* **2020**, *18*, 182. [CrossRef] [PubMed]

121. Estevez, P.; Leao, J.M.; Yasumoto, T.; Dickey, R.W.; Gago-Martinez, A. Caribbean Ciguatoxin-1 Stability under Strongly Acidic Conditions: Characterisation of a New C-CTX1 Methoxy Congener. *Food Addit. Contam. Part A* **2020**, *37*, 519–529. [CrossRef] [PubMed]

122. Lewis, R.J.; Sellin, M.; Poli, M.A.; Norton, R.S.; MacLeod, J.K.; Sheil, M.M. Purification and Characterization of Ciguatoxins from Moray Eel (*Lycodontis Javanicus*, Muraenidae). *Toxicon* **1991**, *29*, 1115–1127. [CrossRef]

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197. Suzuki, T.; Ha, D.V.; Uesugi, A.; Uchida, H. Analytical Challenges to Ciguatoxins. *Curr. Opin. Food Sci.* **2017**, *18*, 37–42. [CrossRef]

198. Reverté, L.; Soliño, L.; Carnicer, O.; Diogène, J.; Campàs, M. Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays. *Mar. Drugs* **2014**, *12*, 5719–5763. [CrossRef]

199. Tosteson, T.R.; Ballantine, D.L.; Durst, H.D. Seasonal Frequency of Ciguatoxic Barracuda in Southwest Puerto Rico. *Toxicon Off. J. Int. Soc. Toxinol.* **1988**, *26*, 795–801. [CrossRef] [PubMed]

200. Lewis, R.J.; Jones, A.; Vernoux, J.-P. HPLC/Tandem Electrospray Mass Spectrometry for the Determination of Sub-Ppb Levels of Pacific and Caribbean Ciguatoxins in Crude Extracts of Fish. *Anal. Chem.* **1999**, *71*, 247–250. [CrossRef]

201. Moreiras, G.; Leão, J.M.; Gago-Martínez, A. Design of Experiments for the Optimization of Electrospray Ionization in the LC-MS/MS Analysis of Ciguatoxins. *J. Mass Spectrom.* **2018**, *53*, 1059–1069. [CrossRef]

202. Sasaki, M.; Iwasaki, K.; Arai, K. Studies toward the Total Synthesis of Caribbean Ciguatoxin C-CTX-1: Synthesis of the LMN-Ring Fragment through Reductive Olefin Cross-Coupling. *Org. Lett.* **2018**, *20*, 7163–7166. [CrossRef]
203. Sasaki, M.; Iwasaki, K.; Arai, K. Synthesis and Structural Implication of the JKLMN-Ring Fragment of Caribbean Ciguatoxin C-CTX-1. *J. Org. Chem.* **2021**, *86*, 4580–4597. [CrossRef]

204. Estevez, P.; Sibat, M.; Leão-Martins, J.M.; Reis Costa, P.; Gago-Martínez, A.; Hess, P. Liquid Chromatography Coupled to High-Resolution Mass Spectrometry for the Confirmation of Caribbean Ciguatoxin-1 as the Main Toxin Responsible for Ciguatera Poisoning Caused by Fish from European Atlantic Coasts. *Toxins* **2020**, *12*, 267. [CrossRef]

205. Kryuchkov, F.; Robertson, A.; Mudge, E.M.; Miles, C.O.; Van Gothem, S.; Uhlig, S. Reductive Amination for LC-MS Signal Enhancement and Confirmation of the Presence of Caribbean Ciguatoxin-1 in Fish. *Toxins* **2022**, *14*, 399. [CrossRef] [PubMed]

206. Mudge, E.M.; Robertson, A.; Leynse, A.K.; McCarron, P.; Miles, C.O. Selective Extraction of Gambierone and Related Metabolites in *Gambierdiscus Silvae* Using m-Aminophenylboronic Acid-Agarose Gel and Liquid Chromatography-High-Resolution Mass Spectrometric Detection. *J. Chromatogr. B* **2022**, *1188*, 123014. [CrossRef] [PubMed]

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208. Yon, T.; Sibat, M.; Robert, E.; Lhaute, K.; Holland, W.C.; Litaker, R.W.; Bertrand, S.; Hess, P.; Réveillon, D. Sulfo-Gambierones, Two New Analogs of Gambierone Produced by *Gambierdiscus Excentricus*. *Mar. Drugs* **2021**, *19*, 657. [CrossRef]

209. Dickey, R.W. *Ciguatera Toxins: Chemistry, Toxicology, and Detection*; CRC Press: New York, NY, USA, 2008; pp. 497–518, ISBN 978-0-429-12850-9.

210. Nagae, M.; Igarashi, T.; Mizukoshi, K.; Kuniyoshi, K.; Oshiro, N.; Yasumoto, T. Development and Validation of an LC-MS/MS Method for the Ultra-Trace Analysis of Pacific Ciguatoxins in Fish. *J. AOAC Int.* **2021**, *104*, 1272–1281. [CrossRef]

211. Sibat, M.; Herrenknecht, C.; Darius, H.T.; Roué, M.; Chinain, M.; Hess, P. Detection of Pacific Ciguatoxins Using Liquid Chromatography Coupled to Either Low or High Resolution Mass Spectrometry (LC-MS/MS). *J. Chromatogr. A* **2018**, *1571*, 16–28. [CrossRef]

212. Estevez, P.; Gago-Martinez, A. Contribution of Mass Spectrometry to the Advances in Risk Characterization of Marine Biotoxins: Towards the Characterization of Metabolites Implied in Human Intoxications. *Toxins* **2023**, *15*, 103. [CrossRef]

213. AOAC Official Method 959.08. Paralytic Shellfish Poison. Biological Method. Final Action. In *AOAC Official Methods for Analysis, Natural Toxins (Chapter 49)*; AOAC International: Gaithersburg, MD, USA, 2005; pp. 79–80.

214. OECD. Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure. In *OECD Guidelines for the Testing of Chemicals, Section 4*; OECD: Paris, France, 2022.

215. Finch, S.C.; Boundy, M.J.; Webb, N.G.; Harwood, D.T. The Effect of Experimental Protocol on the Toxicity of Saxitoxin in Mice. *Toxins* **2023**, *15*, 290. [CrossRef]

216. Turner, A.D.; Tarnovius, S.; Hatfield, R.G.; Teixeira-Alves, M.; Broadwater, M.; Dolah, F.V.; Garcia-Mendoza, E.; Medina, D.; Salhi, M.; Goya, A.B.; et al. Application of Six Detection Methods for Analysis of Paralytic Shellfish Toxins in Shellfish from Four Regions within Latin America. *Mar. Drugs* **2020**, *18*, 616. [CrossRef]

217. Turner, A.D.; Hatfield, R.G.; Maskrey, B.H.; Algoet, M.; Lawrence, J.F. Evaluation of the New European Union Reference Method for Paralytic Shellfish Toxins in Shellfish: A Review of Twelve Years Regulatory Monitoring Using Pre-Column Oxidation LC-FLD. *TrAC Trends Anal. Chem.* **2019**, *113*, 124–139. [CrossRef]

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
203. Sasaki, M.; Iwasaki, K.; Arai, K. Synthesis and Structural Implication of the JKLMN-Ring Fragment of Caribbean Ciguatoxin C-CTX-1. *J. Org. Chem.* **2021**, *86*, 4580–4597. [CrossRef]

204. Estevez, P.; Sibat, M.; Leão-Martins, J.M.; Reis Costa, P.; Gago-Martínez, A.; Hess, P. Liquid Chromatography Coupled to High-Resolution Mass Spectrometry for the Confirmation of Caribbean Ciguatoxin-1 as the Main Toxin Responsible for Ciguatera Poisoning Caused by Fish from European Atlantic Coasts. *Toxins* **2020**, *12*, 267. [CrossRef]

205. Kryuchkov, F.; Robertson, A.; Mudge, E.M.; Miles, C.O.; Van Gothem, S.; Uhlig, S. Reductive Amination for LC-MS Signal Enhancement and Confirmation of the Presence of Caribbean Ciguatoxin-1 in Fish. *Toxins* **2022**, *14*, 399. [CrossRef] [PubMed]

206. Mudge, E.M.; Robertson, A.; Leynse, A.K.; McCarron, P.; Miles, C.O. Selective Extraction of Gambierone and Related Metabolites in *Gambierdiscus Silvae* Using m-Aminophenylboronic Acid-Agarose Gel and Liquid Chromatography-High-Resolution Mass Spectrometric Detection. *J. Chromatogr. B* **2022**, *1188*, 123014. [CrossRef] [PubMed]

207. Estevez, P.; Sibat, M.; Leão-Martins, J.M.; Tudó, A.; Rambla-Alegre, M.; Aligizaki, K.; Diogène, J.; Gago-Martinez, A.; Hess, P. Use of Mass Spectrometry to Determine the Diversity of Toxins Produced by *Gambierdiscus* and *Fukuyoa* Species from Balearic Islands and Crete (Mediterranean Sea) and the Canary Islands (Northeast Atlantic). *Toxins* **2020**, *12*, 305. [CrossRef] [PubMed]

208. Yon, T.; Sibat, M.; Robert, E.; Lhaute, K.; Holland, W.C.; Litaker, R.W.; Bertrand, S.; Hess, P.; Réveillon, D. Sulfo-Gambierones, Two New Analogs of Gambierone Produced by *Gambierdiscus Excentricus*. *Mar. Drugs* **2021**, *19*, 657. [CrossRef]

209. Dickey, R.W. *Ciguatera Toxins: Chemistry, Toxicology, and Detection*; CRC Press: New York, NY, USA, 2008; pp. 497–518, ISBN 978-0-429-12850-9.

210. Nagae, M.; Igarashi, T.; Mizukoshi, K.; Kuniyoshi, K.; Oshiro, N.; Yasumoto, T. Development and Validation of an LC-MS/MS Method for the Ultra-Trace Analysis of Pacific Ciguatoxins in Fish. *J. AOAC Int.* **2021**, *104*, 1272–1281. [CrossRef]

211. Sibat, M.; Herrenknecht, C.; Darius, H.T.; Roué, M.; Chinain, M.; Hess, P. Detection of Pacific Ciguatoxins Using Liquid Chromatography Coupled to Either Low or High Resolution Mass Spectrometry (LC-MS/MS). *J. Chromatogr. A* **2018**, *1571*, 16–28. [CrossRef]

212. Estevez, P.; Gago-Martinez, A. Contribution of Mass Spectrometry to the Advances in Risk Characterization of Marine Biotoxins: Towards the Characterization of Metabolites Implied in Human Intoxications. *Toxins* **2023**, *15*, 103. [CrossRef]

213. AOAC Official Method 959.08. Paralytic Shellfish Poison. Biological Method. Final Action. In *AOAC Official Methods for Analysis, Natural Toxins (Chapter 49)*; AOAC International: Gaithersburg, MD, USA, 2005; pp. 79–80.

214. OECD. Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure. In *OECD Guidelines for the Testing of Chemicals, Section 4*; OECD: Paris, France, 2022.

215. Finch, S.C.; Boundy, M.J.; Webb, N.G.; Harwood, D.T. The Effect of Experimental Protocol on the Toxicity of Saxitoxin in Mice. *Toxins* **2023**, *15*, 290. [CrossRef]

216. Turner, A.D.; Tarnovius, S.; Hatfield, R.G.; Teixeira-Alves, M.; Broadwater, M.; Dolah, F.V.; Garcia-Mendoza, E.; Medina, D.; Salhi, M.; Goya, A.B.; et al. Application of Six Detection Methods for Analysis of Paralytic Shellfish Toxins in Shellfish from Four Regions within Latin America. *Mar. Drugs* **2020**, *18*, 616. [CrossRef]

217. Turner, A.D.; Hatfield, R.G.; Maskrey, B.H.; Algoet, M.; Lawrence, J.F. Evaluation of the New European Union Reference Method for Paralytic Shellfish Toxins in Shellfish: A Review of Twelve Years Regulatory Monitoring Using Pre-Column Oxidation LC-FLD. *TrAC Trends Anal. Chem.* **2019**, *113*, 124–139. [CrossRef]

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.