A Very Deep Graph Convolutional Network for 13C NMR Chemical Shift Calculations with Density Functional Theory Level Performance for Structure Assignment.

Nuclear magnetic resonance (NMR) chemical shift calculations are powerful tools for structure elucidation and have been extensively employed in both natural product and synthetic chemistry. However, density functional theory (DFT) NMR chemical shift calculations are usually time-consuming, while fast data-driven methods often lack reliability, making it challenging to apply them to computationally intensive tasks with a high requirement on quality. Herein, we have constructed a 54-layer-deep graph convolutional network for 13C NMR chemical shift calculations, which achieved high accuracy with low time-cost and performed competitively with DFT NMR chemical shift calculations on structure assignment benchmarks. Our model utilizes a semiempirical method, GFN2-xTB, and is compatible with a broad variety of organic systems, including those composed of hundreds of atoms or elements ranging from H to Rn. We used this model to resolve the controversial J/K ring junction problem of maitotoxin, which is the largest whole molecule assigned by NMR calculations to date. This model has been developed into user-friendly software, providing a useful tool for routine rapid structure validation and assignation as well as a new approach to elucidate the large structures that were previously unsuitable for NMR calculations.

Chemodiversity of Brevetoxins and Other Potentially Toxic Metabolites Produced by Karenia spp. and Their Metabolic Products in Marine Organisms.

In recent decades, more than 130 potentially toxic metabolites originating from dinoflagellate species belonging to the genus Karenia or metabolized by marine organisms have been described. These metabolites include the well-known and large group of brevetoxins (BTXs), responsible for foodborne neurotoxic shellfish poisoning (NSP) and airborne respiratory symptoms in humans. Karenia spp. also produce brevenal, brevisamide and metabolites belonging to the hemi-brevetoxin, brevisin, tamulamide, gymnocin, gymnodimine, brevisulcenal and brevisulcatic acid groups. In this review, we summarize the available knowledge in the literature since 1977 on these various identified metabolites, whether they are produced directly by the producer organisms or biotransformed in marine organisms. Their structures and physicochemical properties are presented and discussed. Among future avenues of research, we highlight the need for more toxin occurrence data with analytical techniques, which can specifically determine the analogs present in samples. New metabolites have yet to be fully described, especially the groups of metabolites discovered in the last two decades (e.g tamulamides). Lastly, this work clarifies the different nomenclatures used in the literature and should help to harmonize practices in the future.

Toxicity Screening of a Gambierdiscus australes Strain from the Western Mediterranean Sea and Identification of a Novel Maitotoxin Analogue.

Dinoflagellate species of the genera Gambierdiscus and Fukuyoa are known to produce ciguatera poisoning-associated toxic compounds, such as ciguatoxins, or other toxins, such as maitotoxins. However, many species and strains remain poorly characterized in areas where they were recently identified, such as the western Mediterranean Sea. In previous studies carried out by our research group, a G. australes strain from the Balearic Islands (Mediterranean Sea) presenting MTX-like activity was characterized by LC-MS/MS and LC-HRMS detecting 44-methyl gambierone and gambieric acids C and D. However, MTX1, which is typically found in some G. australes strains from the Pacific Ocean, was not detected. Therefore, this study focuses on the identification of the compound responsible for the MTX-like toxicity in this strain. The G. australes strain was characterized not only using LC-MS instruments but also N2a-guided HPLC fractionation. Following this approach, several toxic compounds were identified in three fractions by LC-MS/MS and HRMS. A novel MTX analogue, named MTX5, was identified in the most toxic fraction, and 44-methyl gambierone and gambieric acids C and D contributed to the toxicity observed in other fractions of this strain. Thus, G. australes from the Mediterranean Sea produces MTX5 instead of MTX1 in contrast to some strains of the same species from the Pacific Ocean. No CTX precursors were detected, reinforcing the complexity of the identification of CTXs precursors in these regions.

Monitoring the Emergence of Algal Toxins in Shellfish: First Report on Detection of Brevetoxins in French Mediterranean Mussels.

In France, four groups of lipophilic toxins are currently regulated: okadaic acid/dinophysistoxins, pectenotoxins, yessotoxins and azaspiracids. However, many other families of toxins exist, which can be emerging toxins. Emerging toxins include both toxins recently detected in a specific area of France but not regulated yet (e.g., cyclic imines, ovatoxins) or toxins only detected outside of France (e.g., brevetoxins). To anticipate the introduction to France of these emerging toxins, a monitoring program called EMERGTOX was set up along the French coasts in 2018. The single-laboratory validation of this approach was performed according to the NF V03-110 guidelines by building an accuracy profile. Our specific, reliable and sensitive approach allowed us to detect brevetoxins (BTX-2 and/or BTX-3) in addition to the lipophilic toxins already regulated in France. Brevetoxins were detected for the first time in French Mediterranean mussels (Diana Lagoon, Corsica) in autumn 2018, and regularly every year since during the same seasons (autumn, winter). The maximum content found was 345 µg (BTX-2 + BTX-3)/kg in mussel digestive glands in November 2020. None were detected in oysters sampled at the same site. In addition, a retroactive analysis of preserved mussels demonstrated the presence of BTX-3 in mussels from the same site sampled in November 2015. The detection of BTX could be related to the presence in situ at the same period of four Karenia species and two raphidophytes, which all could be potential producers of these toxins. Further investigations are necessary to understand the origin of these toxins.

Method performance verification for the combined detection and quantitation of the marine neurotoxins cyclic imines and brevetoxin shellfish metabolites in mussels (Mytilus edulis) and oysters (Crassostrea gigas) by UHPLC-MS/MS.

A single laboratory method performance verification is reported for a rapid sensitive UHPLC-MS/MS method for the quantification of eight cyclic imine and two brevetoxin analogues in two bivalve shellfish matrices: mussel (Mytilus edulis) and Pacific oyster (Crassostrea gigas). Targeted cyclic imine analogues were from the spirolide, gymnodimine and pinnatoxin groups, namely 20-Me-SPX-C, 13-desMe-SPX-C, 13,19-didesMe-SPX-C, GYM-A, 12-Me-GYM, PnTx-E, PnTx-F and PnTx-G. Brevetoxin analogues consisted of the shellfish metabolites BTX-B5 and S-desoxy-BTX-B2. A rapid dispersive extraction was used as well as a fast six-minute UHPLC-MS/MS analysis. Mobile phase prepared using ammonium fluoride and methanol was optimised for both chromatographic separation and MS/MS response to suit all analytes. Method performance verification checks for both matrices were carried out. Matrix influence was acceptable for the majority of analogues with the MS response for all analogues being linear across an appropriate range of concentrations. In terms of limits of detection and quantitation the method was shown to be highly sensitive when compared with other methods. Acceptable recoveries were found with most analogues, with laboratory precision in terms of intra- and inter-batch precision deemed appropriate. The method was applied to environmental shellfish samples with results showing low concentrations of cyclic imines to be present. The method is fast and highly sensitive for the detection and quantification of all targeted analogues, in both mussel and oyster matrices. Consequently, the method has been shown to provide a useful tool for simultaneous monitoring for the presence or future emergence of these two toxin groups in shellfish.

UPLC-ESI-Q-TOF/MS Based Metabolic Profiling of Protosappanin B in Rat Plasma, Bile, Feces, Urine and Intestinal Bacteria Samples.

BACKGROUND: Caesalpinia sappan L. is a traditional medicinal plant that is used to promote blood circulation and treat stroke in China. Protosappanin B (PTB) is a unique homoisoflavone compound isolated from Sappan Lignum (the heartwood of Caesalpinia sappan L). In a previous study, the metabolic fate of PTB remained unknown. OBJECTIVE: To explore whether PTB is extensively metabolized, the metabolites of PTB in bile, plasma, urine, feces, and intestinal bacteria samples in rats were investigated. METHODS: The biosamples were investigated by ultraperformance liquid chromatography combined with time-offlight mass spectrometry (UPLC-TOF-MS/MS) with MetabolitePilot software. RESULTS: 28 metabolites were identified in the biosamples: 18 metabolites in rat bile, 8 in plasma, 20 in feces, 7 in urine and 2 in intestinal bacteria samples. Both phase I and phase II metabolites were observed. Metabolite conversion occurred via 9 proposed pathways: sulfate conjugation, glucuronide conjugation, bis-glucuronide conjugation, glucose conjugation, dehydration, oxidation, hydrolysis, methylation and hydroxymethylene loss. The metabolic pathways differed among biosamples and exhibited different distributions. Among these pathways, the most important was sulfate and glucuronide conjugation. CONCLUSION: The results showed that the small intestinal and biliary routes play an important role in the clearance and excretion of PTB. The main sites of metabolism in the PTB chemical structure were the phenolic hydroxyl and the side-chains on the eight-element ring.

Characterization of maitotoxin-4 (MTX4) using electrospray positive mode ionization high-resolution mass spectrometry and UV spectroscopy.

RATIONALE: The dinoflagellate genera Gambierdiscus and Fukuyoa are producers of toxins responsible for Ciguatera Poisoning (CP). Although having very low oral potency, maitotoxins (MTXs) are very toxic following intraperitoneal injection and feeding studies have shown they may accumulate in fish muscle. To date, six MTX congeners have been described but two congeners (MTX2 and MTX4) have not yet been structurally elucidated. The aim of the present study was to further characterize MTX4. METHODS: Chemical analysis was performed using liquid chromatography coupled to a diode-array detector (DAD) and positive ion mode high-resolution mass spectrometry (LC/HRMS) on partially purified extracts of G. excentricus (strain VGO792). HRMS/MS studies were also carried out to tentatively explain the fragmentation pathways of MTX and MTX4. RESULTS: The comparison of UV and HRMS (ESI+ ) spectra between MTX and MTX4 led us to propose the elemental formula of MTX4 (C157 H241 NO68 S2 , as the unsalted molecule). The comparison of the theoretical and measured m/z values of the doubly charged ions of the isotopic profile in ESI+ were coherent with the proposed elemental formula of MTX4. The study of HRMS/MS spectra on the tri-ammoniated adduct ([M - H + 3NH4 ]2+ ) of both molecules gave additional information about structural features. The cleavage observed, probably located at C99 -C100 in both MTX and MTX4, highlighted the same A-side product ion shared by the two molecules. CONCLUSIONS: All these investigations on the characterization of MTX4 contribute to highlighting that MTX4 belongs to the same structural family of MTXs. However, to accomplish a complete structural elucidation of MTX4, an NMR-based study and LC/HRMSn investigation will have to be carried out.

Two new epimers of C15-acetogenin, 4-epi-isolaurallene and 4-epi-itomanallene A as diastereomeric model.

Two new C15-acetogenins, 4-epi-isolaurallene (1) and 4-epi-itomanallene A (2) were isolated from a population of marine red alga Laurencia nangii Masuda from Carrington Reef. The structures of these compounds were determined intensively by NMR and HRESIMS data. Their configurations were elucidated by detailed comparison of chemical shifts, germinal protons splitting and NOE correlations with known and synthesized analogues. In addition, antibacterial activities of these compounds were evaluated. These compounds would serve as diastereomeric models for future reference. Since the isolaurallene, neolaurallene, 9-acetoxy-1,10,12-tribromo-4,7:6,13-bisepoxypentadeca-1,2-diene, itomanallene A and laurendecumallene A were isolated, compounds 1 and 2 were the sixth example of C15-acetogenin with dioxabicyclo[7.3.0]dodecene skeleton.

Synthesis of the EFG Framework of Tamulamides A and B.

Synthesis of the fused polycyclic ether motif comprising the EFG rings of the marine ladder polyethers tamulamides A and B has been achieved via two different etherification strategies. Ultimately, a reductive etherification approach proved most successful due to tolerance of the G ring substitution and provided the EFG 6,7,6 ring system in 58% yield.

Immune function in Trachemys scripta following exposure to a predominant brevetoxin congener, PbTx-3, as a model for potential health impacts for sea turtles naturally exposed to brevetoxins.

Many species of marine life in southwestern Florida, including sea turtles, are impacted by blooms of the toxic dinoflagellate, Karenia brevis. Sublethal exposure to toxins produced by K. brevis has been shown to impact sea turtle health. Since all sea turtles in the Gulf of Mexico have protected status, a freshwater turtle, Trachemys scripta, was used as a model for immune system effects following experimental exposure to a predominant brevetoxin congener in K. brevis blooms, PbTx-3. Exposure to PbTx-3 was oral or intratracheal and health effects were assessed using a suite of immune function parameters: innate immune function (phagocytosis, plasma lysozyme activity), adaptive immune function (lymphocyte proliferation), and measures of oxidative stress (superoxide dismutase (SOD) and glutathione-S-transferase (GST) activity in plasma). Inflammation was also measured using plasma protein electrophoresis. In addition, differential expression of genes in peripheral blood leukocytes was determined using suppression subtractive hybridization followed by real-time PCR of specific genes. The primary immune effects of sublethal brevetoxin exposure in T. scripta following PbTx-3 administration, appear to be an increase in oxidative stress, a decrease in lysozyme activity, and modulation of immune function through lymphocyte proliferation responses. Plasma protein electrophoresis showed a decreased A:G ratio which may indicate potential inflammation. Genes coding for oxidative stress, such as thioredoxin and GST, were upregulated in exposed animals. That sublethal brevetoxin exposures impact immune function components suggests potential health implications for sea turtles naturally exposed to toxins. Knowledge of physiological stressors induced by brevetoxins may contribute to the ultimate goal of developing directed treatment strategies in exposed animals for reduced mortality resulting from red tide toxin exposure in sea turtles.

Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels.

Brevetoxins are produced by dinoflagellates such as Karenia brevis in warm-water red tides and cause neurotoxic shellfish poisoning. They bind to voltage-gated sodium channels at neurotoxin receptor 5, making the channels more active by shifting the voltage-dependence of activation to more negative potentials and by slowing the inactivation process. Previous work using photoaffinity labeling identified binding to the IS6 and IVS5 transmembrane segments of the channel α subunit. We used alanine-scanning mutagenesis to identify molecular determinants for brevetoxin binding in these regions as well as adjacent regions IVS5-SS1 and IVS6. Most of the mutant channels containing single alanine substitutions expressed functional protein in tsA-201 cells and bound to the radioligand [42-3H]-PbTx3. Binding affinity for the great majority of mutant channels was indistinguishable from wild type. However, transmembrane segments IS6, IVS5 and IVS6 each contained 2 to 4 amino acid positions where alanine substitution resulted in a 2-3-fold reduction in brevetoxin affinity, and additional mutations caused a similar increase in brevetoxin affinity. These findings are consistent with a model in which brevetoxin binds to a protein cleft comprising transmembrane segments IS6, IVS5 and IVS6 and makes multiple distributed interactions with these α helices. Determination of brevetoxin affinity for Nav1.2, Nav1.4 and Nav1.5 channels showed that Nav1.5 channels had a characteristic 5-fold reduction in affinity for brevetoxin relative to the other channel isoforms, suggesting the interaction with sodium channels is specific despite the distributed binding determinants.

Elucidation and partial NMR assignment of monosulfated maitotoxins from the Caribbean.

Compounds similar to maitotoxin (MTX) have been isolated from several laboratory strains of the dinoflagellate Gambierdiscus spp. from the Caribbean. Mass spectral results suggest that these compounds differ from MTX by the loss of one sulfate group and, in some cases, the loss of one methyl group with the addition of one degree of unsaturation. NMR experiments, using approximately 50 nmol of one of these compounds, have demonstrated that the 9-sulfo group of MTX is still present, suggesting that these compounds are 40-desulfo congeners of MTX.

Structure Elucidation and Biological Evaluation of Maitotoxin-3, a Homologue of Gambierone, from Gambierdiscus belizeanus.

Gambierdiscus species are the producers of the marine toxins ciguatoxins and maitotoxins which cause worldwide human intoxications recognized as Ciguatera Fish Poisoning. A deep chemical investigation of a cultured strain of G. belizeanus, collected in the Caribbean Sea, led to the identification of a structural homologue of the recently described gambierone isolated from the same strain. The structure was elucidated mainly by comparison of NMR and MS data with those of gambierone and ascertained by 2D NMR data analyses. Gratifyingly, a close inspection of the MS data of the new 44-methylgambierone suggests that this toxin would actually correspond to the structure of maitotoxin-3 (MTX3, m/z 1039.4957 for the protonated adduct) detected in 1994 in a Pacific strain of Gambierdiscus and recently shown in routine monitoring programs. Therefore, this work provides for the first time the chemical identification of the MTX3 molecule by NMR. Furthermore, biological data confirmed the similar activities of both gambierone and 44-methylgambierone. Both gambierone and MTX3 induced a small increase in the cytosolic calcium concentration but only MTX3 caused cell cytotoxicity at micromolar concentrations. Moreover, chronic exposure of human cortical neurons to either gambierone or MTX3 altered the expression of ionotropic glutamate receptors, an effect already described before for the synthetic ciguatoxin CTX3C. However, even when gambierone and MTX3 affected glutamate receptor expression in a similar manner their effect on receptor expression differed from that of CTX3C, since both toxins decreased AMPA receptor levels while increasing N-methyl-d-aspartate (NMDA) receptor protein. Thus, further studies should be pursued to clarify the similarities and differences in the biological activity between the known ciguatoxins and the new identified molecule as well as its contribution to the neurological symptoms of ciguatera.

Effectors of thioredoxin reductase: Brevetoxins and manumycin-A.

The activities of two effectors, brevetoxin (PbTx) and manumycin-A (Man-A), of thioredoxin reductase (TrxR) have been evaluated against a series of fourteen TrxR orthologs originating from mammals, insects and protists and several mutants. Man-A, a molecule with numerous electrophilic sites, forms a covalent adduct with most selenocystine (Sec)-containing TrxR enzymes. The evidence also demonstrates that Man-A can form covalent adducts with some non-Sec-containing enzymes. The activities of TrxR enzymes towards various substrates are moderated by Man-A either positively or negatively depending on the enzyme. In general, the reduction of substrates by Sec-containing TrxR is inhibited and NADPH oxidase activity is activated. For non-Sec-containing TrxR the effect of Man-A on the reduction of substrates is variable, but NADPH oxidase activity can be activated even in the absence of covalent modification of TrxR. The effect of PbTx is less pronounced. A smaller subset of enzymes is affected by PbTx. With a single exception, the activities of most of this subset are activated. Although both PbTx variants can react with selenocysteine, a stable covalent adduct is not formed with any of the TrxR enzymes. The key findings from this work are (i) the identification of an alternate mechanism of toxicity for the algal toxin brevetoxin (ii) the demonstration that covalent modification of TrxR is not a prerequisite for the activation of NADPH oxidase activity of TrxR and (iii) the identification of an inhibitor which can discriminate between cytosolic and mitochondrial TrxR.

[Reaction Development Utilizing the Features of Chemical Elements and Synthesis of Marine Natural Products].

Marine natural products and biologically active compounds often contain cyclic ether units. Thus, regio- and stereoselective construction of these structures has long been a topic of interest in organic synthesis. This review summarizes new synthetic approaches to polycyclic ether natural products utilizing the features of chemical elements.

GREEN TREE FROG ( HYLA CINEREA) AND GROUND SQUIRREL ( XEROSPERMOPHILUS SPILOSOMA) MORTALITY ATTRIBUTED TO INLAND BREVETOXIN TRANSPORT AT PADRE ISLAND NATIONAL SEASHORE, TEXAS, USA, 2015.

: On 16 September 2015, a red tide ( Karenia brevis) bloom impacted coastal areas of Padre Island National Seashore Park, Texas, US. Two days later and about 0.9 km inland, 30-40 adult green tree frogs ( Hyla cinerea) were found dead after displaying tremors, weakness, labored breathing, and other signs of neurologic impairment. A rainstorm accompanied by high winds, rough surf, and high tides, which could have aerosolized brevetoxin, occurred on the morning of the mortality event. Frog carcasses were in good body condition but contained significant brevetoxin in tissues. Tissue brevetoxin was also found in two dead or dying spotted ground squirrels ( Xerospermophilus spilosoma) and a coyote ( Canis latrans) found in the area. Rainwater collected from the location of the mortality event contained brevetoxin. Green tree frog and ground squirrel mortality has not been previously attributed to brevetoxin exposure and such mortality suggested that inland toxin transport, possibly through aerosols, rainfall, or insects, may have important implications for coastal species.

Efficient and Accurate Born-Oppenheimer Molecular Dynamics for Large Molecular Systems.

An efficient scheme for the calculation of Born-Oppenheimer molecular dynamics (BOMD) simulations is introduced. It combines the corrected small basis set Hartree-Fock (HF-3c) method by Sure and Grimme [J. Comput. Chem. 2013, 43, 1672], extended Lagrangian BOMD (XL-BOMD) by Niklasson et al. [J. Chem. Phys. 2009, 130, 214109], and the calculation of the two electron integrals on graphics processing units (GPUs) [J. Chem. Phys. 2013, 138, 134114; J. Chem. Theory Comput. 2015, 11, 918]. To explore the parallel performance of our strong scaling implementation of the method, we present timings and extract, as its validation and first illustrative application, high-quality vibrational spectra from simulated trajectories of ß-carotene, paclitaxel, and liquid water (up to 500 atoms). We conclude that the presented BOMD scheme may be used as a cost-efficient and reliable tool for computing vibrational spectra and thermodynamics of large molecular systems including explicit solvent molecules containing 500 atoms and more. Simulating 50 ps of maitotoxin (nearly 500 atoms) employing time steps of 0.5 fs requires ∼3 weeks on 12 CPUs (Intel Xeon E5 2620 v3) with 24 GPUs (AMD FirePro 3D W8100).

Convergent Synthesis of Fused Ring Systems in Large Polycyclic Ethers.

This article reviews our studies on the oxiranyl anion-based, [X+2+Y]-type convergent strategy for the synthesis of polycyclic ether natural products. The strategy is noteworthy for its flexibility, which allows for the generation of different-sized fused ring systems based on a ring expansion reaction. For a precise understanding of this key ring expansion, we first focused on the reaction mechanism, in which an equatorial attack of TMS-diazomethane was determined to be the crucial step. In the later part of this review, the application of our oxiranyl anion-based strategy to large fused ring systems is described. The advantageous flexibility is highlighted in the divergent synthesis of five octacyclic ethers involving the CDEFGHIJ-ring skeleton of yessotoxin and its ring-modified analogs. Total synthesis of gymnocin-A was achieved using the oxiranyl anion convergent strategy, which furnished its large system of fourteen contiguous ether rings.

Syntheses and Biological Activities of the LMNO, ent-LMNO, and NOPQR(S) Ring Systems of Maitotoxin.

Structure-activity relationship studies of maitotoxin (MTX), a marine natural product produced by an epiphytic dinoflagellate, were conducted using chemically synthesized model compounds corresponding to the partial structures of MTX. Both enantiomers of the LMNO ring system were synthesized via aldol reaction of the LM ring aldehyde and the NO ring ketone. These fragments were derived from a common cis-fused pyranopyran intermediate prepared through a sequence involving Nozaki-Hiyama-Kishi reaction, intramolecular oxa-Michael addition, and Pummerer rearrangement. The NOPQR(S) ring system, in which the original seven-membered S ring was substituted with a six-membered ring, was also synthesized through the coupling of the QR(S) ring alkyne and the NO ring aldehyde and the construction of the P ring via 1,4-reduction, dehydration, and hydroboration. The inhibitory activities of the synthetic specimens against MTX-induced Ca2+ influx were evaluated. The LMNO ring system and its enantiomer induced 36 and 18% inhibition, respectively, at 300 µM, whereas the NOPQR(S) ring system elicited no inhibitory activity.

Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus.

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area.