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.

Relative molar response of lipophilic marine algal toxins in liquid chromatography/electrospray ionization mass spectrometry.

RATIONALE: Accurate quantitative analysis of lipophilic toxins by liquid chromatography/mass spectrometry (LC/MS) requires calibration solution reference materials (RMs) for individual toxin analogs. Untargeted analysis is aimed at identifying a vast number of compounds and thus validation of fully quantitative untargeted methods is not feasible. However, a semi-quantitative approach allowing for profiling is still required and will be strengthened by knowledge of the relative molar response (RMR) of analogs in LC/MS with electrospray ionization (ESI). METHODS: RMR factors were evaluated for toxins from the okadaic acid (OA/DTXs), yessotoxin (YTX), pectenotoxin (PTX), azaspiracid (AZA) and cyclic imine (CI) toxin groups, in both solvent standards and environmental sample extracts. Since compound ionization and fragmentation influences the MS response of toxins, RMRs were assessed under different chromatographic conditions (gradient, isocratic) and MS acquisition modes (SIM, SRM, All-ion, target MS/MS) on low- and high-resolution mass spectrometers. RESULTS: In general, RMRs were not significantly impacted by chromatographic conditions (isocratic vs gradient), with the exception of DTX1. MS acquisition modes had a more significant impact, with PnTX-G and SPX differing notably. For a given toxin group, response factors were generally in the range of 0.5 to 2. The cyclic imines were an exception. CONCLUSIONS: Differences in RMRs between toxins of a same chemical base structure were not significant enough to indicate major issues for non-targeted semi-quantitative analysis, where there is limited or no availability of standards for many compounds, and where high degrees of accuracy are not required. Differences in RMRs should be considered when developing methods that use a standard of a single analogue to quantitate other toxins from the same group.

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.

Passive Sampling and High Resolution Mass Spectrometry for Chemical Profiling of French Coastal Areas with a Focus on Marine Biotoxins.

Passive samplers (solid phase adsorption toxin tracking: SPATT) are able to accumulate biotoxins produced by microalgae directly from seawater, thus providing useful information for monitoring of the marine environment. SPATTs containing 0.3, 3, and 10 g of resin were deployed at four different coastal areas in France and analyzed using liquid chromatography coupled to high resolution mass spectrometry. Quantitative targeted screening provided insights into toxin profiles and showed that toxin concentrations and profiles in SPATTs were dependent on the amount of resin used. Between the three amounts of resin tested, SPATTs containing 3 g of resin appeared to be the best compromise, which is consistent with the use of 3 g of resin in SPATTs by previous studies. MassHunter and Mass Profiler Professional softwares were used for data reprocessing and statistical analyses. A differential profiling approach was developed to investigate and compare the overall chemical diversity of dissolved substances in different coastal water bodies. Principal component analysis (PCA) allowed for spatial differentiation between areas. Similarly, SPATTs retrieved from the same location at early, medium, and late deployment periods were also differentiated by PCA, reflecting seasonal variations in chemical profiles and in the microalgal community. This study used an untargeted metabolomic approach for spatial and temporal differentiation of marine environmental chemical profiles using SPATTs, and we propose this approach as a step forward in the discovery of chemical markers of short- or long-term changes in the microbial community structure.

Complex toxin profile of French Mediterranean Ostreopsis cf. ovata strains, seafood accumulation and ovatoxins prepurification.

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 µg·kg-1 of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 µg·kg-1 in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell-1. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.

Quantitative analysis of azaspiracids in Azadinium spinosum cultures.

Azaspiracids (AZAs) are secondary metabolites of Azadinium spinosum that can accumulate in shellfish and cause food poisoning when consumed. We describe here an analytical procedure for the determination of AZAs in cultures of A. spinosum with a focus on the formation of AZA methyl esters as artefacts during extraction and sample pre-treatment. A. spinosum cells were collected from bioreactor cultures using centrifugation or filtration. Different extraction procedures were evaluated for formation of methyl ester artefacts, yield, and matrix effects. Filtration of cultures using glass-fibre filters led to increased formation of methyl esters, and centrifugation is recommended for recovery of cells. The extraction solvent (methanol (MeOH), acetone, and acetonitrile (MeCN)) did not significantly affect the yield of AZAs as long as the organic content was 80% or higher. However, the use of MeOH as extraction solvent led to increased formation of methyl esters. AZA1 recovery over two successive extractions was 100% at the 95% confidence level for acetone and MeOH. In standard-addition experiments, no significant matrix effects were observed in extracts of A. spinosum or Azadinium obesum up to a sample size of 4.5 × 10(9) µm(3). Moreover, experiments carried out to clarify the formation and structure of methylated AZA analogues led to the description of two AZA methyl esters and to the correction of the chemical structures of AZAs29-32.

Production and isolation of azaspiracid-1 and -2 from Azadinium spinosum culture in pilot scale photobioreactors.

Azaspiracid (AZA) poisoning has been reported following consumption of contaminated shellfish, and is of human health concern. Hence, it is important to have sustainable amounts of the causative toxins available for toxicological studies and for instrument calibration in monitoring programs, without having to rely on natural toxin events. Continuous pilot scale culturing was carried out to evaluate the feasibility of AZA production using Azadinium spinosum cultures. Algae were harvested using tangential flow filtration or continuous centrifugation. AZAs were extracted using solid phase extraction (SPE) procedures, and subsequently purified. When coupling two stirred photobioreactors in series, cell concentrations reached 190,000 and 210,000 cell · mL(-1) at steady state in bioreactors 1 and 2, respectively. The AZA cell quota decreased as the dilution rate increased from 0.15 to 0.3 day(-1), with optimum toxin production at 0.25 day(-1). After optimization, SPE procedures allowed for the recovery of 79 ± 9% of AZAs. The preparative isolation procedure previously developed for shellfish was optimized for algal extracts, such that only four steps were necessary to obtain purified AZA1 and -2. A purification efficiency of more than 70% was achieved, and isolation from 1200 L of culture yielded 9.3 mg of AZA1 and 2.2 mg of AZA2 of >95% purity. This work demonstrated the feasibility of sustainably producing AZA1 and -2 from A. spinosum cultures.

Investigation of hydrophilic interaction liquid chromatography coupled with charged aerosol detector for the analysis of tromethamine.

Tromethamine (TMM), often encountered in a final drug product, exhibits interesting chemical properties as a counter ion, buffer, or active ingredient. European and US pharmacopeias propose titration against hydrogen chloride for TMM assays. However, this method can be a hindrance when using drugs containing low concentrations of TMM in complex buffered formulations. Due to the lack of chromophores and the high hydrophilicity of TMM, we performed a simple and reliable hydrophilic interaction chromatography coupled with a charged aerosol detector (HILIC-CAD) separation approach as an alternative for TMM analysis. An amide stationary phase and a mobile phase consisting of a binary mixture of acetonitrile and 10 mM ammonium formate, pH 3 (80/20, V/V) were used. As the CAD response deeply depends on parameters such as stationary phases and pH buffer, we investigated their impact and explored the optimal signal conditions. Including TMM analogs such as tris(hydroxymethyl) nitromethane and 2-amino-2-ethyl-1,3-propanediol allowed us to select these parameters appropriately. The effects of the evaporation temperature, flow rate, and power function value (PFV) on the CAD signal response were also studied and optimized. The method was validated according to the ICH Q2 R1 guidelines. A linear response (mean R2 > 0.997) covering the range for low TMM concentrations (170-520 µg/mL) was achieved. Satisfactory intra-day and inter-day precisions were obtained with RSDs lower than 1.9% and 2.8%, respectively. The trueness ranged from 99.6% to 101.2%, and the LOD was found to be 1.1 µg/mL. The HILIC-CAD method has been applied to a sterile TMM solution for injection.

Hexylacrylate-based mixed-mode monolith, a stationary phase for the nano-HPLC separation of structurally related enkephalins.

The potential of an in situ photopolymerized hexylacrylate-based monolithic stationary phase-bearing sulfonic acid groups was investigated by studying the chromatographic retention of small structurally related peptides (enkephalins) by nano-LC. Several retention mechanisms were highlighted. First, a reverse-phase chromatographic behavior toward neutral solutes due to hexylacrylate-moieties was demonstrated. Second, an evaluation of the influences of buffer pH suggested the involvement of a cation-exchange mechanism due to the presence of 2-acrylamido-2-methyl-1-propanesulfonic acid. This cation-exchange phenomenon was confirmed by the clear influence of Na(+) concentration in the mobile phase on peptide retention.

Rare earth element organotropism in European eel (Anguilla anguilla).

Rare earth elements (REEs) are metallic elements with electronic, magnetic, optical and catalytic properties which make them essential in many industrial and medical fields. REEs are therefore becoming emerging pollutants and it is important to understand their implications for ecosystem health. However, little knowledge of REE bioaccumulation in aquatic organisms is available and especially on their internal distribution in fish. In the present study, REE organotropism was determined in Anguilla anguilla from the Loire estuary (France) by determining burdens in a wide set of tissues, organs and biological fluids. Differences have been observed between life stages and genders. For yellow eels, the most accumulating organ was the gills (126.90 ± 50.78 µg/kg dw) and for silver eels, it was the liver (181.78 ± 62.04 µg/kg dw for males; 203.79 ± 111.86 µg/kg dw for females). The comparison between female silver and yellow eels shown that female silver individuals accumulated significantly more REEs in the urinary system (US), muscles, gonads, spleen and liver, while yellow individuals accumulated more in gills. The comparison between male and female silver eels also highlighted differences, indeed the females accumulated significantly more REEs in the US, gonads, skin and spleen, compared to males which accumulated significantly more in muscles and gills. REEs abundances are also different between organs, life stages and genders. The gonads of female silver eels exhibited a particular profile with the dominance of gadolinium (Gd) (up to 74.2% of ∑REEs). Moreover, the presence of Anguillicola crassus in the swim bladder of organisms seemed to have an impact on REE bioaccumulation: parasitized yellow eels present higher concentrations of REEs in muscles, gills, gonads and liver than non-parasitized individuals. Regarding glass eels, REE contribution profiles in the whole body were close to those of yellow and silver eel skin.

Chemical composition and antimicrobial activity of the essential oils from New Caledonian Citrus macroptera and Citrus hystrix.

The essential oils from the leaves of Citrus macroptera and C. hystrix, collected in New Caledonia, have been analyzed by gas chromatography/mass spectrometry (GC/MS) and evaluated for their antimicrobial activity. A total of 35 and 38 constituents were identified, representing 99.1 and 89.0% of the essential oils, respectively. Both essential oils were rich in monoterpenes (96.1 and 87.0%, resp.), with beta-pinene as major component (33.3 and 10.9%, resp.), and poor in limonene (2.4 and 4.7%, resp.). Other main components of C. macroptera oil were alpha-pinene (25.3%), p-cimene (17.6%), (E)-beta-ocimene (6.7%), and sabinene (4.8%). The essential oil of C. hystrix was characterized by high contents of terpinen-4-ol (13.0%), alpha-terpineol (7.6%), 1,8-cineole (6.4%), and citronellol (6.0%). The antimicrobial activity was evaluated against five bacteria and five fungi strains. Both oils were inactive against bacteria. However, the C. macroptera leaf oil exhibited a pronounced activity against Trichophyton mentagrophytes var. interdigitale, with a minimal-inhibitory concentration (MIC) of 12.5 microg/ml.

Rare earth element bioaccumulation in the yellow and silver European eel (Anguilla anguilla): A case study in the Loire estuary (France).

In the present work, rare earth elements (REEs) were measured in European eel muscles (Anguilla anguilla) from the Loire estuary in France. This study site is characterized by a large anthropogenic pressure with potential activities releasing REEs such as oil refineries, aeronautic and naval industries, wind turbine industries, hospitals with magnetic resonance imaging and coal-fired power plants. These activities may lead to increased REE concentrations in sediments the primary habitat of European eels. In the present work, REE bioaccumulation was evaluated by determining the concentrations in yellow and silver eel muscles sampled at three different locations in the Loire estuary and at two periods (2011/2012 and 2018/2019). The aims of this study were the understanding of the spatio-temporal influences (sampling site and sampling period) and intraspecific variations (age, sex, sexual maturation, length, weight, and parasitism) on the whole REE bioaccumulation. The mean value of the sum of REE concentrations (∑REEs) was 2.91, 6.48 and 12.60 µg/kg of muscle from respectively yellow eels, female silver eels and male silver eels fished in 2018/2019. The results showed that silver males accumulated more REEs than silver females and silver eels accumulate more REEs than yellow ones. Regarding the determination of spatio-temporal variations, an increase of REE concentrations for silver eel muscles between the two periods was observed, certainly related to the increase of REE uses. Finally, a trend of higher contamination of eels sampled in the downstream of Nantes was noticed for yellow eels.

Detection of pacific ciguatoxins using liquid chromatography coupled to either low or high resolution mass spectrometry (LC-MS/MS).

Ciguatera Fish Poisoning (CFP) is primarily caused by consumption of tropical and sub-tropical fish contaminated by Ciguatoxins (CTXs). These lipid-soluble, polyether neurotoxins are produced by dinoflagellates in the genera Gambierdiscus and Fukuyoa. While there is no regulatory level in Europe for CTXs, the European Food Safety Authority (EFSA) adopted the United States guidance level of 0.01 µg P-CTX1B eq.kg-1 of fish. This limit is extremely low and requires significant improvement in the detection of CTXs. In this study, we compared analytical protocols based on liquid chromatography coupled to tandem low or high resolution mass spectrometry (LC-LRMS or HRMS) to find the best conditions for sensitivity and/or selectivity. Different approaches such as LC conditions, ion choice and acquisition modes, were evaluated to detect the Pacific-ciguatoxins (P-CTXs) on a triple quadrupole (API4000 Qtrap, Sciex) or a quadrupole time of flight (QTOF 6550, Agilent Technologies) spectrometer. Moreover, matrix effects were calculated using matrix-matched calibration solutions of P-CTX1B and P-CTX3C prepared in purified fish extract. Subsequently, the method performance was assessed on naturally contaminated samples of seafood and phytoplankton. With LRMS, the ammoniated adduct ion used as a precursor ion showed an advantage for selectivity through confirmatory transitions, without affecting signal-to-noise ratios, and hence limits of detection (LODs). As also reported by some studies in the literature, methanol-based mobile phase gave better selectivity and sensitivity for the detection of P-CTXs. While the LOD for P-CTX1B and P-CTX3C met the EFSA recommendation level when using LRMS, the findings suggested careful evaluation of instrumental parameters for determination of CTXs. LODs were significantly higher for HRMS, which currently results in the need for a significantly higher sample intake. Nevertheless, HRMS allowed for the identification of artefacts and may allow for improved confirmation of the identity of P-CTXs analogues. Consequently, LRMS and HRMS are considered complementary to ensure adequate quantitation and identification of P-CTXs.

Metabolism of 2-substituted quinolines with antileishmanial activity studied in vitro with liver microsomes, hepatocytes and recombinantly expressed enzymes analyzed by LC/MS.

Liver microsome and hepatocyte-mediated biotransformation of three oral antileishmanial 2-substituted quinolines were investigated. One quinoline contains an n-propyl group (1) and the other a propenyl chain functionalized at the gamma position either by a nitrile (2) or an alcohol (3). The different isoforms of rat cytochrome P450 responsible for biotransformation of 1 were also investigated. Compounds 2 and 3 mainly reacted with glutathione, preventing further metabolism. Compound 3 however, the reaction being reversible, could be released from glutathione and take alternative reaction pathways. Microsomal incubations of 1 mainly led to hydroxylation of the side chain, involving many cytochromes, predominantly CYP2B1, CYP2A6 and CYP1A1 (at more than 80%). In contrary, minor metabolites hydroxylated on the quinoline ring involved a few cytochromes. The hydroxylated products of 1 were conjugated with glucuronic acid in rat hepatocyte incubations.

Development of a SPE/HPLC/DAD method for the determination of antileishmanial 2-substituted quinolines and metabolites in rat plasma.

A SPE/HPLC/DAD method was developed for the in vivo monitoring of three new antileishmanial 2-substituted quinolines under study in our laboratory for the development of an oral treatment. Three phase I metabolites were included in this work for the optimization of the method. Trifunctional tC(18) cartridges (resulting from the reaction of trifunctional silanes with silica surface) were selected among four sorbents tested. Two linear gradients were developed to ensure resolution of metabolites. Recovery of quinolines from rat plasma was comprised between 80.6 and 88.2%. In a drug development perspective, apparent pK(a), lipophilicity and solubility were determined, as well as the extent of plasma protein or albumin binding.

Antileishmanial 2-substituted quinolines: in vitro behaviour towards biological components.

Quinolines substituted on their carbon 2 have in vivo antileishmanial activity but some of them could not be detected in plasma when assayed for pharmacokinetic studies, suggesting a sequestration of the drugs by components of the blood compartment. The present study, performed on three quinolines (1, 2 and 3), showed strong affinity for two of them (2 and 3) with red blood cells (RBCs), whereas quinoline 1 did not react with them. This process was saturable, temperature dependant and positively correlated with the in vitro antileishmanial activity of the quinolines. In addition, a rapid and spontaneous reaction with thiol groups was demonstrated for unsaturated quinolines 2 and 3. The reactivity with RBCs could be part of the compounds targeting to the parasite. These results illustrate that derivatives of the quinoline series with similar antileishmanial in vivo activity have different behaviour in the blood compartment.

Antiparasitic activity of some New Caledonian medicinal plants.

Twenty-nine extracts of 18 medicinal plants used in New Caledonia by traditional healers to treat inflammation, fever and in cicatrizing remedies were evaluated in vitro against several parasites (Leishmania donovani, Trypanosoma brucei brucei, Trichomonas vaginalis and Caenorhabditis elegans). Among the selected plants, Scaevola balansae and Premna serratifolia L. were the most active against Leishmania donovani with IC(50) values between 5 and 10microg/ml. The almond and aril extracts from Myristica fatua had an IC(50) value of 0.5-5microg/ml against Trypanosoma brucei brucei. Only Scaevola balansae extract presented a weak activity against Trichomonas vaginalis. The almond extract from Myristica fatua presented significant activity against Caenorhabditis elegans (IC(50) value of 6.6+/-1.2microg/ml).

Hydrophilic interaction liquid chromatography for dalargin separation from its structural analogues and side products.

Retention behaviour of Dalargin and five peptide analogues of Leu-enkephalin, has been extensively studied by hydrophilic interaction liquid chromatography (HILIC) on a bare silica stationary phase (Atlantis® HILIC silica). The influence of buffer pH, ionic strength, and organic modifier content on peptide retentions was examined. Variation of organic modifier content (70-90% ACN) shows that, as expected, the most polar peptide, Dalargin, is the most retained. Moreover, at acidic pH, the retention mechanism for all the peptides studied seems to rely, mainly, on adsorption phenomenon. By varying the pswH buffer (between 4.4-7.5), we observed that the retention of all the peptides was mainly governed by their total number of charges, whatever the variation (increase or decrease) of their retention factor. At pswH 7.5, an increase of the cationic counter-ion concentration (NH4+) lead to a decrease of the retention factor of Dalargin, suggesting a weak cation exchange for this peptide. For the other peptides, the variation of the retention factors was negligible between 5-15mM. Above 15mM, the retention factors of all the peptides increased, probably due to an increase of the water layer thickness at the surface of the stationary phase. In the second part of the study, qualitative analysis of non-purified dalargin, resulting from solid-phase synthesis, was realized. Optimisation of the separation of the target peptide from its side products has been first performed with UV detection. Then, by coupling the HILIC column with ESI-MS, using the optimal separation conditions, it was possible to identify Dalargin and to propose the amino-acids sequence of its side-products.

Toxicity screening of 13 Gambierdiscus strains using neuro-2a and erythrocyte lysis bioassays.

Species in the epi-benthic dinoflagellate genus Gambierdiscus produce ciguatoxins (CTXs) and maitotoxins (MTXs), which are among the most potent marine toxins known. Consumption of fish contaminated with sufficient quantities of CTXs causes Ciguatera Fish Poisoning (CFP), the largest cause of non-bacterial food poisoning worldwide. Maitotoxins, which can be found in the digestive system of fish, could also contribute to CFP if such tissues are consumed. Recently, an increasing number of Gambierdiscus species have been identified; yet, little is known about the variation in toxicity among Gambierdiscus strains or species. This study is the first assessment of relative CTX- and MTX-toxicity of Gambierdiscus species from areas as widespread as the North-Eastern Atlantic Ocean, Pacific Ocean and the Mediterranean Sea. A total of 13 strains were screened: (i) seven Pacific strains of G. australes, G. balechii, G. caribaeus, G. carpenteri, G. pacificus, G. scabrosus and one strain of an undetermined species (Gambierdiscus sp. Viet Nam), (ii) five strains from the North-Eastern Atlantic Ocean (two G. australes, a single G. excentricus and two G. silvae strains), and (iii) one G. carolinianus strain from the Mediterranean Sea. Cell pellets of Gambierdiscus were extracted with methanol and the crude extracts partitioned into a CTX-containing dichloromethane fraction and a MTX-containing aqueous methanol fraction. CTX-toxicity was estimated using the neuro-2a cytoxicity assay, and MTX-toxicity via a human erythrocyte lysis assay. Different species were grouped into different ratios of CTX- and MTX-toxicity, however, the ratio was not related to the geographical origin of species (Atlantic, Mediterranean, Pacific). All strains showed MTX-toxicity, ranging from 1.5 to 86pg MTX equivalents (eq) cell-1. All but one of the strains showed relatively low CTX-toxicity ranging from 0.6 to 50 fg CTX3C eq cell-1. The exception was the highly toxic G. excentricus strain from the Canary Islands, which produced 1426 fg CTX3C eq cell-1. As was true for CTX, the highest MTX-toxicity was also found in G. excentricus. Thus, the present study confirmed that at least one species from the Atlantic Ocean demonstrates similar toxicity as the most toxic strains from the Pacific, even if the metabolites in fish have so far been shown to be more toxic in the Pacific Ocean.

Algal toxin profiles in Nigerian coastal waters (Gulf of Guinea) using passive sampling and liquid chromatography coupled to mass spectrometry.

Algal toxins may accumulate in fish and shellfish and thus cause poisoning in consumers of seafood. Such toxins and the algae producing them are regularly surveyed in many countries, including Europe, North America, Japan and others. However, very little is known regards the occurrence of such algae and their toxins in most African countries. This paper reports on a survey of phytoplankton and algal toxins in Nigerian coastal waters. Seawater samples were obtained from four sites for phytoplankton identification, on three occasions between the middle of October 2014 and the end of February 2015 (Bar Beach and Lekki in Lagos State, Port Harcourt in Rivers State and Uyo in Akwa Ibom State). The phytoplankton community was generally dominated by diatoms and cyanobacteria; however several species of dinoflagellates were also identified: Dinophysis caudata, Lingulodinium polyedrum and two benthic species of Prorocentrum. Passive samplers (containing Diaion(®) HP-20 resin) were deployed for several 1-week periods on the same four sites to obtain profiles of algal toxins present in the seawater. Quantifiable amounts of okadaic acid (OA) and pectenotoxin 2 (PTX2), as well as traces of dinophysistoxin 1 (DTX1) were detected at several sites. Highest concentrations (60 ng OA g(-1) HP-20 resin) were found at Lekki and Bar Beach stations, which also had the highest salinities. Non-targeted analysis using full-scan high resolution mass spectrometry showed that algal metabolites differed from site to site and for different sampling occasions. Screening against a marine natural products database indicated the potential presence of cyanobacterial compounds in the water column, which was also consistent with phytoplankton analysis. During this study, the occurrence of the marine dinoflagellate toxins OA and PTX2 has been demonstrated in coastal waters of Nigeria, despite unfavourable environmental conditions, with regards to the low salinities measured. Hence shellfish samples should be monitored in future to assess the risk for public health through accumulation of such toxins in seafood.