dinophysistoxin-2 and Diarrhea

Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties.

Topics: Animals; Calibration; Chromatography, Liquid; Diarrhea; Humans; Magnetic Resonance Spectroscopy; Marine Toxins; Okadaic Acid; Pyrans; Reference Standards; Shellfish; Shellfish Poisoning; Tandem Mass Spectrometry

Okadaic acid (OA) and its analogs, dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic biotoxins produced by marine algae that can accumulate in shellfish and cause the human illness known as diarrhetic shellfish poisoning (DSP). Regulatory testing of shellfish is required to protect consumers and the seafood industry. Certified reference materials (CRMs) are essential for the development, validation, and quality control of analytical methods, and thus play an important role in toxin monitoring. This paper summarizes work on research and development of shellfish tissue reference materials for OA and DTXs. Preliminary work established the appropriate conditions for production of shellfish tissue CRMs for OA and DTXs. Source materials, including naturally incurred shellfish tissue and cultured algae, were screened for their DSP toxins. This preliminary work informed planning and production of a wet mussel (Mytilus edulis) tissue homogenate matrix CRM. The homogeneity and stability of the CRM were evaluated and found to be fit-for-purpose. Extraction and LC-tandem MS methods were developed to accurately certify the concentrations of OA, DTX1, and DTX2 using a combination of standard addition and matrix-matched calibration to compensate for matrix effects in electrospray ionization. The concentration of domoic acid was also certified. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization. The presence of other toxins in the CRM was also assessed and information values are reported for OA and DTX acyl esters.

Topics: Animals; Calibration; Chromatography, Liquid; Diarrhea; Humans; Marine Toxins; Molecular Conformation; Okadaic Acid; Pyrans; Reference Standards; Shellfish; Shellfish Poisoning; Tandem Mass Spectrometry

Toxins produced by harmful algae are associated with detrimental health effects and mass mortalities of marine mammals. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is generally used to confirm the presence of algal toxins in marine mammals. Sample preparation and LC-MS/MS methods for the determination of three diarrhetic shellfish poisoning (DSP) toxins (okadaic acid, OA; dinophysistoxin-1, DTX1; dinophysistoxin-2, DTX2) and pectenotoxin-2 (PTX2) in bottlenose dolphin (Tursiops truncatus) urine and tissue samples were evaluated using spike-and-recovery tests. Sample clean-up with either reversed-phase silica or polymeric solid-phase extraction (SPE) reduced interference of sample matrices and improved toxin recoveries, with polymeric SPE showing higher sample loading capacity. LC separation on Xbridge C18 columns using acetonitrile/water gradient elutions with ammonia as the additive was chosen for its high detectivity and sensitivity in the MS detection of DSP toxins in negative ion mode. The retention times of OA, DTX1, and DTX2, separated as negative ions, increased with LC column temperature while the retention time of PTX2, separated as the neutral molecule, was weakly affected. At the same column temperature, retention times of OA, DTX1, and DTX2 gradually increased as the mobile phases aged while the retention time of PTX2 remained unchanged; higher column temperatures resulted in a greater increase in the retention time of each DSP toxin with mobile phase aging. Average recoveries of the 4 toxins in bottlenose dolphin samples ranged from 80% to 130% with relative standard deviations of less than 15% using the LC mobile phases prepared within one week at a column temperature of 30°C or 40°C. The preferred column temperature was 30°C, as the retention times of DSP toxins were less affected by mobile phase aging at this temperature. The limit of detection of each toxin analyzed in bottlenose dolphin samples was 2.8 ng/g or less in tissue samples and 0.7 ng/ml or less in urine.

Topics: Animals; Bottle-Nosed Dolphin; Chromatography, Liquid; Diarrhea; Furans; Macrolides; Marine Toxins; Okadaic Acid; Pyrans; Shellfish Poisoning; Solid Phase Extraction; Tandem Mass Spectrometry

Contamination of shellfish from the Portuguese coast with diarrhetic shellfish poisoning (DSP) toxins is a recurrent event, with most of the commercial bivalves contaminated with high percentages of esters of okadaic acid (OA) and dinophysistoxin-2 (DTX2). This report describes the quantification of DSP toxins in unhydrolysed and hydrolysed extracts of several cockle and mussel samples naturally contaminated and the evaluation of their cytotoxicity profiles in V79 cells. The quantification of the acyl esters in the shellfish samples involved the cleavage of the ester bond through alkaline hydrolysis and the release of the parent toxins OA and DTX2. Unhydrolysed and hydrolysed extracts were then analyzed by liquid chromatography (LC) coupled with mass spectrometry (MS) for the detection and quantification of DSP toxins. The cytotoxicity of the analysed extracts was evaluated using the MTT reduction assay and compared with the cytotoxicity presented by different concentrations of OA standard (1-100 nM). OA exhibited marked cytotoxic effects and decreased cell viability in a dose dependent mode, with an IC₅₀ of 27 nM. The cytotoxicity pattern of unhydrolysed extracts was clearly dependent on the concentration of free toxins. Moreover, the cytotoxicity of the esterified toxins present was revealed after their conversion into free toxins by alkaline hydrolysis. For the hydrolysed extracts of cockles and mussels, the cytotoxicity presented was mainly related to the concentration of OA and DTX2.

Topics: Animals; Bivalvia; Cell Survival; CHO Cells; Chromatography, Liquid; Cricetinae; Cricetulus; Diarrhea; Dose-Response Relationship, Drug; Esters; Food Contamination; Formazans; Humans; Hydrogen-Ion Concentration; Hydrolysis; Inhibitory Concentration 50; Marine Toxins; Mass Spectrometry; Molecular Structure; Okadaic Acid; Pyrans; Shellfish; Shellfish Poisoning; Tetrazolium Salts; Tissue Extracts

Diarrheic shellfish poisoning (DSP) is a recurrent gastrointestinal illness in Morocco, resulting from consumption of contaminated shellfish. In order to develop a rapid and reliable technique for toxins detection, we have compared the results obtained by a commercial immunoassay-"DSP-Check" kit" with those obtained by LC-MS. Both techniques are capable of detecting the toxins in the whole flesh extract which was subjected to prior alkaline hydrolysis in order to detect simultaneously the esterified and non esterified toxin forms. The LC-MS method was found to be able to detect a high level of okadaic acid (OA), low level of dinophysistoxin-2 (DTX2), and surprisingly, traces of azaspiracids 2 (AZA2) in mussels. This is the first report of a survey carried out for azaspiracid (AZP) contamination of shellfish harvested in the coastal areas of Morocco. The "DSP-Check" kit was found to detect quantitatively DSP toxins in all contaminated samples containing only OA, provided that the parent toxins were within the range of detection and was not in an ester form. A good correlation was observed between the two methods when appropriate dilutions were performed. The immunoassay kit appeared to be more sensitive, specific and faster than LC-MS for determination of DSP in total shellfish extract.

Topics: Animals; Bivalvia; Chromatography, Liquid; Data Collection; Diarrhea; Enzyme-Linked Immunosorbent Assay; Fisheries; Food Contamination; Furans; Marine Toxins; Mass Spectrometry; Morocco; Okadaic Acid; Pyrans; Reproducibility of Results; Shellfish Poisoning

The aim of this work was to shed light on the anatomical distribution of diarrhetic shellfish poisoning (DSP) toxins in the mussel Mytilus galloprovincialis and to determine any possible changes undergone during the depuration process. To this end, the distribution of two DSP toxins--okadaic acid and DTX2--and some of their derivatives were studied by means of HPLC/MS at different stages of the depuration process. Mussels were collected from mussel farms located in the Galician Rías and they were collected under three types of circumstances: (a) while ingesting toxic phytoplankton cells; (b) 1 week after the toxic cells had disappeared from the water; and (c) after ca. 2 months of depuration. Additionally, in case (b), the distribution among tissues was checked every week over a depuration period of 35 days in the laboratory. DSP toxins were only detected in non-visceral tissues when the extracts were concentrated 20-fold and, even in these cases, the concentrations found were very low. When the maximum possible contribution of non-visceral tissues was computed, taking into account the technique's detection limits and tissue weight, no relevant contribution to the toxin burden of non-visceral tissues was found at any stage of depuration, with the maximum possible contributions usually below 7%. The concentrated samples analysed showed that the actual contribution in all the cases studied was, in fact, less than 1% of the total toxin burden. These findings suggest that (1) when analytical methods are used to monitor DSP toxic mussels, non-visceral tissues should be assumed to be free of toxins in order to precisely compute the toxin concentration of the whole mass of edible tissues and (2) when studying the accumulation kinetics of DSP toxins, transference from the digestive gland to other tissues should not be taken into account, as the other tissues do not contain relevant amounts of DSP toxins.

Topics: Animals; Diarrhea; Marine Toxins; Mytilus; Okadaic Acid; Pyrans; Shellfish Poisoning; Tissue Distribution

Okadaic acid and dinophysistoxin-2 have been found yearly in Portuguese shellfish. Their presence was correlated with the occurrence of Dinophysis spp: Dinophysis acuminata has until now been found to be responsible only for OA contamination, while Dinophysis acuta contributes with OA and the rare diarrhetic toxin DTX2. Differences in toxicity levels may reflect different cell toxicities and different non-toxic phytoplankton availability as food source to shellfish.

Topics: Animals; Diarrhea; Dinoflagellida; Marine Toxins; Okadaic Acid; Pyrans; Seasons; Shellfish

A modification of the high-performance liquid chromatographic method with fluorimetric detection method for the determination of diarrhetic shellfish poisoning toxins was developed to completely avoid the use of dangerous chlorinated solvents. The method was validated for the toxin okadaic acid (OA) over a period of 6 months where 12 calibrations were performed and 72 samples were analyzed. Analysis of toxic and non-toxic mussels, clams and scallops demonstrated its selectivity. Linearity was observed in the tested range of interest for monitoring purposes of edible shellfish, from the limit of detection (0.3 microg OA/g hepatopancreas) to 13 microg OA/g hepatopancreas. Intra-assay precision of the method was 7% RSD at the quantification limit (0.97 microg OA/g hepatopancreas at S/N=10). Accuracy was tested in triplicate recovery experiments from OA-spiked shellfish where recovery ranged from 92 to 106% in the concentration range of 0.8 to 3.6 microg OA/g hepatopancreas. Useful information on critical factors affecting calibration and reproducibility is also reported. Good correlation (R=0.87) was observed between the results of the method and those of the method of Lee, after the analysis of 45 samples of mussels from the galician rias.

Topics: Calibration; Chlorine Compounds; Chromatography, High Pressure Liquid; Diarrhea; Fluorometry; Humans; Mollusk Venoms; Okadaic Acid; Pyrans; Quality Control; Reference Standards; Reproducibility of Results; Shellfish; Solvents

The rare diarrhoeic shellfish poisoning (DSP) toxin, dinophysistoxin-2 (DTX-2), which is an okadaic acid (OA) isomer, has been isolated from a marine phytoplankton biomass that consisted mainly of Dinophysis acuta. Using a large double plankton net (length 5.9 m), bulk phytoplankton samples were collected off the south-west coast of Ireland and extracted with methanol and chloroform. Liquid chromatography coupled with ionspray mass spectrometry and tandem mass spectrometry (LC-MS, LC-MS-MS) showed the sample contained DTX-2 and OA, at a concentration of 80 pg/cell and 60 pg/cell, respectively. Flash chromatography using silica, sephadex LH20 and C18-silica, followed by preparative reversed-phase LC, separated DTX-2 from OA. The efficiency of the separation procedures was substantially improved by the use of a bioscreen to detect DSP toxins in eluate fractions and the application of a new derivatisation procedure for the chromatographic elucidation of toxin profiles with fluorimetric detection (LC-FLD). Thus, 1/1000th aliquots of eluate fractions were assayed using protein phosphatase-2A for the presence of inhibitory compounds. Positive fractions were further analysed for DSP toxins by LC-FLD following derivatisation using the hydrazine reagent, luminarine-3. The identity and purity of the free isolated DTX-2 was confirmed using flow injection analysis (FIA) and liquid chromatography (FIA-MS, LC-MS and LC-MS-MS).

Topics: Animals; Diarrhea; Fluorometry; Gas Chromatography-Mass Spectrometry; Hydrazines; Marine Toxins; Okadaic Acid; Phosphoprotein Phosphatases; Phytoplankton; Protein Phosphatase 2; Pyrans; Shellfish; Stereoisomerism

Liquid chromatography (HPLC) was used to search for esters of DSP toxins in Portuguese bivalves. Hexane-soluble derivatives of okadaic acid (OA) and dinophysistoxin-2 (DTX-2) were found. Presumably they are acyl derivatives, globally known as 'dinophysistoxin-3' (DTX-3). In certain instances DTX-3 content surpassed 50% of the total amount of DSP toxins. A human diarrhetic poisoning (DSP) incident with Donax clams (Donax trunculus) harvested at Fuzeta (Algarve coast) was confirmed where the apolar (DTX-3 type) and other esters remaining in the polar aqueous methanol layer were implicated. The percentage of acyl esters of OA was always higher than those of DTX-2. An enzymic mechanism for the conversion of OA and DTX-2 seems to be implicated in some kind of detoxification process because the percentage of esters increases with the toxin amount ingested by the bivalve and there is some degree of selectivity as DTX-2 seems more difficult to acylate. These findings pose a problem for the current assay methods used to detect DSP because mainly they are able to detect the parent toxins but not their esters. The current bioassay method [Le Baut, C., Bardin, B., Bardouil, M., Bohec, M., Masselin, P., Truquet, P., 1990. Etude de la decontamination de moules toxiques. Rapport IFREMER DERO-90-02 MR. 21 pp.] used in Portugal includes a hexane washing step that prevents interference from free fatty acids. However, it cannot detect the presence of acyl derivatives because they are highly soluble in hexane.

Topics: Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid; Diarrhea; Enzyme Inhibitors; Fluorescence; Humans; Marine Toxins; Okadaic Acid; Portugal; Pyrans; Shellfish; Solubility

Okadaic acid (OA) and dinophysistoxin-2, two of the main diarrhetic shellfish toxins, can be determined by high-performance liquid chromatography coupled to fluorimetry as pyrenacyl esters. Toxin fluorescent derivatives were obtained after quantitative derivatization with 1-bromoacetylpyrene in acetonitrile. An efficient improvement in the silica gel clean-up procedure of the pyrenacyl derivatives is reported. The clean-up cartridge is washed with hexane-dichloromethane (1:1, v/v), dichloromethane-ethyl acetate (8:2, v/v), and finally the pyrenacyl esters were eluted with dichloromethane-methanol (9:1, v/v). We compare this procedure with other methods already described. Good results were obtained with mussels, scallops and clams. The clean-up procedure showed good robustness when checked against silica and solvents activity. Using samples of mussel hepatopancreas with an OA concentration ranging from 0 to 2 micrograms OA/g hepatopancreas, the inter-assay relative standard deviation ranged from 5.5 to 12.6%.

Topics: Animals; Chromatography, High Pressure Liquid; Circadian Rhythm; Diarrhea; Fluorescent Dyes; Foodborne Diseases; Marine Toxins; Mollusca; Okadaic Acid; Pyrans; Pyrenes; Reproducibility of Results; Sensitivity and Specificity; Shellfish; Spectrometry, Fluorescence

A new analogue of okadaic acid (OA), the toxin mainly responsible for diarrhetic shellfish-poisoning (DSP) phenomena in Europe, has been isolated from toxic phytoplankton (Dinophysis acuta) collected in Irish waters. Fluorimetric LC analyses of the extracts of bulk phytoplankton samples using derivatisation with 9-anthryldiazomethane (ADAM) showed a complex toxin profile, with peaks corresponding to OA and dinophysistoxin-2 (DTX-2) as well as a third unidentified compound. This minor unidentified component was isolated by chromatographic techniques such as normal-phase chromatography, gel permeation on Sephadex, solid-phase extraction and reversed-phase separations. Ionspray mass spectrometry (MS) was used for structural investigation on this compound due to the very small amount of isolated material. Flow injection analysis (FIA)-MS of the isolated compound gave positive-ion mass spectrum dominated by the protonated molecule, [M + H]+, at signal m/z 805, whereas the deprotonated molecule [M - H]- was observed in the negative-ion spectrum at signal m/z 803, thus indicating the molecular weight of 804 for the new toxin, the same as OA and its known isomers, DTX-2 and DTX-2B. Collision-induced dissociation (CID) as obtained by positive and negative tandem mass spectrometry (MS-MS) showed a fragmentation pattern for the new compound which was very similar to that of OA, DTX-2 and DTX-2B. Ionspray microLC-MS of a mixture containing the compound under investigation together with OA analogues showed the compound eluted after OA, DTX-2, DTX-2B and before DTX-1. All the chromatographic and mass spectrometric data indicated the compound to be another OA isomer and it was therefore coded DTX-2C. To the best of our knowledge this is the first report on the isolation of a new compound related to DSP toxins from natural communities of toxic phytoplankton.

Topics: Anthracenes; Chromatography, High Pressure Liquid; Diarrhea; Fluorescent Dyes; Fluorometry; Food Analysis; Foodborne Diseases; Marine Toxins; Mass Spectrometry; Okadaic Acid; Phytoplankton; Pyrans

Mussel aquaculture is an important industry for the Galician Rias, located in northwestern Atlantic coast of Spain. Since 1976 this region has been seriously affected by incidents of paralytic and diarrhetic shellfish poisoning (PSP and DSP). A particularly bad episode occurred in 1993, when the toxic event lasted for an unusually long period. Many people were stricken ill with unusual symptoms. In this paper we report on the chemical analysis of toxic 1993 mussel samples, using the techniques of liquid chromatography and capillary electrophoresis coupled with mass spectrometry. These analyses revealed a very complex toxin profile, with both PSP and DSP toxins present. Two DSP toxins, okadaic acid and DTX2, were observed, while the primary PSP toxins were B1 and the decarbamoylated derivatives of saxitoxin, GTX2 and GTX3. Small amounts of saxitoxin and other as yet unidentified PSP toxins were observed.

Topics: Animals; Aquaculture; Bivalvia; Carcinogens; Chromatography, High Pressure Liquid; Diarrhea; Dinoflagellida; Electrophoresis, Capillary; Gas Chromatography-Mass Spectrometry; Marine Toxins; Okadaic Acid; Paralysis; Pyrans; Shellfish Poisoning; Spain; Structure-Activity Relationship

Diarrhoetic shellfish poisoning (DSP) in Europe is due mainly to the presence of the dinoflagellate toxin, okadaic acid (OA). However, analysis of cultivated mussels (Mytilus edulis) from southwest Ireland revealed that an isomer of OA, dinophysistoxin-2, was the major toxin present during DSP episodes. Using fluorimetric HPLC, following derivatisation with 9-anthryldiazomethane, both OA and DTX-2 were found in shellfish during a prolonged toxic episode in 1991. However, examination of similar mussel cultivation locations in 1994 showed that DTX-2 was even more predominant. During this DSP period, OA levels were less than 0.7 microgram/g, whereas maximum DTX-2 levels of 6.3 micrograms/g hepatopancreas were recorded. This toxicity in shellfish occurred soon after high cell counts of Dinophysis acuta were observed. As well as large seasonal variability in toxin levels in rope cultured mussels, substantial variations were also observed, both horizontally and vertically, within the water column.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Diarrhea; Dinoflagellida; Enzyme Inhibitors; Ethers, Cyclic; Ireland; Marine Toxins; Okadaic Acid; Pyrans; Reference Standards; Seawater; Shellfish Poisoning; Stereoisomerism

Two types of low polar derivatives of OA and dinophysitoxins have been reported in shellfish or in phytoplankton: 7-0-acyl esters containing a fatty acyl group attached through the 7-OH group and diol esters in which the carboxylic group of the toxins has been esterified. These compounds cannot be directly detected by liquid chromatography and fluorimetric detection as 9-anthryldiazomethane derivatives, owing in the first case to their low polarity and high molecular weight, and in the second case because they have the carboxylic group esterified. All of them must be hydrolysed before derivatization to be detected as Adam derivatives of the corresponding non-acylated toxins. In the Lee procedure, after extraction of the shellfish digestive glands with 80% methanol, a liquid-liquid partition with a non-polar solvent such as hexane is carried out in order to remove non-polar lipids. The presence of non-polar toxins was investigated in Spanish mussels and confirmed in the hexane layer, usually discarded in conventional extraction procedures, by analysis of the alkaline hydrolysis products. A preferred solubilization of these toxins in a non-polar solvent like hexane is reported. The inclusion of a hydrolytic step of the hexane extract in the general procedure is suggested in order to monitor the contribution of non-polar diarrhoetic shellfish poisons (DSPs) to the total DSP shellfish toxicity. This is the first report of DSPs other than OA and DTX2 in Spanish mussels.

Topics: Animals; Bivalvia; Carcinogens; Chromatography, High Pressure Liquid; Chromatography, Liquid; Diarrhea; Dinoflagellida; Esters; Ethers, Cyclic; Hydrogen-Ion Concentration; Hydrolysis; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Shellfish Poisoning; Solvents; Spain; Structure-Activity Relationship

A novel method for the detection of acylated diarrhetic shellfish poisoning toxins is reported. Direct determination of these compounds is possible using high performance liquid chromatography coupled with ion-spray mass spectrometry. An extract, purified from the digestive glands of toxic mussels (Mytilus edulis) contaminated with okadaic acid, dinophysistoxin-1, and a recently reported analog, dinophysistoxin-2, was also shown to contain small amounts of dinophysistoxin-3, a mixture of 7-O-acyl ester derivatives of dinophysistoxin-1. In addition, acyl ester derivatives of okadaic acid and dinophysistoxin-2 were also detected by direct LC-MS analysis and confirmed by analysis of their hydrolysis products. This is the first report of the detection of other naturally occurring 7-O-acyl esters similar to dinophysistoxin-3.

Topics: Acylation; Animals; Bivalvia; Chromatography, Liquid; Chromatography, Thin Layer; Diarrhea; Ethers, Cyclic; Hydrolysis; Magnetic Resonance Spectroscopy; Marine Toxins; Mass Spectrometry; Okadaic Acid; Pyrans; Shellfish; Spectrophotometry, Ultraviolet; Vasoconstrictor Agents