Investigation of the toxin profile of Greek mussels Mytilus galloprovincialis by liquid chromatography-mass spectrometry.

Samples of Mytilus galloprovincialis were harvested from five different locations in Thermaikos gulf, Greece after harmful algae bloom. All of the mussel samples were found positive by mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins. Liquid chromatography (LC) coupled with mass spectrometry (MS) was used to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and yessotoxins (YTXs). In order to investigate the presence of okadaic acid esters, alkaline hydrolysis was performed for all the samples, and LC-MS analyses were carried out on the samples before and after hydrolysis. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) analyses were also carried out to investigate the presence of domoic acid and paralytic shellfish poisoning (PSP) toxins at trace levels. All of the samples were found to be contaminated only with okadaic acid at levels 0.10-0.20 microg/g.     

Dinoflagellate polyether within the yessotoxin, pectenotoxin and okadaic acid toxin groups: Characterization, analysis and human health implications

Diarrhetic Shellfish Poisoning (DSP) is a specific type of food poisoning, characterized by severe gastrointestinal illness due to the ingestion of filter feeding bivalves contaminated with a specific suite of toxins. It is known that the problem is worldwide and three chemically different groups of toxins have been historically associated with DSP syndrome: okadaic acid (OA) and dinophysistoxins (DTXs), pectenotoxins (PTXs) and yessotoxins (YTXs). PTXs and YTXs have been considered as DSP toxins because they can be detected with the bioassays used for the toxins of the okadaic acid group, but diarrhegenic effects have only been proven for OA and DTXs. Whereas, some PTXs causes liver necrosis and YTXs damages cardiac muscle after intraperitoneal injection into mice. On the other hand, azaspiracids (AZAs) have never been included in the DSP group, but they cause diarrhoea in humans. This review summarizes the origin, characterization, structure, activity, mechanism of action, clinical symptoms, method for analysis, potential risk, regulation and perspectives of DSP and associated toxins produced by marine dinoflagellates.     

Lipophilic toxin profile in Galicia (Spain): 2005 toxic episode

By the end of 2005, a toxic episode of phytoplankton origin in bivalve shellfish led to the closing down of several shellfish production areas in Galicia (northwestern region of Spain). During this time, different kinds of shellfish were collected and analysed by LC–MS/MS to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and spirolides. Samples were analysed before alkaline hydrolysis in order to investigate the presence of free OA and DTXs, AZAs, PTXs and spirolides, and after alkaline hydrolysis to detect OA and DTXs esters. All of the samples were found to be contaminated with OA and/or DTX-2, as well as esterified forms of these diarrhetic shellfish poison (DSP) toxins, at levels around and above European regulatory limit (160 μg of okadaic acid equivalents/kg). The analyses of mussels and razor clam also revealed the presence of 13-desmethyl spirolide C (SPX-1) at levels below 31 μg/kg. Likewise, in many of the samples different levels of pectenotoxin-2 secoacid (PTX-2sa) were detected. DSP toxin esters represent practically the 100% of the total OA equivalents for scallops, clams, razor clams and cockles.     

Studies of polyether toxins in the marine phytoplankton, Dinophysis acuta, in Ireland using multiple tandem mass spectrometry

Diarretic shellfish poisoning (DSP) is a toxic syndrome associated with the consumption of bivalve molluscs. The DSP toxins are polyether compounds, which include okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs) and pectenotoxin seco acids (PTX2SAs). These toxins originate in marine dinoflagellates, including Dinophysis spp. Phytoplankton samples were collected from the southwest coast of Ireland and D. acuta was the predominant species. Monocultures of D. acuta cells were prepared by hand picking from microscope slides in order to confirm their toxin profiles. There was a remarkable consistency in the toxin profiles in all of the phytoplankton samples collected during the summer months, irrespective of location, depth or mesh size. Analysis using liquid chromatography—multiple tandem mass spectrometry (LC-MS/MS) revealed that DTX2 and OA were the predominant toxins at a consistent ratio. The average toxin composition was: DTX2 (53±5%), OA (26.5±2.3%) and total pectenotoxins (20.8±4.7%). Toxin profiles in D. acuta from Europe were distinctly different from those found in New Zealand, where PTX2 was the predominant toxin and DTX2 was absent.     

A comparison of the mouse bioassay with liquid chromatography-mass spectrometry for the detection of lipophilic toxins in shellfish from Scottish waters.

Some lipophilic shellfish toxins (LSTs) can cause human illness due to eating shellfish that have become naturally contaminated following filter feeding on toxin producing algae. A mouse bioassay (MBA) is widely used to detect LSTs in regulatory monitoring of shellfish. However, the MBA is imprecise giving only a positive or negative result and is prone to interference from other compounds. In this study, liquid chromatography-mass spectrometry (LC-MS) was compared to the MBA, with the aim of substituting the in vivo assay for monitoring shellfish from Scottish waters. Overall, it was not feasible to demonstrate equivalence of LC-MS with the MBA, but due to the detection of a range of LSTs, it is considered that LC-MS methods capable of detecting multiple analogues are accepted by international markets of shellfish to assure consumer protection.     

First report on azaspiracid and yessotoxin groups detection in French shellfish.

The French Phytoplankton and Phycotoxins monitoring network (REPHY) recently found positive or dubious negative shellfish samples using lipophilic toxins mouse bioassay. These samples were analyzed by liquid chromatography (LC) in combination with mass spectrometry (MS) to detect the following toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirolides (SPXs) and gymnodimines (GYMs). Over the 2006-2007 period, chemical analyses revealed various lipophilic toxin profiles according to shellfish sampling locations. In addition to OA and/or PTX-2 and their derivatives, several other compounds were found for the first time in France: (1) during the summer of 2006, AZA-1 and AZA-2 in Queen scallops (Aequipecten opercularis) from Northern Brittany; (2) during the summer of 2007, YTX and its major metabolites (45-hydroxy-YTX, homo-YTX, carboxy-YTX) in shellfish from the Mediterranean coast. Regarding YTX-group, the toxin profiles evolution in mussels during summer showed that: (i) the carboxy-YTX depuration rate was much slower than the YTX and 45-hydroxy-YTX ones; (ii) the homo-YTX concentration, which was initially very weak, increased significantly during the last depuration phase, which seems to reveal a YTX-group high metabolisation level in mussels. This paper reports for the first time on AZA and YTX-groups detection in French shellfish.     

Comparative determination of paralytic shellfish toxins (PSTs) using five different toxin detection methods in shellfish species collected in the Aleutian Islands, Alaska

Paralytic shellfish poisoning (PSP), a human illness caused by the ingestion of shellfish contaminated with paralytic shellfish toxins (PSTs), has been reported in Alaska for decades. These poisoning incidents have resulted in losses to local economies due to shellfish harvest closures. Thus the development of an effective biotoxin monitoring program designed specifically for the remote regions of Alaska would provide protection for public health and allow for a viable shellfish industry. The present study provides data useful for the development of an effective toxin screening protocol by comparing PST levels quantified in shellfish by many of the currently available PST detection techniques. Seven bivalve species were collected along beaches of the Aleutian Islands from June 2006 to September 2007. The concentration of PSTs was quantified and compared using five different analytical methods: the mouse bioassay, high performance liquid chromatography (HPLC), receptor-binding assay, the commercially available Jellett Rapid PSP Test strips, and an enzyme linked immunosorbent assay technique. The Association of Official Analytical Chemists (AOAC)-approved HPLC method proved to be valuable for characterizing the suite of individual PSTs in each species for research purposes, but was not considered practical for rapid toxin screening in remote Alaskan regions due to its time-consuming nature and requirement of expensive equipment and considerable expertise. In the present study, Jellett test strips were shown to be an effective tool for rapid screening, however due to the high percentage of false positives, subsequent validation via AOAC-approved methods would be required to prevent unnecessary closures.     

Confirmation by LC–MS/MS of azaspiracids in shellfish from the Portuguese north-western coast

The search for azaspiracids (AZAs) in shellfish on the Portuguese coast started in 2002, but the presence of these toxins could not be demonstrated until the summer of 2006. Analysis by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) allowed the confirmation of AZA2 as a dominant compound, followed by AZA1, in blue mussel (Mytilus galloprovincialis), common cockle (Cerastoderma edule), clams (Venerupis senegalensis, Ruditapes decussatus), razor clam (Solen marginatus) and oyster (Crassostrea spp). Traces of AZA3 were found only in blue mussel. Total levels of AZA1-3 determined in the whole flesh by LC-MS/MS ranged from 1.6 to 6.1 microg/kg. The finding of low levels of AZAs since 2002 suggests a low risk level when compared with the highest risks posed by diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP) toxins. However, the limited number of years studied might generate a misleading conclusion. The contamination with PSP is an example, as no contamination occurred for an extended period of time between 1996 and 2004, despite high levels having occurred outside this period. Thus, there appears overall a moderate likelihood of occurrence of AZAs in the range that may be relevant to consumers.     

Determination of paralytic shellfish poisoning toxins in Norwegian shellfish by liquid chromatography with fluorescence and tandem mass spectrometry detection.

A novel extraction and clean-up method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish samples. Raw shellfish material was extracted with an acidic acetonitrile/water (80:20, v/v) solution, whilst being homogenised. During the homogenisation the sample extraction solution was cooled with ice water. Subsequently, the extract was frozen at -20 degrees C for at least 4h. During freezing, two layers were formed, only the lower predominantly aqueous layer was used for the determination. The final extract solution was cleaned-up using a combination of Oasis HLB and Carbograph activated carbon SPE columns. The developed extraction and clean-up methods combined with gradient elution liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS) has resulted in a method which can determine the analogs GTX 1-5, C1-2, DcGTX 2-3, DcSTX, Neo, STX in a single analysis with an overall detection limit of 313mug STXdiHCL-eq./kg shellfish meat. The use of the developed extraction method with post-column high performance liquid chromatography (HPLC) with fluorescence detection (FLD) method provided an overall limit of detection of 89mug STXdiHCL-eq./kg shellfish meat for the same toxins. Both post-column HPLC-FLD and LC-MS/MS was used to investigate the Norwegian PSP toxin profile. It was found that the PSP toxins could be detected in shellfish samples from the Norwegian coastline for 10 months of the year, from March till December. The toxin profile consisted mainly of the carbamate toxins, GTX 1-4, Neo and STX, in terms of both concentrations and contribution to the overall toxicity. In addition, several of the n-sulfo-carbamoyl toxins were either detected in the samples at relatively low concentrations or their presence in the samples were indicated but could not be confirmed by the post-column HPLC-FLD and LC-MS/MS analyses.     

First U.S. report of shellfish harvesting closures due to confirmed okadaic acid in Texas Gulf coast oysters

Between March 7 and April 12, 2008, several bay systems on the east (Gulf of Mexico) coast of Texas, USA were closed to the harvesting of oysters (Crassostrea virginica) due to the presence of the DSP (Diarrheic Shellfish Poisoning) toxin okadaic acid in excess of the 20 μg/100 g tissue FDA regulatory guidance level. This was the first shellfish harvesting closure due to the confirmed presence of DSP toxins in US history. Light microscopic cell counts were performed on water samples collected from numerous sampling sites along the Texas Gulf coast where shellfish harvesting occurs. Ultra performance liquid chromatography, electrospray ionization, selected reaction monitoring, mass spectrometry (UPLC/ESI/SRM/MS) was used to detect DSP toxins in oysters. The closures were associated with an extensive bloom of the dinoflagellate Dinophysis cf. ovum. Only okadaic acid (OA) and OA acyl esters were found in shellfish tissues (max. OA eq. levels 47 μg/100 g tissue). OA was also confirmed in a bloom water sample. No illnesses were reported associated with this event. DSP toxins now add to a growing list of phycotoxins, which include those responsible for PSP (paralytic shellfish poisoning), NSP (neurotoxic shellfish poisoning), and ASP (amnesic shellfish poisoning) which must now be monitored for in US coastal waters where shellfish are harvested.     

Seasonality of diarrhetic shellfish poisoning at a coastal lagoon in Portugal: rainfall patterns and folk wisdom.

Of the three types of toxicity known so far in Portuguese shellfish, only diarrhetic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP) are produced by microalgae that seem to have been present in the last decades or centuries. The most important paralytic shellfish poisoning (PSP) producer, Gymnodinium catenatum, is hypothesised to have been introduced quite recently as only in 1976 PSP toxicity was detected for the first time in shellfish from Galicia, NW Iberian Peninsula. While ASP presents very short episodes of contamination, the concentration of DSP toxins in some years surpasses human safety values for much longer periods. It is traditionally stated that shellfish should be consumed in 'months with R' (September-April). A retrospective study of the maximum monthly DSP levels attained in mussels from a coastal lagoon-Ria de Aveiro-between 1994 and 2001, showed that the highest frequency of months with concentrations surpassing the safety level of 2 microg/g digestive glands were found in June-September, followed by May and October. These months correspond with the months of lowest historical average rainfall in the period 1941-1998.Oscillations in the rainfall pattern coincided with earliest (or latest) detection by HPLC of DSP toxins in mussel in the years studied. In a semi-closed lagunar environment prone to in situ growth of DSP-producer microalgae, like Dinophysis acuminata, rainfall affects river output, lowering salinity and disrupting water column stability that favours Dinophysis growth. The seasonality of DSP recurrence may be connected to the folk adage on safety of shellfish consumption, after many years of empirical observations by coastal populations of diarrhoea episodes in summertime.     

Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry

Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of ±1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD).     

贝类毒素检验及排除方法的研究进展

简要介绍了麻痹性贝类毒素、腹泻性贝类毒素、神经性贝类毒素及健忘性贝类毒素等贝类毒素的来源、性质、组成及中毒机理，并探讨了贝类毒素的检验及其排除方法。     

Acute Cardiotoxicity Evaluation of the Marine Biotoxins OA,DTX-1 and YTX

Phycotoxins are marine toxins produced by phytoplankton that can get accumulated in filter feeding shellfish. Human intoxication episodes occur due to contaminated seafood consumption. Okadaic acid (OA) and dynophysistoxins (DTXs) are phycotoxins responsible for a severe gastrointestinal syndrome called diarrheic shellfish poisoning (DSP). Yessotoxins (YTXs) are marine toxins initially included in the DSP class but currently classified as a separated group. Food safety authorities from several countries have regulated the content of DSPs and YTXs in shellfish to protect human health. In mice, OA and YTX have been associated with ultrastructural heart damage in vivo. Therefore, this study explored the potential of OA, DTX-1 and YTX to cause acute heart toxicity. Cardiotoxicity was evaluated in vitro by measuring hERG (human èter-a-go-go gene) channel activity and in vivo using electrocardiogram (ECG) recordings and cardiac damage biomarkers. The results demonstrated that these toxins do not exert acute effects on hERG channel activity. Additionally, in vivo experiments showed that these compounds do not alter cardiac biomarkers and ECG in rats acutely. Despite the ultrastructural damage to the heart reported for these toxins, no acute alterations of heart function have been detected in vivo, suggesting a functional compensation in the short term.     

Integrative Monitoring of Marine and Freshwater Harmful Algae in Washington State for Public Health Protection

The more frequent occurrence of both marine and freshwater toxic algal blooms and recent problems with new toxic events have increased the risk for illness and negatively impacted sustainable public access to safe shellfish and recreational waters in Washington State. Marine toxins that affect safe shellfish harvest in the state are the saxitoxins that cause paralytic shellfish poisoning (PSP), domoic acid that causes amnesic shellfish poisoning (ASP) and the first ever US closure in 2011 due to diarrhetic shellfish toxins that cause diarrhetic shellfish poisoning (DSP). Likewise, the freshwater toxins microcystins, anatoxin-a, cylindrospermopsins, and saxitoxins have been measured in state lakes, although cylindrospermopsins have not yet been measured above state regulatory guidance levels. This increased incidence of harmful algal blooms (HABs) has necessitated the partnering of state regulatory programs with citizen and user-fee sponsored monitoring efforts such as SoundToxins, the Olympic Region Harmful Algal Bloom (ORHAB) partnership and the state’s freshwater harmful algal bloom passive (opportunistic) surveillance program that allow citizens to share their observations with scientists. Through such integrated programs that provide an effective interface between formalized state and federal programs and observations by the general public, county staff and trained citizen volunteers, the best possible early warning systems can be instituted for surveillance of known HABs, as well as for the reporting and diagnosis of unusual events that may impact the future health of oceans, lakes, wildlife, and humans.     

Studies in the use of magnetic microspheres for immunoaffinity extraction of paralytic shellfish poisoning toxins from shellfish

Paralytic shellfish poisoning (PSP) is a potentially fatal human health condition caused by the consumption of shellfish containing high levels of PSP toxins. Toxin extraction from shellfish and from algal cultures for use as standards and analysis by alternative analytical monitoring methods to the mouse bioassay is extensive and laborious. This study investigated whether a selected MAb antibody could be coupled to a novel form of magnetic microsphere (hollow glass magnetic microspheres, brand name Ferrospheres-N) and whether these coated microspheres could be utilized in the extraction of low concentrations of the PSP toxin, STX, from potential extraction buffers and spiked mussel extracts. The feasibility of utilizing a mass of 25 mg of Ferrospheres-N, as a simple extraction procedure for STX from spiked sodium acetate buffer, spiked PBS buffer and spiked mussel extracts was determined. The effects of a range of toxin concentrations (20-300 ng/mL), incubation times and temperature on the capability of the immuno-capture of the STX from the spiked mussel extracts were investigated. Finally, the coated microspheres were tested to determine their efficiency at extracting PSP toxins from naturally contaminated mussel samples. Toxin recovery after each experiment was determined by HPLC analysis. This study on using a highly novel immunoaffinity based extraction procedure, using STX as a model, has indicated that it could be a convenient alternative to conventional extraction procedures used in toxin purification prior to sample analysis.     

Risk Assessment of Pectenotoxins in New Zealand Bivalve Molluscan Shellfish, 2009–2019

Pectenotoxins (PTXs) are produced by Dinophysis spp., along with okadaic acid, dinophysistoxin 1, and dinophysistoxin 2. The okadaic acid group toxins cause diarrhetic shellfish poisoning (DSP), so are therefore regulated. New Zealand currently includes pectenotoxins within the DSP regulations. To determine the impact of this decision, shellfish biotoxin data collected between 2009 and 2019 were examined. They showed that 85 samples exceeded the DSP regulatory limit (0.45%) and that excluding pectenotoxins would have reduced this by 10% to 76 samples. The incidence (1.3%) and maximum concentrations of pectenotoxins (0.079 mg/kg) were also found to be low, well below the current European Food Safety Authority (EFSA) safe limit of 0.12 mg/kg. Inclusion within the DSP regulations is scientifically flawed, as pectenotoxins and okadaic acid have a different mechanism of action, meaning that their toxicities are not additive, which is the fundamental principle of grouping toxins. Furthermore, evaluation of the available toxicity data suggests that pectenotoxins have very low oral toxicity, with recent studies showing no oral toxicity in mice dosed with the PTX analogue PTX2 at 5000 µg/kg. No known human illnesses have been reported due to exposure to pectenotoxins in shellfish, a fact which combined with the toxicity data indicates that they pose negligible risk to humans. Regulatory policies should be commensurate with the level of risk, thus deregulation of PTXs ought to be considered, a stance already adopted by some countries.     

Paralytic shellfish poisoning: post-mortem analysis of tissue and body fluid samples from human victims in the Patagonia fjords.

In July 5, 2002 fishermen working in harvesting sea urchin (Loxechinus albus) in the Patagonia Chilean fjords were intoxicated by consumption of filter-feeder bivalve Aulacomya ater. After the ingestion of 7-9 ribbed mussel, two fishermen died 3-4 h after shellfish consumption. The forensic examination in both victims did not show pathological abnormalities with the exception of the lungs conditions, crackling to the touch, pulmonary congestion and edema. The toxic mussel sample showed a toxicity measured by mouse bioassay of 8575 microg of STX (saxitoxin) equivalent by 100 g of shellfish meat. Using post-column derivatization HPLC method with fluorescent on line detection was possible to measure mass amount of each paralytic shellfish poisoning (PSP) toxin yielding individual toxin concentrations. These PSP toxins were identified in the gastric content, body fluids (urine, bile and cerebrospinal fluid) and tissue samples (liver, kidney, lung, stomach, spleen, heart, brain, adrenal glands, pancreas and thyroids glands). The toxin profiles of each body fluid and tissue samples and the amount of each PSP toxin detected are reported. The PSP toxins found in the gastric content, were STX and the gonyautoxins (GTX4, GTX1, GTX5, GTX3 and GTX2) which showed to be the major amount of PSP toxins found in the victims biological samples. The PSP toxin composition in urine and bile showed as major PSP toxins neoSaxitoxin (neoSTX) and GTX4/GTX1 epimers, both STX analogues with an hydroxyl group (-OH) in the N(1) of the tetrahydropurine nucleus. The neoSTX was not present in the gastric content sample, indicating that the oxidation of N(1) in the STX tetrahydropurine nucleus resulted neoSTX, in a similar way that GTX3/GTX2 epimers were transformed in GTX4/GTX1 epimers. Beside this metabolic transformation, also the hydrolysis of carbamoyl group from STX to form its decarbomoyl analogue decarbamoylsaxitoxin was detected in liver, kidney and lung. These two findings show that PSP toxins went under metabolic transformation during the 3-4 h of human intoxication period, in which PSP toxins showed enzymatic oxidation of N(1) in the tetrahydropurine nucleus, producing neoSTX and GTX4/GTX1 epimers starting from STX and GTX3/GTX2 epimers, respectively. This study conclude, that PSP toxins are metabolically transformed by humans and that they are removed from the body by excretion in the urine and feces like any other xenobiotic compound.     

Efficient isolation of the rare diarrhoeic shellfish toxin, dinophysistoxin-2, from marine phytoplankton.

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).     

Comparative analysis of pre- and post-column oxidation methods for detection of paralytic shellfish toxins

Paralytic shellfish poisoning (PSP) toxins are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. Shellfish contaminated with these toxins create significant public health threat and economic losses to the shellfish industry. For this reason, several methods of high performance liquid chromatography (HPLC) with fluorescence detection have been developed in order to gain better knowledge of toxins profiles in shellfish and dinoflagellates samples. These methods have been subjected to continuous modifications to improve and shorten the run time of analysis in the routine monitoring control. In this paper, different samples are analyzed by pre- and post- column HPLC methods to compare toxin profiles. All PSP toxins were individually identified and quantified within the post-column oxidation method. However, although the pre-column oxidation method is significantly more sensitive and detects lower toxin levels, it provides a total amount of toxins that co-elute together, as GTX2 and 3, GTX1 and 4 and dcGTX2 and dcGTX3. The results obtained by both HPLC methods showed similar toxin concentration (expressed in μg/mL) in mussel samples, however when dinoflagellates samples were analyzed the toxin profile and concentration were different. In summary, the post-column oxidation method is accurate to determine the amount of each individual PSP toxin and to know the real toxic profile of non-transformed samples. In addition, this method is easy and faster to screen a large number of samples. The pre-column HPLC method is useful when mussel samples are analyzed even though the time required to prepare the samples is longer.