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A major challenge for LC-MS analysis is the ability to compare data between laboratories and across instrument platforms. Currently, mycotoxin determination relies on dereplication strategies based on physicochemical properties such as the m/z of the precursor and product ions. Unlike these intrinsic properties, retention time (tR) is an extrinsic property impacted by LC conditions, including mobile phases and column chemistry, making exchange of data between groups difficult. To address this, we are introducing the concept of incorporating an electrospray compatible, retention index (RI) system based on a series of N-alkylpyridinium-3-sulfonates (NAPS) into routine mycotoxin determination. These standards of differing alkyl chain length span RI units from 100 to 2000, are UV active and have fixed positive and negative charges for electrospray ionization in either mode. Using high resolution LC-MS/MS, the RIs of 96 mycotoxins and fungal secondary metabolites were normalized as a proof of concept with the NAPS RI system under multiple pH, column and gradient chromatographic conditions. This method was then applied to the analysis of a crude extract of Penicillium roqueforti, where we were able to decrease the number of false positives by implementing an RI filter as well as a secondary correction factor. Additionally, we developed software that allows users to convert published RIs to a predicted tR values. Integration of the NAPS RI system into routine LC-MS analysis will improve compound identifications and help facilitate ease of data sharing.

Paralytic shellfish toxins (PSTs) are a group of over 50 closely related naturally occurring tetrahydropurine toxins with saxitoxin (STX) as one of the most common and potent. Saxitoxin was first isolated and characterised from the butter clam Saxidomus giganteus as the dihydrochloride salt (Schantz et al., 1975). It is traded as a stand-alone reference material, or included in diagnostic test kits, mainly as the dihydrochloride salt or diacetate salt. This is primarily due to the saxitoxin hydrate (free base) form having poor stability. However, the certificate of analysis for various reference materials may state the concentration as either the salt or free base equivalent, and care must be taken to ensure the value is traceable and used correctly in subsequent calculations.

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.

Polar marine toxins are more challenging to analyze by mass spectrometry-based methods than lipophilic marine toxins, which are now routinely measured in shellfish by multiclass reversed-phase liquid chromatography–tandem mass spectrometry (MS/MS) methods. Capillary electrophoresis (CE)–MS/MS is a technique that is well suited for the analysis of polar marine toxins, and has the potential of providing very high resolution separation. Here, we present a CE–MS/MS method developed, with use of a custom-built interface, for the sensitive multiclass analysis of paralytic shellfish toxins, tetrodotoxins, and domoic acid in seafood. A novel, highly acidic background electrolyte (5 M formic acid) was designed to maximize protonation of analytes and to allow a high degree of sample stacking to improve the limits of detection. The method was applied to a wide range of regulated and less common toxin analogues, and exhibited a high degree of selectivity between toxin isomers and matrix interference. The limits of detection in mussel tissue were 0.0052 mg/kg for tetrodotoxins, 0.160 mg/kg for domoic acid, and between 0.0018 and 0.120 mg/kg for paralytic shellfish toxins, all of which showed good linearity. Minimal ionization suppression was observed when the response from neat and mussel-matrix-matched standards was corrected with multiple internal standards. Analysis of shellfish matrix reference materials and spiked samples demonstrated good accuracy and precision. Finally, the method was transferred to a commercial CE–MS/MS system to demonstrate its widespread applicability for use in both R & D and routine regulatory settings. The approach of using a highly acidic background electrolyte is of broad interest, and can be considered generally applicable to simultaneous analysis of other classes of small, polar molecules with differing pKa values.

Two methods were developed for the analysis of algal biotoxins in complex biological and environmental samples to demonstrate the concept of isotope-labelling derivatisation for quantitation. These methods are based on dansyl chloride derivatisation of samples and dansyl-d6 chloride derivatisation of toxin standards. Derivatised sample and standard are then mixed to achieve isotope dilution calibration in liquid chromatography–tandem mass spectrometry analyses. Quantitation of the marine toxin domoic acid (DA) in mussel tissues and the freshwater toxins anatoxin-a (ATX) and homoanatoxin-a (hATX) in cyanobacteria is demonstrated. For DA, isotope-labelling was incorporated into existing dansylation methodology using inexpensive and commercially available reagents. For ATXs, a novel sample preparation procedure is presented that involves solid phase extraction on a mixed reverse phase/weak anion exchange column that facilitates simultaneous clean-up of the derivatised toxins and removal of excess dansylation reagent through covalent bonding. The challenge of achieving co-elution in LC between deuterated and non-deuterated dansylated toxins was addressed by modifying separation conditions from the usual reverse phase (RP) separation to hydrophilic interaction liquid chromatography in the case of DA and a shortened RP separation with high organic modifier content in the case of the ATXs. The new methods gave limits of detection between 10 and 60 μg kg−1 and allowed for precise, accurate and fast determination of toxins in spiked control samples and matrix reference materials. This work demonstrates that isotope-labelling derivatisation is broadly applicable to the field of algal toxin analysis where derivatisation is well established but isotopically-labelled standards are not available.

The non-protein amino acid β-methylamino-L-alanine (BMAA) has been linked to neurodegenerative disease and reported throughout the environment. Proposed mechanisms of bioaccumulation, trophic transfer and chronic toxicity of BMAA rely on the hypothesis of protein misincorporation. Poorly selective methods for BMAA analysis have led to controversy. Here, a recently reported highly selective method for BMAA quantitation using hydrophilic interaction liquid chromatography-differential mobility spectrometry-tandem mass spectrometry (HILIC-DMS-MS/MS) is expanded to include proteinogenic amino acids from hydrolyzed biological samples. For BMAA quantitation, we present a double spiking isotope dilution approach using D3-BMAA and 13C15N2-BMAA. These methods were applied to study release of BMAA during acid hydrolysis under a variety of conditions, revealing that the majority of BMAA can be extracted along with only a small proportion of protein. A time course hydrolysis of BMAA from mussel tissue was carried out to assess the recovery of BMAA during sample preparation. The majority of BMAA measured by typical methods was released before a significant proportion of protein was hydrolyzed. Little change was observed in protein hydrolysis beyond typical hydrolysis times but the concentration of BMAA increased linearly. These findings demonstrate protein misincorporation is not the predominant form of BMAA in cycad and shellfish.

Contamination of economic bivalves with paralytic shellfish toxins (PST) occurs frequently in many parts of the world, which potentially threatens consumer health and the marine aquaculture economy. It is the objective of this study to develop a suitable technology for accelerating detoxification of PST from shellfish using activated carbon (AC). The adsorption efficiency of PST by eight different AC materials and by different particle sizes of wood-based AC (WAC) were tested and compared. Then WAC particles (37–48 µm) were fed to mussels Mytilus galloprovincialis and scallops Chlamys farreri previously contaminated with PST through feeding with dinoflagellate Alexandrium tamarense ATHK. Results showed that the maximum adsorption ratio (65%) of PST was obtained by WAC. No significant differences in adsorption ratios were found between different particle sizes of WAC. The toxicity of mussels decreased by 41% and 68% after detoxification with WAC for 1 d and 3 d, respectively. Meanwhile, the detoxification ratio of mussels was approximately 3 times higher than that of scallops. This study suggests that the WAC could be used to accelerate the detoxification of PST by shellfish.

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by marine dinoflagellates that are responsible for paralytic shellfish poisoning (PSP) in humans. This work highlights our ongoing efforts to develop quantitative methods for PSTs using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). Compared with the commonly used method of liquid chromatography with post-column oxidation and fluorescence detection (LC-ox-FLD), HILIC-MS/MS has the potential of being more robust, sensitive and straightforward to operate, and provides unequivocal confirmation of toxin identity. The main driving force for the present work was the need for a complementary method to LC-ox-FLD to assign values to shellfish tissue matrix reference materials for PSTs. Method parameters that were optimized included LC mobile and stationary phases, electrospray ionization (ESI) conditions, and MS/MS detection parameters. The developed method has been used in the detection and identification of a wide range of PSTs including less common analogues and metabolites in a range of shellfish and algal samples. We have assessed the matrix effects of shellfish samples and have evaluated dilution, standard addition and matrix matched calibration as means of mitigating them. Validation on one LC-MS/MS system for nine common PST analogues (GTX1-4, dcGTX2&3, STX, NEO, and dcSTX) was completed using standard addition. The method was then transferred to a more sensitive LC-MS/MS system, expanded to include five more PSTs (C1&2, dcNEO and GTX5&6) and validated using matrix matched calibration. Limits of detection of the validated method ranged between 6 and 280 nmol/kg tissue using standard addition in extracts of blue mussels, with recoveries between 92 and 108%. Finally, this method was used in combination with the AOAC Official Method based on LC-ox-FLD to measure PSTs in a new mussel tissue matrix reference material.

Azaspiracids (AZAs) are lipophilic biotoxins produced by marine algae that can contaminate shellfish and cause human illness. The European Union (EU) regulates the level of AZAs in shellfish destined for the commercial market, with liquid chromatography-mass spectrometry (LC-MS) being used as the official reference method for regulatory analysis. Certified reference materials (CRMs) are essential tools for the development, validation, and quality control of LC-MS methods. This paper describes the work that went into the planning, preparation, characterization, and certification of CRM-AZA-Mus, a tissue matrix CRM, which was prepared as a wet homogenate from mussels (Mytilus edulis) naturally contaminated with AZAs. The homogeneity and stability of CRM-AZA-Mus were evaluated, and the CRM was found to be fit for purpose. Extraction and LC-MS/MS methods were developed to accurately certify the concentrations of AZA1 (1.16 mg/kg), AZA2 (0.27 mg/kg), and AZA3 (0.21 mg/kg) in the CRM. Quantitation methods based on standard addition and matrix-matched calibration were used to compensate for the matrix effects in LC-MS/MS. Other toxins present in this CRM at lower levels were also measured with information values reported for okadaic acid, dinophysistoxin-2, yessotoxin, and several spirolides.]

The implementation of instrumental analytical methods such as LC-MS for routine monitoring of toxins requires the availability of accurate calibration standards. This is a challenge because many toxins are rare, expensive, dangerous to handle, and/or unstable, and simple gravimetric procedures are not reliable for establishing accurate concentrations in solution. NMR has served as one method of qualitative and quantitative characterization of toxin calibration solution Certified Reference Materials (CRMs). LC with chemiluminescence N detection (LC-CLND) was selected as a complementary method for comprehensive characterization of CRMs because it provides a molar response to N. Here we report on our investigation of LC-CLND as a method suitable for quantitative analysis of nitrogenous toxins. It was demonstrated that a wide range of toxins could be analyzed quantitatively by LC-CLND. Furthermore, equimolar responses among diverse structures were established and it was shown that a single high-purity standard such as caffeine could be used for instrument calibration. The limit of detection was approximately 0.6 ng N. Measurement of several of Canada's National Research Council toxin CRMs with caffeine as the calibrant showed precision averaging 2% RSD and accuracy ranging from 97 to 102%. Application of LC-CLND to the production of calibration solution CRMs and the establishment of traceability of measurement results are presented.

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.

The concentration of the saxitoxin analogue LWTX-1 was quantified in samples of the benthic filamentous cyanobacterium Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck collected in two fluvial lakes of the St. Lawrence River (Canada) over the 2006–2013 period. The study was aimed at documenting the spatial (between fluvial lakes, between sites within each lake) and temporal (interannual, monthly) variations of toxin concentration in relation with hydrological (water level), physical (water temperature, conductivity, transparency), chemical (nutrients in overlying water) and biological (L. wollei biomass and mat condition) characteristics. Toxin concentration was hypothesized to vary seasonally with biomass accumulation and environmental conditions. Toxin concentrations measured in Lake Saint-Louis (51 +/- 40 mg LWTX-1 g-1 DM, N = 29 days in 2007, 2009–2011) were double those in Lake Saint-Pierre (25 +/- 31 mg LWTX-1 g-1 DM, N = 26 days in 2006–2008, 2012–2013); however, August 2007 measurements taken from both lakes did not differ significantly. Ten of the twelve highest values (>100 mg LWTX-1 g-1 DM) were obtained from Lake Saint-Louis, between April and October in 2007, 2010 or 2011. Under ice samples showed intermediate concentrations of LWTX-1 (42 +/- 9 mg LWTX-1 g-1 DM, N = 2). Concentrations of LWTX-1 were positively correlated with Secchi depth (r = 0.59, p < 0.001), L. wollei biomass (Spearman r = 0.31, p < 0.01) and %N in filaments (r = 0.48, p < 0.001), suggesting toxin production was linked to mat growth and metabolism rather than water quality. Although LWTX-1 has been reported to have a low toxicity, monitoring of L. wollei abundance is required to assess the environmental and human health risks posed by this taxon in the St. Lawrence - Great Lakes system.

Samples of toxic scallop (Patinopecten yessoensis) and clam (Saxidomus purpuratus) collected on the northern coast of China from 2008 to 2009 were analysed. High-performance liquid chromatography with post-column oxidation and fluorescence detection was used to determine the profile of the main paralytic shellfish poisoning (PSP) toxins in these samples and their total toxicity. Hydrophilic interaction liquid ion chromatography with mass spectrometric detection confirmed the toxin profile and detected several metabolites in the shellfish. Results show that C1/2 toxins were the most dominant toxins in the scallop and clam samples. However, GTX1/4 and GTX2/3 were also present. M1 was the predominant metabolite in all the samples, but M3 and M5 were also identified, along with three previously unreported presumed metabolites, M6, M8 and M10. The results indicate that the biotransformation of toxins was species specific. It was concluded that the reductive enzyme in clams is more active than in scallops and that an enzyme in scallops is more apt to catalyse hydrolysis of both the sulfonate moiety at the N-sulfocabamoyl of C toxins and the 11-hydroxysulfate of C and GTX toxins to produce metabolites. This is the first report of new metabolites of PSP toxins in scallops and clams collected in China.

Four metabolites of okadaic acid were generated by incubation with human recombinant cytochrome P450 3A4. The structures of two of the four metabolites have been determined by MS/MS experiments and 1D and 2D NMR methods using 94 and 133 μg of each metabolite. The structure of a third metabolite was determined by oxidation to a metabolite of known structure. Like okadaic acid, the metabolites are inhibitors of protein phosphatase PP2A. Although one of the metabolites does have an α,β unsaturated carbonyl with the potential to form adducts with an active site cysteine, all of the metabolites are reversible inhibitors of PP2A.

The presence of cyanotoxins in benthic Lyngbya wollei algae samples collected in a fluvial lake along the St. Lawrence River, Canada, was investigated using a multi-toxins method. Hydrophilic interaction liquid chromatography (HILIC) and reverse phased liquid chromatography (RPLC) were coupled to triple quadrupole mass spectrometry (LC-QqQMS) for quantification and to quadrupole-time of flight mass spectrometry (LC-QqTOFMS) for screening and confirmation. The presence of two saxitoxin analogues, LWTX-1 and LWTX-6, was confirmed in benthic Lyngbya wollei algae samples. Concentration of LWTX-1 was between 209 ± 5 and 279 ± 9 μg g −1 . No other targeted cyanotoxin (such as anatoxin-a, nodularin, microcystin-LR, microcystins-RR and saxitoxin) was found in the samples. The presence of LWTX-6 was observed by using a screening approach based on an in-house database of cyanotoxins, an algorithm of identification and high resolution mass spectrometry measurements on the precursor and product ions. This work demonstrates the need for more research on the fate of benthic cyanotoxins in aquatic ecosystems such the St. Lawrence River.

Spirolides are marine phycotoxins produced by the dinoflagellates Alexandrium ostenfeldii and A. peruvianum. Here we report that 13-desmethyl spirolide C shows little cytotoxicity when incubated with various cultured mammalian cell lines. When administered to mice by intraperitoneal (ip) injection, however, this substance was highly toxic, with an LD(50) value of 6.9 µg/kg body weight (BW), showing that such in vitro cytotoxicity tests are not appropriate for predicting the in vivo toxicity of this toxin. Four other spirolides, A, B, C, and 20-methyl spirolide G, were also toxic to mice by ip injection, with LD(50) values of 37, 99, 8.0 and 8.0 µg/kg BW respectively. However, the acute toxicities of these compounds were lower by at least an order of magnitude when administration by gavage and their toxic effects were further diminished when administered with food. These results have implications for future studies of the toxicology of these marine toxins and the risk assessment of human exposure.

Azaspiracids (AZAs) are an important group of regulated lipophilic biotoxins that cause shellfish poisoning. Currently, the only widely available analytical method for quantitation of AZAs is liquid chromatography-mass spectrometry (LC-MS). Alternative methods for AZA analysis are needed for detailed characterization work required in the preparation of certified reference materials (CRMs) and by laboratories not equipped with LC-MS. Chemical derivatization of the amine and carboxyl groups on AZAs was investigated for the purpose of facilitating analysis by LC with fluorescence detection (FLD). Experiments towards chemical modification of AZA1 at the amine achieved only limited success. Derivatization of the carboxyl group, on the other hand, proved successful using the 9-anthryldiazomethane (ADAM) method previously applied to the okadaic acid (OA) group toxins. Extraction and clean-up methods were investigated for shellfish tissue samples and a post-reaction solid phase extraction procedure was developed for the AZA ADAM derivatives. Chromatographic separations were developed for the LC-FLD analysis of derivatized AZAs alone or in the presence of other derivatized toxins. This new analytical method for analysis of AZAs enabled verification of AZA1-3 concentrations in recently certified reference materials. The method demonstrated good linearity, repeatability and accuracy showing its potential as an alternative to LC-MS for measurement of AZAs.

Sixteen laboratories participated in a collaborative study to evaluate method performance parameters of a liquid chromatographic method of analysis for paralytic shellfsh toxins (PST) in blue mussels (Mytilus edulis), soft shell clams (Mya arenaria), sea scallops (Placopectin magellanicus), and American oysters (Crassostrea virginicus). The specifc analogs tested included saxitoxin, neosaxitoxin, gonyautoxins-1 to -5, decarbamoyl-gonyautoxins-2 and -3, decarbamoyl-saxitoxin, and N-sulfocarbamoylgonyautoxin-2 and -3. This instrumental technique has been developed as a replacement for the current AOAC biological method (AOAC Offcial MethodSM 959.08) and an alternative to the pre-column oxidation LC method (AOAC Offcial MethodSM 2005.06). The method is based on reversed-phase liquid chromatography with post-column oxidation and fluorescence detection (excitation 330 nm and emission 390 nm). The shellfsh samples used in the study were prepared from the edible tissues of clams, mussels, oysters, and scallops to contain concentrations of PST representative of low, medium, and high toxicities and with varying profles of individual toxins. These concentrations are approximately equivalent to ½ maximum level (ML), ML, or 2×ML established by regulatory authorities (0.40, 0.80, and 1.60 mg STX·diHCl eq/kg, respectively). Recovery for the individual toxins ranged from 104 to 127%, and recovery of total toxin averaged 116%. Horwitz Ratio (HorRat) values for individual toxins in the materials included in the study were generally within the desired range of 0.3 to 2.0. For the estimation of total toxicity in the test materials, the reproducibility relative standard deviation ranged from 4.6 to 20%. A bridging study comparing the results from the study participants using the post-column oxidation (PCOX) method with the results obtained in the study director’s laboratory on the same test materials using the accepted reference method, the mouse bioassay (MBA; AOAC Offcial MethodSM 959.08), showed that the average ratio of results obtained from the two methods was 1.0. A good match of values was also achieved with a new certifed reference material. The results from this study demonstrated that the PCOX method is a suitable method of analysis for PST in shellfsh tissue and provides both an estimate of total toxicity, equivalent to that determined using the MBA AOAC Offcial MethodSM 959.08, and a detailed profle of the individual toxin present in the sample.

A candidate certified reference material (CRM) for multiple shellfish toxins (domoic acid, okadaic acid and dinophysistoxins, pectenotoxins, yessotoxin, azaspiracids and spirolides) has been prepared as a freeze-dried powder from mussel tissues (Mytilus edulis). Along with the certified values, the most important characteristics for a reference material to be fit-for-purpose are homogeneity and stability. Acceptable between-bottle homogeneity was found for this CRM. Within-bottle homogeneity was assessed using domoic acid, and it was shown that repeated subsampling of the CRM can be performed precisely down to 0.35 g. Both short- and long-term stability studies carried out under isochronous conditions demonstrated excellent stability of the various toxins present in the material. While degradation of some analytes was observed at +60°C in short-term studies, it was determined that shipping at ambient temperature is adequate. No instability was detected in long-term stability studies, and it was shown that the material can be held at +18°C safely for up to 1 year. To guarantee stability of the CRM over its lifetime the stock will be maintained at -20°C. The results of the homogeneity and stability testing show that CRM-FDMT1 is appropriate for its intended use in quality assurance and quality control of shellfish toxin analysis methods.

A refined version of the pre-column oxidation liquid chromatography with fluorescence detection (ox-LC-FLD) official method AOAC 2005.06 was developed in the UK and validated for the determination of paralytic shellfish poisoning toxins in UK shellfish. Analysis was undertaken here for the comparison of PSP toxicities determined using the LC method for a range of UK bivalve shellfish species against the official European reference method, the PSP mouse bioassay (MBA, AOAC 959.08). Comparative results indicated a good correlation in results for some species (mussels, cockles and clams) but a poor correlation for two species of oysters (Pacific oysters and native oysters), where the LC results in terms of total saxitoxin equivalents were found to be on average more than double the values determined by MBA. With the potential for either LC over-estimation or MBA under-estimation, additional oyster and mussel samples were analysed using MBA and ox-LC-FLD together with further analytical and functional methodologies: a post-column oxidation LC method (LC-ox-FLD), an electrophysiological assay and hydrophilic interaction liquid chromatography with tandem mass spectrometric detection. Results highlighted a good correlation among non-bioassay results, indicating a likely cause of difference was the under-estimation in the MBA, rather than an over-estimation in the LC results. [Figure not available: see fulltext.

The fates of a phenolic contaminant and its hydrocarbon precursor have rarely been compared, especially in an invertebrate species. Two groups of Buccinum undatum were exposed to equimolar amounts of pyrene and 1-hydroxypyrene over 15 d through their diets. Tissue extracts from the muscle and visceral mass were analyzed by liquid chromatography with fluorescence and mass spectrometry detection. Nine biotransformation products were detected in animals from both exposures. These included 1-hydroxy- pyrene, pyrene-1-sulfate, pyrene-1-glucuronide, pyrene glucose sulfate, two isomers each of pyrenediol sulfate and pyrenediol disulfate, and one isomer of pyrenediol glucuronide sulfate. These compounds represent a more complex metabolic pathway for pyrene than is typically reported. Diconjugated metabolites were as important in animals exposed to pyrene as in those exposed to 1-hydroxypyrene. Biotransformation products represented >90% of the material detected in the animals and highlight the importance of analyzing metabolites when assessing exposure. A mean of only 2 to 3% of the body burden was present in muscle compared with the visceral mass of both groups. The analytical methods were sufficiently sensitive to detect biotransformation products both in laboratory control whelks and in those sampled offshore. The tissue distribution of ( 14 C)pyrene was also studied by autoradiography. Radioactivity was present primarily in the digestive and excretory system of the whelks and not in the gonads or muscle tissue. Environ. Toxicol. Chem. 2010;29:779-788. # 2009 SETAC Keywords—Dietary uptake Polycyclic aromatic hydrocarbons Metabolite Phenolic Invertebrate

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Okadaic acid (OA) and dinophysistoxins-1 and -2 (DTX1, DTX2), the toxins responsible for incidents of diarrhetic shellfish poisoning (DSP), can occur as complex mixtures of ester derivatives in both plankton and shellfish. Alkaline hydrolysis is usually employed to release parent OA/DTX toxins, and analyses are conducted before and after hydrolysis to determine the concentrations of nonesterified and esterified toxins. Recent research has shown that other toxins, including pectenotoxins and spirolides, can also exist as esters in shellfish, but these toxins cannot survive alkaline hydrolysis. A promising alternative approach is enzymatic hydrolysis. In this study, two enzymatic methods were developed for the hydrolysis of 7-O-acyl esters, “DTX3,” and the carboxylate esters of OA, “diol-esters.” Porcine pancreatic lipase induced complete conversion of DTX3 to OA and DTXs within one hour for reference solutions. The presence of mussel tissue matrix reduced the rate of hydrolysis, but an optimized lipase concentration resulted in greater than 95% conversion within four hours. OA-diol-ester was hydrolyzed by porcine liver esterase and was completely converted to OA in less than 30 min, even in the presence of mussel tissue matrix. Esters and OA/DTX toxins were all monitored by LC–MS. Further experiments with pectenotoxin esters indicated that enzymatic hydrolysis could also be applied to esters of other toxins. Enzymatic hydrolysis has excellent potential as an alternative to the conventional alkaline hydrolysis procedure used in the preparation of shellfish samples for the analysis of toxins.

A novel analytical protocol for the determination of free fatty acids (FFAs; saturated, monounsaturated and polyunsaturated) in shellfish using electrospray ionisation and liquid chromatography-mass spectrometry (LC-MS) is described. Total lipids were extracted from four commercially important shellfish species using chloroform-methanol in a modification of the traditionally used Bligh and Dyer method. FFAs were recovered from lipidic shellfish extracts by solid-phase extraction (SPE) on an aminopropyl-silica column using a 98:2 v/v diethyl ether (DEE)-acetic acid solution. Ether extracts containing the FFAs were evaporated and reconstituted in 70:30 v/v methanol-chloroform before analysis by LC-MS. The limits of quantification (LOQs) of the method ranged from 60 to 560 microgg(-1) wet weight depending on the different FFAs determined with selected ion monitoring (SIM). Results demonstrate that LC-MS is well suited for identification and quantification of FFAs in shellfish and negates the use of sample derivatisation required in gas chromatographic analysis.

This paper describes a new method for sensitive determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP), in shellfish. The method involves extraction of tissue homogenates with 50% methanol followed by a highly selective strong anion exchange solid phase extraction and a derivatization with dansyl chloride (DNS-Cl) to form the dansyl derivative of domoic acid (DNS-DA). Reaction times were very rapid and proceeded under ambient conditions to yield stable derivatives. A study of the collision-induced dissociation of ESI-produced protonated DNS-DA was carried out to identify the most sensitive transitions to use in development of a selected reaction monitoring detection method. Compared with un-derivatized DA, DNS-DA showed a 5-fold increase in sensitivity of MS/MS detection and improved retention on a reversed phase LC stationary phase. Resolution of DNS-DA and its isomers was achieved using isocratic elution in 15 min. A quantitative verification of the new method was carried out by analyzing a mussel tissue certified reference material (CRM) containing 49 mg kg−1 DA, as well as a toxin-free mussel tissue CRM spiked at levels ranging from 0.003 to 10 mg kg−1. Results showed good recovery (83–107%) with a between-sample variability of ≤5% RSD. The LC-MS/MS method presented is suitable for DA analysis over a broad range of concentrations spanning from above the regulatory limit of 20 mg DA per kg tissue down to near the method detection limit of 1.1 μg DA per kg mussel tissue. The resulting method serves as a confirmatory method with alternative selectivity to existing methods. It is also suitable for quantification of low levels of DA in shellfish as an early warning sign for toxic events or in forensic applications after intoxication has occurred.

A thirteen-laboratory comparative study tested the performance of four methods as alternatives to mouse bioassay for the determination of brevetoxins in shellfish. The methods were N2a neuroblastoma cell assay, two variations of the sodium channel receptor binding assay, competitive ELISA, and LC/MS. Three to five laboratories independently performed each method using centrally prepared spiked and naturally incurred test samples. Competitive ELISA and receptor binding (96-well format) compared most favorably with mouse bioassay. Between-laboratory relative standard deviations (RSDR) ranged from 10 to 20% for ELISA and 14 to 31% for receptor binding. Within-laboratory (RSDr) ranged from 6 to 15% for ELISA, and 5 to 31% for receptor binding. Cell assay was extremely sensitive but data variation rendered it unsuitable for statistical treatment. LC/MS performed as well as ELISA on spiked test samples but was inordinately affected by lack of toxin-metabolite standards, uniform instrumental parameters, or both, on incurred test samples. The ELISA and receptor binding assay are good alternatives to mouse bioassay for the determination of brevetoxins in shellfish.

The neurotoxin β-N-methylamino-L-alanine (BMAA) has been reported in cyanobacteria and shellfish, raising concerns about widespread human exposure. However, inconsistent results for BMAA analysis have led to controversy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the most appropriate method for analysis of BMAA, but the risk of interference from isomers, other sample components, and the electrospray background is still present. We have investigated differential mobility spectrometry (DMS) as an ion filter to improve selectivity in the hydrophilic interaction liquid chromatographic (HILIC)-MS/MS determination of BMAA. We obtained standards for two BMAA isomers not previously analyzed by HILIC-MS, β-amino-N-methylalanine and 3,4-diaminobutanoic acid, and the typically used 2,4-diaminobutanoic acid and N-(2-aminoethyl)glycine. DMS separation of BMAA from these isomers was achieved and optimized conditions were used to develop a sensitive and highly selective multidimensional HILIC-DMS-MS/MS method. This work revealed current technical limitations of DMS for trace quantitation, and practical solutions were implemented. Accurate control of low levels of DMS carrier gas modifier was essential, but required external metering. The linearity of our optimized method was excellent from 0.01 to 6 μmol L(-1). The instrumental LOD was 0.4 pg BMAA injected on-column and the estimated method LOD was 20 ng g(-1) dry weight for BMAA in sample matrix. The method was used to analyze cycad plant tissue, a cyanobacterial reference material, and mussel tissues, by use of isotope-dilution quantitation with deuterated BMAA. This confirmed the presence of BMAA and several of its isomers in cycad and mussel tissues, including commercially available mussel tissue reference materials certified for other biotoxins. Graphical Abstract Differential Mobility Spectrometry is used to increases the selectivity of BMAA analysis by HILIC-MS/MS.

The marine dinoflagellate Gymnodinium catenatum has been associated with paralytic shellfish poisoning (PSP) outbreaks in Portuguese waters for many years. PSP syndrome is caused by consumption of seafood contaminated with paralytic shellfish toxins (PSTs), a suite of potent neurotoxins. Gymnodinium catenatum was frequently reported along the Portuguese coast throughout the late 1980s and early 1990s, but was absent between 1995 and 2005. Since this time, G. catenatum blooms have been recurrent, causing contamination of fishery resources along the Atlantic coast of Portugal. The aim of this study was to evaluate the toxin profile of G. catenatum isolated from the Portuguese coast before and after the 10-year hiatus to determine changes and potential impacts for the region. Hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was utilized to determine the presence of any known and emerging PSTs in sample extracts. Several PST derivatives were identified, including the N-sulfocarbamoyl analogues (C1–4), gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6), and decarbamoyl derivatives, decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNeo) and decarbamoyl gonyautoxin 3 (dcGTX3). In addition, three known hydroxy benzoate derivatives, G. catenatum toxin 1 (GC1), GC2 and GC3, were confirmed in cultured and wild strains of G. catenatum. Moreover, two presumed N-hydroxylated analogues of GC2 and GC3, designated GC5 and GC6, are reported. This work contributes to our understanding of the toxigenicity of G. catenatum in the coastal waters of Portugal and provides valuable information on emerging PST classes that may be relevant for routine monitoring programs tasked with the prevention and control of marine toxins in fish and shellfish.

The worldwide increase in cyanobacterial contamination of freshwater lakes and rivers is of great concern as many cyanobacteria produce potent hepatotoxins and neurotoxins (cyanotoxins). Such toxins pose a threat to aquatic ecosystems, livestock, and drinking water supplies. In addition, dietary supplements prepared from cyanobacteria can pose a risk to consumers if they contain toxins. Analytical monitoring for toxins in the environment and in consumer products is essential for the protection of public health. Reference materials (RMs) are an essential tool for the development and validation of analytical methods and are necessary for ongoing quality control of monitoring operations. Since the availability of appropriate RMs for cyanotoxins has been very limited, the present study was undertaken to examine the feasibility of producing a cyanobacterial matrix RM containing various cyanotoxins. The first step was large-scale culturing of various cyanobacterial cultures that produce anatoxins, microcystins, and cylindrospermopsins. After harvesting, the biomass was lyophilized, blended, homogenized, milled, and bottled. The moisture content and physical characteristics were assessed in order to evaluate the effectiveness of the production process. Toxin levels were measured by liquid chromatography with tandem mass spectrometry and ultraviolet detection. The reference material was found to be homogeneous for toxin content. Stability studies showed no significant degradation of target toxins over a period of 310 days at temperatures up to +40 °C except for the anatoxin-a, which showed some degradation at +40 °C. These results show that a fit-for-purpose matrix RM for cyanotoxins can be prepared using the processes and techniques applied in this work.

The need for reference materials for quality control of analysis of foodstuffs has been stressed frequently. This has been particularly true in the phycotoxins field, where there is a great shortage of both pure calibration standards and reference materials. Worldwide there are very few independent bodies that produce certified reference materials for phycotoxins, the main producers currently being the National Research Council of Canada and the Japanese Food Research Laboratory. Limited availability of contaminated shellfish and algae, as well as the time and knowledge necessary for the production of adequate reference materials, continuously lead to limited editions of certified reference materials and even more limited production of in-house reference materials. The restricted availability of in-house quality control materials promotes the rapid use of the limited certified reference materials, which in turn hampers the production of the suite of materials required globally for complete protection of public health. This paper outlines the various options that analysts can pursue in the use of reference materials for internal and external quality control, with a view to optimising the efforts of both reference materials users and reference materials producers. For this purpose, the logical sequence is reviewed from the discovery of a new bioactive compound in shellfish, through initial method development up to regulation for food safety purposes including accepted reference methods. Subsequently, the requirements for and efforts typically spent in the production and characterisation of laboratory reference materials, certified reference materials and other test materials used in inter-laboratory studies or proficiency testing, in the area of marine biotoxins are evaluated. Particular emphasis is put on practical advice for the preparation of in-house reference materials. The intricate link between reference material characterisation and method performance is outlined to give guidance on the appropriate in-house method validation in the rapidly developing field of phycotoxins.

A solid-phase extract from Protoceratium reticulatum was partitioned between water and butanol and the two fractions purified on an alumina column. Fractionation was monitored by ELISA and LC–MS. Results indicate that while almost all yessotoxin (1) was extracted into butanol, large amounts of yessotoxin analogs remained in the aqueous extract along with lesser amounts in the butanolic extract. NMR analysis of selected fractions from reverse-phase chromatography of the extracts confirmed the presence of yessotoxin analogs, although structure determinations were not possible due to the complexity of the mixtures. Analysis of fractions with LC–MS3 and neutral-loss LC–MS/MS indicated the presence of more than 90 yessotoxin analogs, although structures for most of these have not yet been determined. These analogs provide a mechanism to rationalise the discrepancy between ELISA and LC–MS analyses of algae and shellfish.

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol–water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate–acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13–250 μg/kg for OA, PTX-2, and AZA-1 and 100–400 μg/kg for YTX. Recovery and precision ranged between 72–120 and 1–22%, respectively, over a fortification range of 40–160 μg/kg for OA, PTX-2, and AZA-1 and 100–400 μg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 on their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.

44,55-Dihydroxyyessotoxin (1) was isolated from extracts of Protoceratium reticulatum and identified by analysis of its one- and two-dimensional NMR and mass spectra. In addition, LC–MS methods revealed the presence of compounds tentatively identified as (44-R,S)-44,55-dihydroxy-41a-homoyessotoxin (2) and (44-R,S)-44,55-dihydroxy-9-methyl-41a-homoyessotoxin (3). LC–MS analyses indicate that 1 is a constituent of P. reticulatum in New Zealand and Norway, and it was present in three species of mussels from New Zealand, Norway, and Canada.

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05–0.20 mg/kg, recoveries were 71–99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10–24%. Limits of detection were

We examine the use of external standards for quantitative measurement by 1H NMR of solution concentrations of natural products and other low molecular weight, hydrogen-containing compounds and show that precision and accuracy ca. 1% is obtainable with a commercial 11.7 T spectrometer when standards and analytes are contained in separate but identical sealed precision glass NMR tubes. Numerous factors contributing to the intensity of the NMR signals are evaluated. Precise measurements of 360 degrees pulse lengths for each sample provide direct corrections for variations in probe Q-factor that enable samples in different solvents to be compared, provided single-coil excitation and detection is used throughout. Samples need not be prepared in deuterated solvents if the 1H spectra of the solvents are simple enough for peak suppression by presaturation. The approach is particularly suitable for hazardous materials kept in sealed tubes and for the preparation of certified calibration solution reference materials for use with LC-MS and other techniques where deuterated solvents should be avoided.

A new method for sensitive, specific and direct determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP) syndrome, in shellfish is proposed herein. It is based on combination of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC-MS). The high percentage of organic modifier in the mobile phase and the omission of ion pairing reagents, both favoured in HILIC, result in enhanced detection limits with MS detection. The new method was set up either on a ionspray (ISI)-ion trap MS instrument operating in MS and MS/MS scanning acquisition modes, or on a turboionspray (TSI)-triple quadrupole MS system operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes. Positive and negative ion experiments were performed. Multiple reaction monitoring experiments are recommended for screening contaminated shellfish tissue and for quantitative analyses due to highest sensitivity and selectivity. The minimum detection levels for the toxin in tissue were found to be 63 and 190 ng/g in positive and negative MRM experiments, respectively, which are well below the regulatory limit of DA in tissue (20 ug/g). Application to shellfish samples collected in the Adriatic Sea (Italy) in the period 2000-2004, demonstrated for the first time in Italy the presence of domoic acid as a new toxin that has entered the Adriatic Mytilus galloprovincialis toxin profile.

We have developed a simple and effective method for isolating pectenotoxin-2 (PTX-2) from Dinophysis cells collected from a natural bloom. A two-step extraction procedure followed by two column chromatography steps produced PTX-2 in high purity suitable for use as an analytical standard and for toxicological studies. Incubation of purified PTX-2 with the supernatant from ultracentrifuged blue (Mytilus edulis) or Greenshell (Perna canaliculus) mussel hepatopancreas homogenate caused rapid conversion to pectenotoxin-2 seco acid (PTX-2 SA). Purification of PTX-2 SA was achieved by solvent extraction followed by column chromatography. PTX-2 and PTX-2 SA were fully characterized by LC-MS and NMR, and full (1)H and (13)C NMR assignments were obtained. Okadaic acid C(8)-diol ester was isolated during the purification of PTX-2, and its identity confirmed by NMR and LC-MS analyses. Pectenotoxin-2 seco acid methyl ester, identified by LC-MS, was also produced during the hydrolytic procedure due to the presence of methanol. PTX-2 was acutely toxic to mice by i.p. injection (LD(50)=219 microg/kg) but no effects were seen with PTX-2 SA at 5000 microg/kg. Neither PTX-2 nor PTX-2 SA was overtly toxic to mice by the oral route at doses up to 5000 microg/kg. No diarrhea was observed in mice dosed with either compound, suggesting that pectenotoxins do not belong in the diarrhetic shellfish poison group.

In 1991, routine biotoxin monitoring of bivalve molluscs at aquaculture sites along the eastern shore of Nova Scotia, Canada revealed a group of novel seafood toxins called spirolides, whose origin was the dinoflagellate Alexandrium ostenfeldii. Result from this preliminary study in rodents demonstrates a highly toxic lethal response in rats and mice after intraperitoneal injections of lipophilic extracts. To elucidate the modes of action and toxicologic pathology, brain and internal organs were examined by histology and various biomarkers of neural injury were monitored by immunohistochemistry (IH) and/or transcriptional analysis. The histological and transcriptional data showed that the effects of spirolides are species dependent for mice and rats. Histopathology showed that in the mouse brain, the hippocampus and brain stem appeared to be the major target regions but no histological changes were observed in the rat. Transcriptional analysis in the mouse brain showed no alterations in the biomarkers whereas in the rat brain there were major changes in the markers of neuronal injury. These biomarkers included the early injury markers HSP-72, c-jun and c-fos which are essential for converting stimuli into intracellular changes within neurons. The potential effects of spirolides were also evaluated with respect to different subtypes of the acetylcholine receptors (AChRs) since earlier reports showed these as putative targets. Both the muscarinic and nicotinic AChRs were found to be upregulated. Hence, transcriptional and immunohistochemical analysis does provide insight to the molecular mechanisms of this novel group of shellfish toxins. No histological changes were observed in other tissues.

In a recent survey of paralytic shellfish poisoning (PSP) toxins in Gymnodinium catenatum Graham extracts, using LC with postcolumn oxidation and fluorescence detection, three novel saxitoxin analogues were revealed in isolates from several locations, including Australian waters. We have named them as G. catenatum toxins, GC1 (1), GC2 (2), and GC3 (3). The compounds were isolated from a culture of the Australian strain by LC-MS-guided fractionation employing a C18-silica column and hydrophilic interaction chromatography. The unusual structures of these novel compounds were characterized by low- and high-resolution MS, MS/MS, and NMR spectroscopy. GC3 (3) was found to be the 4-hydroxybenzoate ester derivative of decarbamoylsaxitoxin, while GC1 (1) and GC2 (2) are the epimeric 11-hydroxysulfate derivatives of GC3 (3).

An overview is given of the different approaches that have been used to identify toxins responsible for seafood poisoning incidents, to investigate the origins of toxins, and to monitor seafood on a routine basis. It is shown that advancements in our knowledge of toxins and our ability to protect the public have often followed key developments in separation and analysis technologies. Specific examples of research in this field are presented to illustrate the significant role that chromatographic methods play. The presentation will be given in an order that reflects the typical sequence of investigations that follow a new toxin episode.

Per-agallo has attracted much attention in recent years be-cause some species within this genus have the poten-tial to produce the neurotoxic amino acid, domoicacid (DA). DA was identified as the causative toxin inan episode of amnesic shellfish poisoning in easternCanada during November to December 1987 (Wrightet al. 1989). Three people died and over 100 becameill after consuming blue mussels (

The toxins associated with paralytic shellfish poisoning (PSP) are potent neurotoxins produced by natural populations of the marine dinoflagellate Alexandrium tamarense. In early June 2000, a massive bloom (>7×105 cells l−1) of this dinoflagellate coincided with an unusually high mortality of farmed salmon in sea cages in southeastern Nova Scotia. Conditions in the water column in the harbour were characterised by the establishment of a sharp pycnocline after salinity stratification due to abundant freshwater runoff. In situ fluorescence revealed a high sub-surface (2–4 m depth) chlorophyll peak related to the plankton bloom. The intense bloom was virtually monospecific and toxicity was clearly related to the concentration of Alexandrium cells in plankton size fractions. Cultured clonal isolates of A. tamarense from the aquaculture sites were very toxic on a per cell basis and yielded a diversity of PSP toxin profiles, some of which were similar to those from plankton concentrates from the natural bloom population. The toxin profile of plankton concentrates from the 21–56 μm size fraction was complex, dominated by the N-sulfocarbamoyl derivative C2, with levels of other PSP toxins GTX4, NEO, GTX5 (=B1), GTX3, GTX1, STX, C1, and GTX2, in decreasing order of relative abundance. Although no PSP toxin was found systemically in the fish tissues (liver, digestive tract) from this salmon kill event, the detection of Alexandrium cells and low levels of PSP toxins in salmon gills provide evidence that the enhanced mortalities were caused by direct exposure to toxic Alexandrium cells and/or to soluble toxins released during the bloom.

Following heavy precipitation, we observed an intense algal bloom in the St. Lawrence Estuary (SLE) that coincided with an unusually high mortality of several species of marine fish, birds and mammals, including species designated at risk. The algal species was identified as Alexandrium tamarense and was determined to contain a potent mixture of paralytic shellfish toxins (PST). Significant levels of PST were found in the liver and/or gastrointestinal contents of several carcasses tested as well as in live planktivorous fish, molluscs and plankton samples collected during the bloom. This provided strong evidence for the trophic transfer of PST resulting in mortalities of multiple wildlife species. This conclusion was strengthened by the sequence of mortalities, which followed the drift of the bloom along the coast of the St. Lawrence Estuary. No other cause of mortality was identified in the majority of animals examined at necropsy. Reports of marine fauna presenting signs of neurological dysfunction were also supportive of exposure to these neurotoxins. The event reported here represents the first well-documented case of multispecies mass mortality of marine fish, birds and mammals linked to a PST-producing algal bloom.

This recent paper by Otero and co-workers presents some data from analysis of okadaic acid group toxins by liquid chromatography tandem mass spectrometry (LC MS/MS) using different instruments, operating parameters, and solvent conditions. They question the suitability of this tool for quantitative analysis. This paper reveals a lack of understanding of critical factors for the successful use of LC MS methodology in general as well as some specific proficiency issues with the work reported on the three toxins. We show that there are problems with the conduct and reporting of the experiments, including possible injector carry-over and lack of quality assurance/quality control (QA/QC) controls. Therefore the specific conclusions they draw from their data are considered invalid.

A new species of Pseudo-nitzschia (Bacillariophyceae) is described from plankton samples collected from Port Dickson (Malacca Strait, Malaysia) and Manzanillo Bay (Colima, Mexico). The species possesses a distinctive falcate cell valve, from which they form sickle-like colonies in both environmental samples and cultured strains. Detailed observation of frustules under TEM revealed ultrastructure that closely resembles P. decipiens, yet the new species differs by the valve shape and greater ranges of striae and poroid densities. The species is readily distinguished from the curve-shaped P. subcurvata by the presence of a central interspace. The morphological distinction is further supported by phylogenetic discrimination. We sequenced and analyzed the nuclear ribosomal RNA genes in the LSU and the second internal transcribed spacer, including its secondary structure, to infer the phylogenetic relationship of the new species with its closest relatives. The results revealed a distinct lineage of the new species, forming a sister cluster with its related species, P. decipiens and P. galaxiae, but not with P. subcurvata. We examined the domoic acid (DA) production of five cultured strains from Malaysia by Liquid chromatography-mass spectrometry (LC-MS), but they showed no detectable DA. Here, we present the taxonomic description of the vegetative cells, document the sexual reproduction, and detail the molecular phylogenetics of Pseudo-nitzschia sabit sp. nov.