Experimental accumulation and depuration kinetics and natural occurrence of microcystin-LR in basil (Ocimum basilicum L.).

Microcystin-LR (MC-LR) is a hepatotoxic metabolite that naturally occurs during some cyanobacterial blooms in eutrophic waterbodies, and irrigation of edible plants with MC-LR-contaminated water causes bioaccumulation of the toxin. However, sufficient information about accumulation and depuration mechanics in hydroculture-grown herb plants is still lacking. This work aimed at 1) investigating bioaccumulation and depuration of MC-LR in basil, 2) verifying the possible MC-LR detoxification mechanisms in the plant, and 3) detecting the natural occurrence of MC-LR in basil (n = 50) collected from the Belgian market. Basil plants grown in a hydroculture were exposed to MC-LR (5, 20, and 50 µg L-1) spiked in a Hoagland solution for seven days. MC-LR depuration was also studied by transferring the plants to a non-contaminated Hoagland solution after exposure to MC-LR for another seven days. MC-LR concentrations in Hoagland solution, basil leaves, and roots were quantified using a validated UHPLC-MS/MS method. In addition, ELISA and LC-HRMS (only basil leaves) were used for confirmation. The results showed an increase in the accumulated levels of MC-LR at higher exposure doses, with higher MC-LR levels in roots than in leaves for all the treatment conditions. For MC-LR depuration, significant reductions were observed in all the treatment conditions for roots only. No MC-LR conjugates, potentially related to metabolism, were detected by LC-HRMS. Finally, MC-LR was detected in one store-bought basil sample, representing the first occurrence of cyanotoxins in an edible crop from Belgium.

Microcystin profiles in European noble crayfish Astacus astacus and water in Lake Steinsfjorden, Norway.

Lake Steinsfjorden, an important noble crayfish (Astacus astacus) habitat, is often affected by blooms of Planktothrix spp. that produce microcystins (MCs). A poor correlation between MCs by ELISA in the water and in crayfish tissue in a study in 2015 prompted further investigation by LC-HRMS. LC-HRMS analyses of filters from water samples and on selected crayfish tissue extracts from the 2015 study revealed the presence of known and previously unreported MCs. Crayfish samples from May and June 2015 were dominated by MCs from the Planktothrix bloom, whereas in September novel MCs that appeared to be metabolites of MC-LR were dominant, even though neither these nor MC-LR were detected in the water in 2015. A water sample from October 2016 also showed MCs typical of Planktothrix (i.e., [d-Asp3]- and [d-Asp3,Dhb7]MC-RR and -LR), but low levels of MC-RR and MC-LR were detected in the lake water for the first time. In late summer and autumn, the MC profiles of crayfish were dominated by the homonorvaline (Hnv) variant MC-LHnv, a putative metabolite of MC-LR. Taken together, ELISA, LC-HRMS and previous PCR analyses showed that although Planktothrix was part of the crayfish diet, it was not the sole source of MCs in the crayfish. Possibly, crayfish in Lake Steinsfjorden may be ingesting MCs from benthic cyanobacteria or from contaminated prey. Therefore, information on the cyanobacterial or MC content in the water column cannot safely be used to make predictions about MC concentrations in the crayfish in Lake Steinsfjorden. Interestingly, the results also show that targeted LC-MS analysis of the crayfish would at times have underestimated their MC content by nearly an order of magnitude, even if all previously reported MC variants had been included in the analysis.

Structure and toxicity of AZA-59, an azaspiracid shellfish poisoning toxin produced by Azadinium poporum (Dinophyceae).

To date, the putative shellfish toxin azaspiracid 59 (AZA-59) produced by Azadinium poporum (Dinophyceae) has been the only AZA found in isolates from the Pacific Northwest coast of the USA (Northeast Pacific Ocean). Anecdotal reports of sporadic diarrhetic shellfish poisoning-like illness, with the absence of DSP toxin or Vibrio contamination, led to efforts to look for other potential toxins, such as AZAs, in water and shellfish from the region. A. poporum was found in Puget Sound and the outer coast of Washington State, USA, and a novel AZA (putative AZA-59) was detected in low quantities in SPATT resins and shellfish. Here, an A. poporum strain from Puget Sound was mass-cultured and AZA-59 was subsequently purified and structurally characterized. In vitro cytotoxicity of AZA-59 towards Jurkat T lymphocytes and acute intraperitoneal toxicity in mice in comparison to AZA-1 allowed the derivation of a provisional toxicity equivalency factor of 0.8 for AZA-59. Quantification of AZA-59 using ELISA and LC-MS/MS yielded reasonable quantitative results when AZA-1 was used as an external reference standard. This study assesses the toxic potency of AZA-59 and will inform guidelines for its potential monitoring in case of increasing toxin levels in edible shellfish.

Preparation and characterization of an immunoaffinity column for the selective extraction of azaspiracids.

The presence of azaspiracids (AZAs) in shellfish may cause food poisoning in humans. AZAs can accumulate in shellfish filtering seawater that contains marine dinoflagellates such as Azadinium and Amphidoma spp. More than 60 AZA analogues have been identified, of which AZA1, AZA2 and AZA3 are regulated in Europe. Shellfish matrices may complicate quantitation by ELISA and LC-MS methods. Polyclonal antibodies have been developed that bind specifically to the C-26-C-40 domain of the AZA structure and could potentially be used for selectively extracting compounds containing this substructure. This includes almost all known analogues of AZAs, including AZA1, AZA2 and AZA3. Here we report preparation of immunoaffinity chromatography (IAC) columns for clean-up and concentration of AZAs. The IAC columns were prepared by coupling polyclonal anti-AZA IgG to CNBr-activated sepharose. The columns were evaluated using shellfish extracts, and the resulting fractions were analyzed by ELISA and LC-MS. The columns selectively bound over 300 ng AZAs per mL of gel without significant leakage, and did not retain the okadaic acid, cyclic imine, pectenotoxin and yessotoxin analogues that were present in the applied samples. Furthermore, 90-92% of the AZAs were recovered by elution with 90% MeOH, and the columns could be re-used without significant loss of performance.

A Feasibility Study into the Production of a Mussel Matrix Reference Material for the Cyanobacterial Toxins Microcystins and Nodularins.

Microcystins and nodularins, produced naturally by certain species of cyanobacteria, have been found to accumulate in aquatic foodstuffs such as fish and shellfish, resulting in a risk to the health of the seafood consumer. Monitoring of toxins in such organisms for risk management purposes requires the availability of certified matrix reference materials to aid method development, validation and routine quality assurance. This study consequently targeted the preparation of a mussel tissue reference material incurred with a range of microcystin analogues and nodularins. Nine targeted analogues were incorporated into the material as confirmed through liquid chromatography with tandem mass spectrometry (LC-MS/MS), with an additional 15 analogues detected using LC coupled to non-targeted high resolution mass spectrometry (LC-HRMS). Toxins in the reference material and additional source tissues were quantified using LC-MS/MS, two different enzyme-linked immunosorbent assay (ELISA) methods and with an oxidative-cleavage method quantifying 3-methoxy-2-methyl-4-phenylbutyric acid (MMPB). Correlations between the concentrations quantified using the different methods were variable, likely relating to differences in assay cross-reactivities and differences in the abilities of each method to detect bound toxins. A consensus concentration of total soluble toxins determined from the four independent test methods was 2425 ± 575 µg/kg wet weight. A mean 43 ± 9% of bound toxins were present in addition to the freely extractable soluble form (57 ± 9%). The reference material produced was homogenous and stable when stored in the freezer for six months without any post-production stabilization applied. Consequently, a cyanotoxin shellfish reference material has been produced which demonstrates the feasibility of developing certified seafood matrix reference materials for a large range of cyanotoxins and could provide a valuable future resource for cyanotoxin risk monitoring, management and mitigation.

In Vitro Metabolism of Azaspiracids 1-3 with a Hepatopancreatic Fraction from Blue Mussels (Mytilus edulis).

Azaspiracids (AZAs) are a group of biotoxins produced by the marine dinoflagellates Azadinium and Amphidoma spp. that can accumulate in shellfish and cause food poisoning in humans. Of the 60 AZAs identified, levels of AZA1, AZA2, and AZA3 are regulated in shellfish as a food safety measure based on occurrence and toxicity. Information about the metabolism of AZAs in shellfish is limited. Therefore, a fraction of blue mussel hepatopancreas was made to study the metabolism of AZA1-3 in vitro. A range of AZA metabolites were detected by liquid chromatography-high-resolution tandem mass spectrometry analysis, most notably the novel 22α-hydroxymethylAZAs AZA65 and AZA66, which were also detected in naturally contaminated mussels. These appear to be the first intermediates in the metabolic conversion of AZA1 and AZA2 to their corresponding 22α-carboxyAZAs (AZA17 and AZA19). α-Hydroxylation at C-23 was also a prominent metabolic pathway, producing AZA8, AZA12, and AZA5 as major metabolites of AZA1-3, respectively, and AZA67 and AZA68 as minor metabolites via double-hydroxylation of AZA1 and AZA2, but only low levels of 3ß-hydroxylation were observed in this study. In vitro generation of algal toxin metabolites, such as AZA3, AZA5, AZA6, AZA8, AZA12, AZA17, AZA19, AZA65, and AZA66 that would otherwise have to be laboriously purified from shellfish, has the potential to be used for the production of standards for analytical and toxicological studies.

Microcystin Toxins at Potentially Hazardous Levels in Algal Dietary Supplements Revealed by a Combination of Bioassay, Immunoassay, and Mass Spectrometric Methods.

Microcystins (MCs) are hepatotoxic heptapeptides produced by cyanobacteria and are potent inhibitors of protein phosphatases in eukaryotic cells. Algae for dietary supplements are harvested from outdoor environments and can be contaminated with MCs. Monitoring of MCs in these products is necessary but is complicated by their structural diversity (>250 congeners). We used a combination of protein phosphatase inhibition assay (PPIA), ELISA, LC-MS/MS, and nontargeted LC-high-resolution MS (LC-HRMS) with thiol derivatization to characterize the total MCs in 18 algal dietary supplements. LC-MS/MS revealed that some products contained >40 times the maximum acceptable concentration (MAC) of 1 µg/g MCs, but ELISA and PPIA showed up to 50-60 times the MAC. LC-HRMS identified all congeners targeted by LC-MS/MS plus MC-(H4)YR contributing up to 18% of total MCs, along with numerous minor MCs. Recommended dosages of the products greater than the MAC would result in 2.6-75 times the tolerable daily intake, presenting a risk to consumers. This study confirms the need for monitoring these products and presents strategies to fully describe the total MC pool in environmental samples and algal products.

A Practical ELISA for Azaspiracids in Shellfish via Development of a New Plate-Coating Antigen.

Azaspiracids (AZAs) are a group of biotoxins that appear periodically in shellfish and can cause food poisoning in humans. Current methods for quantifying the regulated AZAs are restricted to LC-MS but are not well suited to detecting novel and unregulated AZAs. An ELISA method for total AZAs in shellfish was reported recently, but unfortunately, it used relatively large amounts of the AZA-1-containing plate-coating conjugate, consuming significant amounts of pure AZA-1 per assay. Therefore, a new plate-coater, OVA-cdiAZA1 was produced, resulting in an ELISA with a working range of 0.30-4.1 ng/mL and a limit of quantification of 37 µg/kg for AZA-1 in shellfish. This ELISA was nearly twice as sensitive as the previous ELISA while using 5-fold less plate-coater. The new ELISA displayed broad cross-reactivity toward AZAs, detecting all available quantitative AZA reference materials as well as the precursors to AZA-3 and AZA-6, and results from shellfish analyzed with the new ELISA showed excellent correlation ( R2 = 0.99) with total AZA-1-10 by LC-MS. The results suggest that the new ELISA is suitable for screening samples for total AZAs, even in cases where novel AZAs are present and regulated AZAs are absent, such as was reported recently from Puget Sound and the Bay of Naples.

Analysis of free and metabolized microcystins in samples following a bird mortality event.

In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL-1 of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener (m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu1]MC-LR using NMR spectroscopy, LC-HRMS and LC-MS2, which slowly converted to [D-Leu1,Glu(OMe)6]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC-MS2 in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard (Anas platyrhynchos) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC-MS2 were used to measure free MCs, while 'total' MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63-112 ng g-1) accounted for 33-41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu1]MC-LR was quantitated in tissues at 25-67 ng g-1 (LC-MS2). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present.

Selective Extraction and Purification of Azaspiracids from Blue Mussels ( Mytilus edulis) Using Boric Acid Gel.

Azaspiracids belong to a family of more than 50 polyether toxins originating from marine dinoflagellates such as Azadinium spinosum. All of the azaspiracids reported thus far contain a 21,22-dihydroxy group. Boric acid gel can bind selectively to compounds containing vic-diols or α-hydroxycarboxylic acids via formation of reversible boronate complexes. Here we report use of the gel to selectively capture and release azaspiracids from extracts of blue mussels. Analysis of the extracts and fractions by liquid chromatography-tandem mass spectrometry (LC-MS) showed that this procedure resulted in an excellent cleanup of the azaspiracids in the extract. Analysis by enzyme-linked immunoasorbent assay (ELISA) and LC-MS indicated that most azaspiracid analogues were recovered in good yield by this procedure. The capacity of boric acid gel for azaspiracids was at least 50 µg/g, making this procedure suitable for use in the early stages of preparative purification of azaspiracids. In addition to its potential for concentration of dilute samples, the extensive cleanup provided by boric acid gel fractionation of azaspiracids in mussel samples almost eliminated matrix effects during subsequent LC-MS and could be expected to reduce matrix effects during ELISA analysis. The method may therefore prove useful for quantitative analysis of azaspiracids as part of monitoring programs. Although LC-MS data showed that okadaic acid analogues also bound to the gel, this was much less efficient than for azaspiracids under the conditions used. The boric acid gel methodology is potentially applicable to other important groups of natural toxins containing diols including ciguatoxins, palytoxins, pectenotoxins, tetrodotoxin, trichothecenes, and toxin glycosides.

Occurrence of cyclic imines in European commercial seafood and consumers risk assessment.

Cyclic imines constitute a quite recently discovered group of marine biotoxins that act on neural receptors and that bioaccumulate in seafood. They are grouped together due to the imino group functioning as their common pharmacore, responsible for acute neurotoxicity in mice. Cyclic imines (CIs) have not been linked yet to human poisoning and are not regulated in the European Union (EU), although the European Food Safety Authority (EFSA) requires more data to perform conclusive risk assessment for consumers. Several commercial samples of bivalves including raw and processed samples from eight countries (Italy, Portugal, Slovenia, Spain, Ireland, Norway, The Netherlands and Denmark) were obtained over 2 years. Emerging cyclic imine concentrations in all the samples were analysed on a LC-3200QTRAP and LC-HRMS QExactive mass spectrometer. In shellfish, two CIs, pinnatoxin G (PnTX-G) and 13-desmethylspirolide C (SPX-1) were found at low concentrations (0.1-12µg/kg PnTX-G and 26-66µg/kg SPX-1), while gymnodimines and pteriatoxins were not detected in commercial (raw and processed) samples. In summary, SPX-1 (n: 47) and PnTX-G (n: 96) were detected in 9.4% and 4.2% of the samples, respectively, at concentrations higher than the limit of quantification (LOQ), and in 7.3% and 31.2% of the samples at concentrations lower than the LOQ (25µg/kg for SPX-1 and 3µg/kg for PnTX-G), respectively. For the detected cyclic imines, the average exposure and the 95th percentile were calculated. The results obtained indicate that it is unlikely that a potential health risk exists through the seafood diet for CIs in the EU. However, further information about CIs is necessary in order to perform a conclusive risk assessment.

Immunorecognition magnetic supports for the development of an electrochemical immunoassay for azaspiracid detection in mussels.

As azaspiracids (AZAs) are being reported from the coastal waters of an increasing number of countries on a global scale, the need for rapid, simple and cost-effective methods to detect these marine toxins and protect seafood consumers' health is becoming evident. A magnetic bead (MB)-based direct immunoassay for the detection of AZAs, using protein G-coated MBs as supports for antibody immobilisation and peroxidase-labelled AZA as a tracer is detailed. A colorimetric approach was first developed to optimise the experimental parameters and establish the cross-reactivity factors for AZA-1-10. The subsequent combination of the immunorecognition MBs with 8-electrode arrays enabled the multiplexed electrochemical detection of AZAs. Naturally-contaminated mussel samples were analysed and the results obtained showed an excellent correlation with LC-MS/MS analysis. The MB-based immunoassay facilitated the quantification of a wide range of AZA concentrations (120-2875µg AZA-1 equiv./kg), with a limit of detection (63µg AZA-1 equiv./kg) below the European regulatory threshold, using a protocol that requires very few steps and a short analysis time (~ 15min). The simplicity, cost-effectiveness, rapidity, robustness, selectivity and precision of the assay provide a valuable tool for the detection of all regulated AZAs and other toxic AZA analogues, suitable for end users in the field of food safety.

Development of an ELISA for the Detection of Azaspiracids.

Azaspiracids (AZAs) are a group of biotoxins that cause food poisoning in humans. These toxins are produced by small marine dinoflagellates such as Azadinium spinosum and accumulate in shellfish. Ovine polyclonal antibodies were produced and used to develop an ELISA for quantitating AZAs in shellfish, algal cells, and culture supernatants. Immunizing antigens were prepared from synthetic fragments of the constant region of AZAs, while plate coating antigen was prepared from AZA-1. The ELISA provides a sensitive and rapid analytical method for screening large numbers of samples. It has a working range of 0.45-8.6 ng/mL and a limit of quantitation for total AZAs in whole shellfish at 57 µg/kg, well below the maximum permitted level set by the European Commission. The ELISA has good cross-reactivity to AZA-1-10, -33, and -34 and 37-epi-AZA-1. Naturally contaminated Irish mussels gave similar results whether they were cooked or uncooked, indicating that the ELISA also detects 22-carboxy-AZA metabolites (e.g., AZA-17 and AZA-19). ELISA results showed excellent correlation with LC-MS/MS analysis, both for mussel extract spiked with AZA-1 and for naturally contaminated Irish mussels. The assay is therefore well suited to screening for AZAs in shellfish samples intended for human consumption, as well as for studies on AZA metabolism.

Multihapten approach leading to a sensitive ELISA with broad cross-reactivity to microcystins and nodularin.

Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp3]MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 µg/L, well below the WHO's maximum recommendation of 1 µg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs.

Combined oral toxicity of azaspiracid-1 and yessotoxin in female NMRI mice.

For many years, the presence of yessotoxins (YTXs) in shellfish has contributed to the outcome of the traditional mouse bioassay and has on many occasions caused closure of shellfisheries. Since YTXs do not appear to cause diarrhoea in man and exert low oral toxicity in animal experiments, it has been suggested that they should be removed from regulation. Before doing so, it is important to determine whether the oral toxicity of YTXs is enhanced when present together with shellfish toxins known to cause damage to the gastrointestinal tract. Consequently, mice were given high doses of YTX, at 1 or 5 mg/kg body weight, either alone or together with azaspiracid-1 (AZA1) at 200 µg/kg. The latter has been shown to induce damage to the small intestine at this level. The combined exposure caused no clinical effects, and no pathological changes were observed in internal organs. These results correspond well with the very low levels of YTX detected in internal organs by means of LC-MS/MS and ELISA after dosing. Indeed, the very low absorption of YTX when given alone remained largely unchanged when YTX was administered in combination with AZA1. Thus, the oral toxicity of YTX is not enhanced in the presence of sub-lethal levels of AZA1.

A convenient and cost-effective method for monitoring marine algal toxins with passive samplers.

Passive sampling disks were developed based on the method of MacKenzie, L, Beuzenberg, V., Holland, P., McNabb, P., Selwood, A. [2004. Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves. Toxicon 44, 901-918] and protocols were formulated for recovering toxins from the adsorbent resin via elution from small columns. The disks were used in field studies to monitor in situ toxin dynamics during mixed algal blooms at Flødevigen in Norway. Examples are given from time-integrated sampling using the disks followed by extraction and high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis for azaspiracids, okadaic acid analogues, pectenotoxins, yessotoxins and spirolides. Profiles of accumulated toxins in the disks and toxin profiles in blue mussels (Mytilus edulis) were compared with the relative abundance of toxin-producing algal species. Results obtained showed that passive sampling disks correlate with the toxin profiles in shellfish. The passive sampling disks were cheap to produce and convenient to use and, when combined with HPLC-MS or enzyme-linked immunosorbent assay (ELISA) analysis, provide detailed time-averaged information on the profile of lipophilic toxin analogues in the water. Passive sampling is therefore a useful tool for monitoring the exposure of shellfish to the toxigenic algae of concern in northern Europe.

Clarification of the C-35 stereochemistries of dinophysistoxin-1 and dinophysistoxin-2 and its consequences for binding to protein phosphatase.

Okadaic acid analogues are well known as protein phosphatase inhibitors and occur naturally in marine shellfish feeding on dinoflagellates of the genus Dinophysis, leading to diarrhetic shellfish poisoning of shellfish consumers. Knowledge of the correct structures for these toxins is important in understanding their toxicology, biochemistry, and biosynthesis. We have performed extensive NMR analyses on okadaic acid (1), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) obtained from natural sources. Consequently, we were able to unambiguously deduce the stereochemistries at C-35 for DTX-1 and DTX-2 based on analysis of NMR coupling constants and NOE interactions. Our results revealed that DTX-2 (3) has a stereochemistry opposite to that of DTX-1 (2) at C-35. Molecular modeling of the docking of 1-3 with protein phosphatase-1 and protein phosphatase 2A (PP2A) suggested that the reduced affinity of DTX-2 for PP2A may be due to the newly defined stereochemistry at the 35-methyl group. The implications of these findings for biosynthesis and toxicology are discussed.

Antibodies with broad specificity to azaspiracids by use of synthetic haptens.

The development of general, sensitive, portable, and quantitative assays for the azaspiracid (AZA) class of marine toxins is urgently needed. Use of a synthetic hapten containing rings F-I of AZA to generate antibodies that cross-react with the AZAs via their common C28-C40 domain and use of these antibodies in ELISA and immunoaffinity columns are reported. This approach has many advantages over using intact azaspiracids (AZAs) derived from environmental samples or total synthesis as haptens for antibody development. A derivative of the levorotatory C28-C40 azaspiracid domain (1) was synthesized efficiently using a one-pot Staudinger reduction/intramolecular aza-Wittig reaction-imine capture sequence to form the H-I ring spiroaminal and a double intramolecluar hetero-Michael addition to assemble the F-G ring ketal. Conjugation of the hapten 1 to cBSA and immunization in sheep generated antibodies that recognized and bound to ovalbumin-conjugated 1 in the absence of AZA1. This binding was inhibited by 1 in a concentration-dependent manner. A mixture of AZA1, AZA2, AZA3, and AZA6 caused a degree of inhibition of antibody binding consistent with its total AZA content, rather than just its content of AZA1. This result suggests that the antibodies also have a similar affinity for AZA2, AZA3, and AZA6 as they do for AZA1 and that such antibodies are suitable for analysis of AZAs in shellfish samples.

Isolation and identification of (44-R,S)-44,55-dihydroxyyessotoxin from Protoceratium reticulatum, and its occurrence in extracts of shellfish from New Zealand, Norway and Canada.

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.

Comparison of ELISA and LC-MS analyses for yessotoxins in blue mussels (Mytilus edulis).

Blue mussels (Mytilus edulis) collected from Flødevigen Bay, Norway, in 2001 and 2002 were analysed for yessotoxins (YTXs) by ELISA and yessotoxin (YTX), 45-hydroxyYTX, and carboxyYTX by LC-MS. Results from the two methods were compared to evaluate the ELISA. The response in the ELISA was 3-13 times higher than LC-MS, probably due to the antibodies binding to other YTX analogues not included in the LC-MS analysis. Nevertheless, the correlation between ELISA and LC-MS was good, with r2 values> or =0.8. The results indicate that the ELISA is a reliable method for estimating the total level of YTXs in mussels, and are consistent with extensive metabolism of algal YTXs in mussels. YTX was a minor component in the blue mussels at all times compared to 45-hydroxyYTX and especially carboxyYTX, except when the P. reticulatum bloom occurred. The results also indicate the presence of significant amounts of YTX analogues in addition to those measured by LC-MS. All samples below 4 mg/kg by ELISA were below the current EU regulatory limit of 1 mg/kg by LC-MS. Therefore, we propose using ELISA as a screening tool with a cut-off limit at 4 mg/kg for negative samples, whereas samples above this limit would be reanalyzed by LC-MS.