Improved Isolation Procedure for Azaspiracids from Shellfish, Structural Elucidation of Azaspiracid-6, and Stability Studies

Azaspiracids are a group of lipophilic polyether toxins produced by the small dinoflagellate <i>Azadinium spinosum</i>. They may accumulate in shellfish and can result in illnesses when consumed by humans. Research into analytical methods, chemistry, metabolism, and toxicology of azaspiracids has been severely constrained by the scarcity of high-purity azaspiracids. Consequently, since their discovery in 1995, considerable efforts have been made to develop methods for the isolation of azaspiracids in sufficient amounts and purities for toxicological studies, in addition to the preparation of standard reference materials. A seven-step procedure was improved for the isolation of azaspiracids-1–3 (<b>1</b>, <b>2</b>, and <b>3</b>) increasing recoveries 2-fold as compared to previous methods and leading to isolation of sufficiently purified azaspiracid-6 (<b>6</b>) for structural determination by NMR spectroscopy. The procedure, which involved a series of partitioning and column chromatography steps, was performed on 500 g of <i>Mytilus edulis</i> hepatopancreas tissue containing ∼14 mg of <b>1</b>. Overall yields of <b>1</b> (52%), <b>2</b> (43%), <b>3</b> (43%), and <b>6</b> (38%) were good, and purities were confirmed by NMR spectroscopy. The structure of <b>6</b> was determined by one- and two-dimensional NMR spectroscopy and mass spectrometry. The stability of <b>6</b> relative to <b>1</b> was also assessed in three solvents in a short-term study that demonstrated the greatest stability in aqueous acetonitrile

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-<i>epi</i>-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

Epimers of Azaspiracids: Isolation, Structural Elucidation, Relative LC-MS Response, and <i>in Vitro</i> Toxicity of 37-<i>epi</i>-Azaspiracid‑1

Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ∼2–16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-<i>epi</i>-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-<i>epi</i>-AZA2 and 37-<i>epi</i>-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-<i>epi</i>-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-<i>epi</i>-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an <i>in vitro</i> cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-<i>epi</i>-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC₅₀ values in the subnanomolar range. On the basis of EC₅₀ comparisons, 37-<i>epi</i>-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes

Structure Elucidation, Relative LC–MS Response and In Vitro Toxicity of Azaspiracids <b>7</b>–<b>10</b> Isolated from Mussels (Mytilus edulis)

Azaspiracids (AZAs) are marine biotoxins produced by dinoflagellates that can accumulate in shellfish, which if consumed can lead to poisoning events. AZA7–10, <b>7</b>–<b>10</b>, were isolated from shellfish and their structures, previously proposed on the basis of only LC–MS/MS data, were confirmed by NMR spectroscopy. Purified AZA4–6, <b>4</b>–<b>6</b>, and <b>7</b>–<b>10</b> were accurately quantitated by qNMR and used to assay cytotoxicity with Jurkat T lymphocyte cells for the first time. LC–MS­(MS) molar response studies performed using isocratic and gradient elution in both selected ion monitoring and selected reaction monitoring modes showed that responses for the analogues ranged from 0.3 to 1.2 relative to AZA1, <b>1</b>. All AZA analogues tested were cytotoxic to Jurkat T lymphocyte cells in a time- and concentration-dependent manner; however, there were distinct differences in their EC₅₀ values, with the potencies for each analogue being: AZA6 > AZA8 > AZA1 > AZA4 ≈ AZA9 > AZA5 ≈ AZA10. This data contributes to the understanding of the structure–activity relationships of AZAs

Isolation, Structure Elucidation, Relative LC-MS Response, and in Vitro Toxicity of Azaspiracids from the Dinoflagellate <i>Azadinium spinosum</i>

We identified three new azaspiracids (AZAs) with molecular weights of 715, 815, and 829 (AZA33 (<b>3</b>), AZA34 (<b>4</b>), and AZA35, respectively) in mussels, seawater, and <i>Azadinium spinosum</i> culture. Approximately 700 μg of <b>3</b> and 250 μg of <b>4</b> were isolated from a bulk culture of <i>A. spinosum</i>, and their structures determined by MS and NMR spectroscopy. These compounds differ significantly at the carboxyl end of the molecule from known AZA analogues and therefore provide valuable information on structure–activity relationships. Initial toxicological assessment was performed using an in vitro model system based on Jurkat T lymphocyte cytotoxicity, and the potencies of <b>3</b> and <b>4</b> were found to be 0.22- and 5.5-fold that of AZA1 (<b>1</b>), respectively. Thus, major changes in the carboxyl end of <b>1</b> resulted in significant changes in toxicity. In mussel extracts, <b>3</b> was detected at low levels, whereas <b>4</b> and AZA35 were detected only at extremely low levels or not at all. The structures of <b>3</b> and <b>4</b> are consistent with AZAs being biosynthetically assembled from the amino end