Rapid Quantitation of Anatoxins in Benthic Cyanobacterial Mats Using Direct Analysis in Real-Time–High-Resolution Tandem Mass Spectrometry

Toxic benthic cyanobacterial mats are increasingly reported worldwide as being responsible for animal mortalities due to their production of the potent neurotoxin anatoxin-a (ATX) and its analogues. Improved analytical methods for anatoxins are needed to address public health and watershed management challenges arising from extremely high spatial and temporal variability within impacted systems. We present the development, validation, and application of a direct analysis in real-time–high-resolution tandem mass spectrometry (DART–HRMS/MS) method for analysis of anatoxins in cyanobacterial field samples, including a simplified sample preparation approach. The method showed excellent sensitivity and selectivity for ATX, homoanatoxin-a, and dihydroanatoxin-a. Isotopically labeled ATX was used as an internal standard for all three analogues and successfully corrected for the matrix effects observed (86 ± 16% suppression). The limit of detection and recovery for ATX was estimated as 5 ng/g and 88%, respectively, using spiked samples. The total analysis time was ∼2 min, and excellent agreement was observed with results from a liquid chromatography–HRMS reference method. Finally, the DART–HRMS/MS method was applied to a set of 45 Microcoleus-dominated benthic cyanobacterial mat samples from the Wolastoq near Fredericton, Canada, demonstrating its power and applicability in enabling broad-scale field studies of ATX distribution

Identification of Pinnatoxins and Discovery of Their Fatty Acid Ester Metabolites in Mussels (Mytilus edulis) from Eastern Canada

Pinnatoxins are a group of fast-acting cyclic imine toxins previously identified in shellfish from Asia, the southern Pacific, and northern Europe. In this work pinnatoxins were detected in mussels from locations across the eastern coast of Canada. Pinnatoxin G (<b>6</b>) was the major structural variant present, sometimes at levels >80 μg/kg, whereas much lower levels of pinnatoxin A (<b>1</b>) were detected in some samples. Increased concentrations were observed following base hydrolysis of extracts, leading to the discovery by LC-MS of a range of fatty acid esters of <b>6</b>. Information on the structures of these acylated derivatives was provided through a series of mass spectrometric experiments, supported by partial synthesis, and it is proposed that the compounds are 28-<i>O</i>-acyl esters of <b>6</b>. Although acyl esters of a range of other phycotoxins are known to form as metabolites in shellfish, this is the first report of their existence for this particular toxin class. The occurrence of pinnatoxins in North American shellfish further highlights the international distribution of these toxins

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