LC/MS methodology for the analysis of domoic acid and lipophilic toxins in shellfish was developed using a hybrid triple quadrupole linear ion trap mass spectrometer. For routine quantitation a scheduled selected reaction monitoring (SRM) method for the analysis of domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxins, yessotoxins, gymnodimines, spirolides, and pinnatoxins was developed and validated. The method performed well in terms of LOD, linearity, precision, and trueness. Taking advantage of the high instrument sensitivity, matrix effects were mitigated by reducing the amount of sample introduced to the mass spectrometer. Optionally, samples can be analyzed using information dependent acquisition (IDA) methods, either in positive or negative mode, which can provide an extra level of confirmation by matching the full product ion spectra acquired for a sample with those from a specially constructed spectral library. Methods were applied to the analysis df a new certified reference material and Canadian mussels (Mytilus edulis) implicated in a 2011 diarrhetic shellfish poisoning (DSP) incident. The scheduled SRM method enabled the screening and quantitation of multiple phycotoxins. As DSP had not previously been observed in this area of Canada, positive identification of putative toxins was accomplished using the IDA and spectral search method. Analysis of the 2011 toxic mussel samples revealed the presence of high levels of dinophysistoxin-1, which explained the DSP symptoms, as well as pectenotoxins, yessotoxins, and variety of cyclic imines.

Marine shellfish exposed to the microalgae Karenia selliformis can accumulate gymnodimines (GYM). Shellfish samples collected from Beihai City in Guangxi Autonomous Region, and Ningde City in Fujian Province, in the South China Sea, as well as mussels Mytilus galloprovincialis fed on K. selliformis under laboratory conditions were analyzed. Gymnodimines and various fatty acid ester metabolites were detected in the clam Antigona lamellaris and pen shell Atrina pectinata, while no esters were found in the oyster Crassostrea sp. and the gastropod Batillaria zonalis despite positive detection of free GYM in both species. When present, the predominant acyl esters observed were 18:0-GYM-A and 20:1-GYM-A. Under laboratory conditions GYM-A was accumulated and metabolized to fatty acid esters in mussels exposed to K. selliformis, with 16:0-GYM-A and 20:1-GYM-A as the major variants. A novel compound with the same accurate mass as GYM-A and its 16:0 fatty acid ester were observed in the experimental mussels but was not present in the microalgal strain to which mussels were exposed. No significant differences of reactive oxygen species (ROS) levels and antioxidant enzymes were found between mussels fed on K. selliformis or GYM-free microalgae Isochrysis galbana. This suggests the accumulation of GYM and its metabolites does not significantly impact the physiological status of mussels. While it is currently not proven that GYM affects human health, risk assessments should consider the presence of GYM esters in naturally contaminated shellfish as part of exposure analysis. Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site.

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.

Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n'arrivez pas à les repérer, communiquez avec nous à PublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. Questions? Contact the NRC Publications Archive team atPublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. If you wish to email the authors directly, please see the first page of the publication for their contact information. NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. NRC Publications Record / Notice d'Archives des publications de CNRC:http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/view/object/?id=2d72a8c8-d862-4bae-ae12-3ba2bf19d83e http://nparc.cisti-icist.nrc-cnrc.gc.ca/fra/voir/objet/?id=2d72a8c8-d862-4bae-ae12-3ba2bf19d83e Abstract A freeze-dried mussel tissue (Mytilus edulis)reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatographybased analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13 ± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57 ± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation.

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Microcystins (MCs) are a potent class of hepatotoxins produced by several species of cyanobacteria, which have been responsible for illness and death in livestock and humans.

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Okadaic acid (OA) group toxins may accumulate in shellfish and can result in diarrhetic shellfish poisoning when consumed by humans, and are therefore regulated. Purified toxins are required for the production of certified reference materials used to accurately quantitate toxin levels in shellfish and water samples, and for other research purposes. An improved procedure was developed for the isolation of dinophysistoxin 2 (DTX2) from shellfish (M. edulis), reducing the number of purification steps from eight to five, thereby increasing recoveries to ~68%, compared to ~40% in a previously reported method, and a purity of >95%. Cell densities and toxin production were monitored in cultures of Prorocentrum lima, that produced OA, DTX1, and their esters, over ~1.5 years with maximum cell densities of ~70,000 cells mL−1 observed. Toxin accumulation progressively increased over the study period, to ~0.7 and 2.1 mg L−1 of OA and DTX1 (including their esters), respectively, providing information on appropriate harvesting times. A procedure for the purification of OA and DTX1 from the harvested biomass was developed employing four purification steps, with recoveries of ~76% and purities of >95% being achieved. Purities were confirmed by LC-HRMS, LC-UV, and NMR spectroscopy. Additional stability observations led to a better understanding of the chemistry of these toxins.