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Quantitative analysis of azaspiracids in Azadinium spinosum cultures

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Abstract

Azaspiracids (AZAs) are secondary metabolites of Azadinium spinosum that can accumulate in shellfish and cause food poisoning when consumed. We describe here an analytical procedure for the determination of AZAs in cultures of A. spinosum with a focus on the formation of AZA methyl esters as artefacts during extraction and sample pre-treatment. A. spinosum cells were collected from bioreactor cultures using centrifugation or filtration. Different extraction procedures were evaluated for formation of methyl ester artefacts, yield, and matrix effects. Filtration of cultures using glass-fibre filters led to increased formation of methyl esters, and centrifugation is recommended for recovery of cells. The extraction solvent (methanol (MeOH), acetone, and acetonitrile (MeCN)) did not significantly affect the yield of AZAs as long as the organic content was 80% or higher. However, the use of MeOH as extraction solvent led to increased formation of methyl esters. AZA1 recovery over two successive extractions was 100% at the 95% confidence level for acetone and MeOH. In standard-addition experiments, no significant matrix effects were observed in extracts of A. spinosum or Azadinium obesum up to a sample size of 4.5 × 109 μm3. Moreover, experiments carried out to clarify the formation and structure of methylated AZA analogues led to the description of two AZA methyl esters and to the correction of the chemical structures of AZAs29–32.

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Acknowledgements

This study was carried out under the Sea Change strategy with the support of the Marine Institute and the Marine Research Sub-Programme of the National Development Plan 2007–2013, co-financed by the European Regional Development Fund (ASTOX2). Further funding was obtained through Ifremer from the French Ministry of Education, Research and Technology through Programme 187 of the National Finance Law. The authors would like to thank John Lee and Dr. Thomas Glauner of Agilent Technologies for their collaboration on Q-TOF Technology. The authors would also like to thank all the members of the laboratory EMP/PHYC at the Atlantic Centre of Ifremer for their help and technical advice during this study.

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Authors and Affiliations

  1. IFREMER, Laboratoire EMP/PHYC, Rue de l’Ile d’Yeu, 44311, Nantes, France

    Thierry Jauffrais, Véronique Séchet, Manoella Sibat, Zouher Amzil & Philipp Hess

  2. Nantes Atlantique Université, MMS EA2160, 9 rue Bias, 44035, Nantes, France

    Christine Herrenknecht

  3. Alfred Wegener Institute, Am Handelshafen 12, 27570, Bremerhaven, Germany

    Urban Tillmann & Bernd Krock

  4. Marine Institute, Rinville, Oranmore, Co., Galway, Ireland

    Jane Kilcoyne

  5. Norwegian Veterinary Institute, P. O. Box 750 Sentrum, 0106, Oslo, Norway

    Christopher O. Miles

  6. National Research Council Canada, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada

    Pearse McCarron

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  1. Thierry Jauffrais
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  2. Christine Herrenknecht
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  3. Véronique Séchet
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  9. Pearse McCarron
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  10. Zouher Amzil
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  11. Philipp Hess
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Correspondence to Thierry Jauffrais or Philipp Hess.

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Jauffrais, T., Herrenknecht, C., Séchet, V. et al. Quantitative analysis of azaspiracids in Azadinium spinosum cultures. Anal Bioanal Chem 403, 833–846 (2012). https://doi.org/10.1007/s00216-012-5849-2

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