Analytical and Bioanalytical Chemistry

, Volume 400, Issue 3, pp 821–833 | Cite as

A mussel tissue certified reference material for multiple phycotoxins. Part 1: design and preparation

  • Pearse McCarron
  • Håkan Emteborg
  • Cíara Nulty
  • Thomas Rundberget
  • Jared I. Loader
  • Katharina Teipel
  • Christopher O. Miles
  • Michael A. Quilliam
  • Philipp Hess
Original Paper


The development of multi-analyte methods for lipophilic shellfish toxins based on liquid chromatography–mass spectrometry permits rapid screening and analysis of samples for a wide variety of toxins in a single run. Validated methods and appropriate certified reference materials (CRMs) are required to ensure accuracy of results. CRMs are essential for accurate instrument calibration, for assessing the complete analytical method from sample extraction to data analysis and for verifying trueness. However, CRMs have hitherto only been available for single toxin groups. Production of a CRM containing six major toxin groups was achieved through an international collaboration. Preparation of this material, CRM-FDMT1, drew on information from earlier studies as well as improved methods for isolation of toxins, handling bulk tissues and production of reference materials. Previous investigations of stabilisation techniques indicated freeze-drying to be a suitable procedure for preparation of shellfish toxin reference materials and applicable to a wide range of toxins. CRM-FDMT1 was initially prepared as a bulk wet tissue homogenate containing domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxin-2, yessotoxin and 13-desmethylspirolide C. The homogenate was then freeze-dried, milled and bottled in aliquots suitable for distribution and analysis. The moisture content and particle size distribution were measured, and determined to be appropriate. A preliminary toxin analysis of the final material showed a comprehensive toxin profile.


Bottling of CRM-FDMT1


CRM-FDMT1 Certified reference material Shellfish toxins Phycotoxins Accuracy Precision Liquid chromatography–mass spectrometry 



The staff of the processing sector at IRMM, Jean Charoud-Got, Andrea Bau, Marie-France Tumba-Tshilumba, Paul De Vos and Albert Oostra are gratefully acknowledged for their technical support as well as Hendrik Emons (also at IRMM) for facilitating and supporting this collaboration. The help of the Biotoxin Chemistry team at the Marine Institute is appreciated. We thank Morten Sandvik of the Norwegian Veterinary Institute and Allan D. Hawkes of AgResearch Ltd. for assistance with supply of PTX2 and YTX, respectively, and Jeremy Melanson of NRC for reviewing this manuscript.

This study was supported by the ASTOX project (funded by The Marine Institute and National Development Plan (NDP; ST/02/02, 2003–2006)), by the BIOTOX project (partly funded by the European Commission, through Sixth Framework Programme contract no. 514074, priority Food Quality and Safety), by the Norwegian Research Council grant 139593/140, by the New Zealand Foundation for Research, Science and Technology (FRST), by the International Investment Opportunities Fund (IIOF contract number C10X0406) and by a FRST Post-Doctoral Fellowship (contract number AGRX0402). The routine shellfish toxin monitoring program in Ireland is funded by the Irish government.

This is NRCC publication number 51794.

Supplementary material

216_2011_4786_MOESM1_ESM.pdf (77 kb)
ESM 1 (PDF 77.4 kb)


  1. 1.
    Wright JLC, Boyd RK, De Freitas ASW, Falk M, Foxall RA, Jamieson WD, Laycock MV, McCulloch AW, McInnes AG, Odense P, Pathak VP, Quilliam MA, Ragan MA, Sim PG, Thibault P, Walter JA, Gilgan M, Richard DJA, Dewar D (1989) Identification of domoic acid, a neuroexcitatory amino acid, in toxic mussels from eastern Prince Edward Island. Can J Chem 67:481–490CrossRefGoogle Scholar
  2. 2.
    Yasumoto T, Murata M, Lee JA, Torigoe K (1978) Occurrence of a new type of toxic shellfish poisoning in the Tohoku district. Bull Jpn Soc Sci Fish 44:1249–1255Google Scholar
  3. 3.
    Satake M, Ofuji K, Naoki H, James K, Furey A, McMahon T, Silke J (1998) Azaspiracid, a new marine toxin having unique spiro ring assemblies, isolated from Irish mussels, Mytilus edulis. J Am Chem Soc 120:9967–9968CrossRefGoogle Scholar
  4. 4.
    CRLMB (2009) EU harmonised SOP for the detection of lipophilic biotoxins by mouse bioassay, Version 5, June 2009. Community Reference Laboratory for Marine Biotoxins (CRLMB), Vigo, Spain. Available at Accessed 4 Feb 2011
  5. 5.
    Hess P, Grune B, Anderson DB, Aune T, Botana LM, Caricato P, van Egmond HP, Halder M, Hall S, Lawrence JF, Moffat C, Poletti R, Richmond J, Rossini GP, Seamer C, Serratosa Vilageliu J (2006) Three Rs approaches in marine biotoxin testing—the report and recommendations of a joint ECVAM/DG SANCO Workshop (ECVAM Workshop 55). Altern Labor Anim (ATLA) 34:193–224Google Scholar
  6. 6.
    Lee SO, Yanagi T, Kenma R, Yasumoto T (1987) Fluorometric determination of diarrhetic shellfish toxins by high-performance liquid chromatography. Agric Biol Chem 51:877–881Google Scholar
  7. 7.
    Quilliam MA, Sim PG, McCulloch AW, McInnes AG (1989) High performance liquid chromatography of domoic acid, a marine neurotoxin, with application to shellfish and plankton. Int J Environ Anal Chem 36:139–154CrossRefGoogle Scholar
  8. 8.
    Suzuki T, Mitsuya T, Matsubara H, Yamasaki M (1998) Determination of pectentoxin-2 after solid phase extraction from seawater and from the dinoflagellate Dinophysis fortii by liquid chromatography with electrospray mass spectrometry and ultraviolet detection. Evidence of oxidation of pectenotoxin-2 to pectenotoxin-6 in scallops. J Chromatogr A 815:155–160CrossRefGoogle Scholar
  9. 9.
    Ofuji K, Satake M, Oshima Y, McMahon T, James KJ, Yasumoto T (1999) A sensitive and specific determination method for azaspiracids by liquid chromatography mass spectrometry. Nat Toxins 7:247–250CrossRefGoogle Scholar
  10. 10.
    Suzuki T, Horie Y, Koike K, Satake M, Oshima Y, Iwataki M, Yoshimatsu S (2007) Yessotoxin analogues in several strains of Protoceratium reticulatum in Japan determined by liquid chromatography–hybrid triple quadrupole/linear ion trap mass spectrometry. J Chromatogr A 1142:172–177CrossRefGoogle Scholar
  11. 11.
    Quilliam MA, Hess P, Dell'Aversano C (2001) Recent developments in the analysis of phycotoxins by liquid chromatography–mass spectrometry. In: De Koe WJ, Samson RA, van Egmond HP, Gilbert J, Sabino M (eds) Proceedings of the Xth International IUPAC Symposium on Mycotoxins and Phycotoxins, 21–25 May, 2000, Guaruja, Brazil, pp 383–391Google Scholar
  12. 12.
    Stobo LA, Lacaze JP, Scott AC, Gallacher S, Smith EA, Quilliam MA (2005) Liquid chromatography with mass spectrometry-detection of lipophilic shellfish toxins. J AOAC Int 88:1371–1382Google Scholar
  13. 13.
    Fux E, McMillan D, Bire R, Hess P (2007) Development of an ultra-performance liquid chromatography–mass spectrometry method for the detection of lipophilic marine toxins. J Chromatogr A 1157:273–280CrossRefGoogle Scholar
  14. 14.
    McNabb P, Selwood AI, Holland PT, Aasen J, Aune T, Eaglesham G, Hess P, Igarishi M, Quilliam M, Slattery D, van de Riet J, van Egmond H, Van den Top H, Yasumoto T (2005) Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study. J AOAC Int 88:761–772Google Scholar
  15. 15.
    ISO-Guide-30 (1992) Terms and definitions used in connection with reference materials. ISO/IEC, Geneva, SwitzerlandGoogle Scholar
  16. 16.
    Emons H, Fajgelj A, Veen AMH, Watters R (2006) New definitions on reference materials. Accredit Qual Assur 10:576–578CrossRefGoogle Scholar
  17. 17.
    Emons H (2006) The ‘RM family’—identification of all of its members. Accredit Qual Assur 10:690–691CrossRefGoogle Scholar
  18. 18.
    Hess P, McCarron P, Quilliam MA (2006) Fit-for-purpose shellfish reference materials for internal and external quality control in the analysis of phycotoxins. Anal Bioanal Chem 387:2463–2474CrossRefGoogle Scholar
  19. 19.
    Boenke A (1998) Activities and current research of the European Community standards, measurements and testing programme (SMT) in the area of phycotoxins. In: Reguera B, Blanco J, Fernández ML, Wyatt T (eds) Harmful Algae. Xunta De Galicia and Intergovernmental Oceanographic Commission (IOC) of UNESCO, Proceedings of the VIII International Conference on Harmful Algae, Vigo, Spain, 25–29 June 1997, pp 211–215Google Scholar
  20. 20.
    Goto H, Igarashi T, Sekiguchi K, Tanno K, Satake M, Oshima Y, Yasumoto T (1998) A Japanese project for the production and distribution of shellfish toxins as calibrants for HPLC analysis. In: Reguera B, Blanco J, Fernández ML, Wyatt T (eds) Harmful Algae. Xunta De Galicia and Intergovernmental Oceanographic Commission (IOC) of UNESCO, Proceedings of the VIII International Conference on Harmful Algae, Vigo, Spain, 25–29 June 1997, pp 216–219Google Scholar
  21. 21.
    van den Top HJ, Boenke A, Burdaspal PA, Bustos J, van Egmond HP, Legarda T, Mesego A, Mourino A, Paulsch WE, Salgado C (2001) The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study. Food Addit Contam 18:810–824Google Scholar
  22. 22.
    Quilliam MA, Wright JLC (1989) The amnesic shellfish poisoning mystery. Anal Chem 61:1053–1060CrossRefGoogle Scholar
  23. 23.
    Hardstaff WR, Jamieson WD, Milley JE, Quilliam MA, Sim PG (1990) Reference materials for domoic acid, a marine neurotoxin. Fresenius J Anal Chem 338:520–525CrossRefGoogle Scholar
  24. 24.
    CRMP Website. Available at Accessed 22 Nov 2010
  25. 25.
    McCarron P, Hess P (2006) Tissue distribution and effects of heat treatments on the content of domoic acid in blue mussels, Mytilus edulis. Toxicon 47:473–479CrossRefGoogle Scholar
  26. 26.
    McCarron P, Kilcoyne J, Hess P (2008) Effects of cooking and heat treatment on concentration and tissue distribution of okadaic acid and dinophysistoxin-2 in mussels (Mytilus edulis). Toxicon 51:1081–1089CrossRefGoogle Scholar
  27. 27.
    NRC-CNRC (2005) NRC CRM ASP-Mus-c: mussel tissue reference material for domoic acid. Certificate of analysis. Institute for Marine Biosciences, Halifax, NS, CanadaGoogle Scholar
  28. 28.
    NRC-CNRC (2005) NRC CRM-DSP-Mus-b: mussel tissue reference material for DSP toxins. Certificate of analysis. Institute for Marine Biosciences, Halifax, NS, CanadaGoogle Scholar
  29. 29.
    Quilliam MA, Reeves K, MacKinnon S, Craft C, Whyte H, Walter J, Stobo L, Gallacher S (2006) Preparation of reference materials for azaspiracids. In: Deegan B, Butler C, Cusack C, Henshilwood K, Hess P, Keaveney S, McMahon T, O'Cinneide M, Lyons D, Silke J (eds) 5th International Conference of Molluscan Shellfish Safety, 14–18 June 2004, The Marine Institute, Galway, Ireland, pp 111–115, ISBN: 1 902895-33-9Google Scholar
  30. 30.
    McCarron P, Kotterman M, Boer J, Rehmann N, Hess P (2007) Feasibility of gamma irradiation as a stabilisation technique in the preparation of tissue reference materials for a range of shellfish toxins. Anal Bioanal Chem 387:2487–2493CrossRefGoogle Scholar
  31. 31.
    McCarron P, Burrell S, Hess P (2006) Effect of addition of antibiotics and an antioxidant on the stability of tissue reference materials for domoic acid, the amnesic shellfish poison. Anal Bioanal Chem 387:2495–2502CrossRefGoogle Scholar
  32. 32.
    Linsinger TPJ, Pauwels J, Van der Veen AMH, Schimmel H, Lamberty A (2001) Homogeneity and stability of reference materials. Accredit Qual Assur 6:20–25CrossRefGoogle Scholar
  33. 33.
    McCarron P, Emteborg H, Hess P (2007) Freeze-drying for the stabilisation of shellfish toxins in mussel tissue (Mytilus edulis) reference materials. Anal Bioanal Chem 387:2475–2486CrossRefGoogle Scholar
  34. 34.
    Huhn J, Jeffrey PD, Larsen K, Rundberget T, Rise F, Cox NR, Arcus V, Shi Y, Miles CO (2009) A structural basis for the reduced toxicity of dinophysistoxin-2. Chem Res Toxicol 22:1782–1786CrossRefGoogle Scholar
  35. 35.
    Hess P, McCarron P, Rehmann N, Kilcoyne J, McMahon T, Ryan G, Ryan MP, Twiner M, Doucette G, Satake M, Ito E, Yasumoto T (2007) Isolation and purification of AZAs from naturally contaminated material, and evaluation of their toxicological effects (ASTOX). Marine Environment & Health Series, ISSN: 1649 0053Google Scholar
  36. 36.
    McCarron P, Kilcoyne J, Miles CO, Hess P (2009) Formation of azaspiracids-3, -4, -6, and -9 via decarboxylation of carboxyazaspiracid metabolites from shellfish. J Agric Food Chem 57:160–169CrossRefGoogle Scholar
  37. 37.
    McCarron P (2008) Studies in the development of reference materials for phycotoxins, with a focus on azaspiracids. University College Dublin, IrelandGoogle Scholar
  38. 38.
    Loader JI, Hawkes AD, Beuzenberg V, Jensen DJ, Cooney JM, Wilkins AL, Fitzgerald JM, Briggs LR, Miles CO (2007) Convenient large-scale purification of yessotoxin from Protoceratium reticulatum culture and isolation of novel furanoyessotoxin. J Agric Food Chem 55:11093–11100CrossRefGoogle Scholar
  39. 39.
    Cembella A, Lewis N, Quilliam MA (2000) The marine dinoflagellate Alexandrium ostenfeldii (Dinophyceae) as the causative organism of spirolides shellfish toxins. Phycologia 39:67–74CrossRefGoogle Scholar
  40. 40.
    Rundberget T, Sandvik M, Larsen K, Pizarro GM, Reguera B, Castberg T, Gustad E, Loader J, Rise F, Wilkins AL, Miles CO (2007) Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway, and isolation of okadaic acid and dinophysistoxin-2. Toxicon 50:960–970CrossRefGoogle Scholar
  41. 41.
    Quilliam MA, Xie M, Hardstaff W (1995) A rapid extraction and cleanup procedure for the liquid chromatographic determination of domoic acid in unsalted seafood. J AOAC Int 78:543–554Google Scholar
  42. 42.
    Hess P, Kilcoyne J, Swords D, Mulcahy N, McCarron M, Keogh M, Gibbons B, Ronan J (2006) Impact of HPLC-UV methods (solid-phase extraction/UV detection and photodiode-array detection) for the determination of domoic acid on quality of results and sample turnaround time. In: Deegan B, Butler C, Cusack C, Henshilwood K, Hess P, Keaveney S, McMahon T, O'Cinneide M, Lyons D, Silke J (eds) 5th International Conference of Molluscan Shellfish Safety, 14–18 June 2004, The Marine Institute, Galway, Ireland, pp 77–80, ISBN: 1 902895-33-9Google Scholar
  43. 43.
    Mountfort DO, Suzuki T, Truman P (2001) Protein phosphatase inhibition adapted for determination of total DSP in contaminated mussel. Toxicon 39:383–390CrossRefGoogle Scholar
  44. 44.
    Kestens V, Charoudgot J, Bau A, Bernreuther A, Emteborg H (2008) Online measurement of water content in candidate reference materials by acousto-optical tuneable filter near-infrared spectrometry (AOTF-NIR) using pork meat calibrants controlled by Karl Fischer titration. Food Chem 106:1359–1365CrossRefGoogle Scholar
  45. 45.
    Anonymous (2004) Regulation (EC) No 853/2004 of the European parliament and of the council of 29 April 2004 laying down specific hygiene rules for food of animal origin. Official Journal of the European Union L 139 of 30 April 2004Google Scholar
  46. 46.
    Carmody EP, James KJ, Kelly SS (1996) Dinophysistoxin-2: the predominant diarrhoetic shellfish toxin in Ireland. Toxicon 34:351–359CrossRefGoogle Scholar
  47. 47.
    Miles CO, Wilkins AL, Munday R, Dines MH, Hawkes AD, Briggs LR, Sandvik M, Jensen DJ, Cooney JM, Holland PT, Quilliam MA, MacKenzie AL, Beuzenberg V, Towers NR (2004) Isolation of pectenotoxin-2 from Dinophysis acuta and its conversion to pectenotoxin-2 seco acid, and preliminary assessment of their acute toxicities. Toxicon 43:1–9CrossRefGoogle Scholar
  48. 48.
    Flink JM (1975). In: Goldblith SM, Rey L, Rothmayr WW (eds) Freeze drying and advanced food technology, chapter 20. Academic press, New York, pp 319–320, ISBN: 0-12-288450-7Google Scholar
  49. 49.
    Quevauviller P, Maier EA (1999) Interlaboratory studies and certified reference materials for environmental analysis: the BCR approach. In: Techniques and instrumentation in analytical chemistry. Elsevier Science B.V., Amsterdam, p 119, ISBN: 0-444-82389-1Google Scholar

Copyright information

© Her Majesty the Queen in Right of Canada 2011

Authors and Affiliations

  • Pearse McCarron
    • 1
    • 2
  • Håkan Emteborg
    • 3
  • Cíara Nulty
    • 2
  • Thomas Rundberget
    • 4
  • Jared I. Loader
    • 5
    • 6
  • Katharina Teipel
    • 3
  • Christopher O. Miles
    • 4
    • 5
  • Michael A. Quilliam
    • 1
  • Philipp Hess
    • 2
    • 7
  1. 1.National Research Council Canada, Institute for Marine BiosciencesHalifaxCanada
  2. 2.Marine InstituteOranmoreIreland
  3. 3.European Commission, Joint Research Centre, Institute for Reference Materials and MeasurementsGeelBelgium
  4. 4.National Veterinary InstituteOsloNorway
  5. 5.AgResearch Ltd.HamiltonNew Zealand
  6. 6.FDA Gulf Coast Seafood LaboratoryDauphin IslandUSA
  7. 7.IfremerNantes Cedex 03France

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