Analytical and Bioanalytical Chemistry

, Volume 400, Issue 2, pp 577–585 | Cite as

Screening for multiple classes of marine biotoxins by liquid chromatography–high-resolution mass spectrometry

  • Pearl Blay
  • Joseph P. M. Hui
  • James Chang
  • Jeremy E. MelansonEmail author
Original Paper


Marine biotoxins pose a significant food safety risk when bioaccumulated in shellfish, and adequate testing for biotoxins in shellfish is required to ensure public safety and long-term viability of commercial shellfish markets. This report describes the use of a benchtop Orbitrap system for liquid chromatography–mass spectrometry (LC-MS) screening of multiple classes of biotoxins commonly found in shellfish. Lipophilic toxins such as dinophysistoxins, pectenotoxins, and azaspiracids were separated by reversed phase LC in less than 7 min prior to MS data acquisition at 2 Hz with alternating positive and negative scans. This approach resulted in mass accuracy for analytes detected in positive mode (gymnodimine, 13-desmethyl spirolide C, pectenotoxin-2, and azaspiracid-1, -2, and -3) of less than 1 ppm, while those analytes detected in negative mode (yessotoxin, okadaic acid, and dinophysistoxin-1 and -2) exhibited mass errors between 2 and 4 ppm. Hydrophilic toxins such as domoic acid, saxitoxin, and gonyautoxins were separated by hydrophilic interaction LC (HILIC) in less than 4 min, and MS data was collected at 1 Hz in positive mode, yielding mass accuracy of less than 1 ppm error at a resolving power of 100,000 for the analytes studied (m/z 300–500). Data were processed by extracting 5 ppm mass windows centered around the calculated masses of the analytes. Limits of detection (LOD) for the lipophilic toxins ranged from 0.041 to 0.10 μg/L (parts per billion) for the positive ions, 1.6–5.1 μg/L for those detected in negative mode, while the domoic acid and paralytic shellfish toxins yielded LODs ranging from 3.4 to 14 μg/L. Toxins were detected in mussel tissue extracts free of interference in all cases.


Marine biotoxins Paralytic shellfish toxins High-resolution mass spectrometry Accurate mass screening LC-MS HILIC 



The authors wish to thank Dr. Pearse McCarron for reviewing this manuscript and for providing extracts of the reference material containing lipophilic toxins. Loan of the Exactive™ mass spectrometer from Thermo Fisher Scientific is also gratefully acknowledged. This is NRC publication number 51791.

Supplementary material

216_2011_4772_MOESM1_ESM.pdf (320 kb)
ESM 1 (PDF 320 kb)


  1. 1.
    Moestrup Ø, Akselman R, Cronberg G, Elbraechter M, Fraga S, Halim Y, Hansen G, Hoppenrath M, Larsen J, Lundholm N, Nguyen LN, Zingone A (2009) IOC-UNESCO taxonomic reference list of harmful micro algae. Available online at Accessed on 25 Oct 2010
  2. 2.
    Commission Regulation (EC) No. 2074/2005 of 5 December 2005 laying down implementing measures for certain products under Regulation (EC) No. 853/2004 of the European Parliament and of the Council and for the organisation of official controls under Regulation (EC) No. 854/2004 of the European Parliament and of the Council and Regulation (EC) No. 882/2004 of the European Parliament and of the Council, derogating from Regulation (EC) No. 852/2004 of the European Parliament and of the Council and amending Regulations (EC) No. 853/2004 and (EC) No. 854/2004 (2005). Official Journal of the European Union L 338/48Google Scholar
  3. 3.
    (2009) Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European Commission on Marine Biotoxins in Shellfish—summary on regulated marine biotoxins. The EFSA Journal 1306:1–23Google Scholar
  4. 4.
    Hess P (2010) Requirements for screening and confirmatory methods for the detection and quantification of marine biotoxins in end-product and official control. Anal Bioanal Chem 397:1683–1694CrossRefGoogle Scholar
  5. 5.
    McNabb P, Selwood AI, Holland PT (2005) Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study. J AOAC Int 88:761–772Google Scholar
  6. 6.
    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
  7. 7.
    Gerssen A, Mulder PPJ, McElhinney MA, de Boer J (2009) Liquid chromatography-tandem mass spectrometry method for the detection of marine lipophilic toxins under alkaline conditions. J Chromatogr A 1216:1421–1430CrossRefGoogle Scholar
  8. 8.
    Gerssen A, Van Olst EHW, Mulder PPJ, De Boer J (2010) In-house validation of a liquid chromatography tandem mass spectrometry method for the analysis of lipophilic marine toxins in shellfish using matrix-matched calibration. Anal Bioanal Chem 397:3079–3088CrossRefGoogle Scholar
  9. 9.
    Quilliam MA, Xie M, Hardstaff WR (1995) Rapid extraction and cleanup for liquid chromatographic determination of domoic acid in unsalted seafood. J AOAC Int 78:543–546Google Scholar
  10. 10.
    Mafra LL Jr, Leger C, Bates SS, Quilliam MA (2009) Analysis of trace levels of domoic acid in seawater and plankton by liquid chromatography without derivatization, using UV or mass spectrometry detection. J Chromatogr A 1216:6003–6011CrossRefGoogle Scholar
  11. 11.
    Lawrence JF, Niedzwiadek B (2001) Quantitative determination of paralytic shellfish poisoning toxins in shellfish by using prechromatographic oxidation and liquid chromatography with fluorescence detection. J AOAC Int 84:1099–1108Google Scholar
  12. 12.
    Lawrence JF, Niedzwiadek B, Menard C (2005) Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: collaborative study. J AOAC Int 88:1714–1732Google Scholar
  13. 13.
    Rourke WA, Murphy CJ, Pitcher G, Van De Riet JM, Burns BG, Thomas KM, Quilliam MA (2008) Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue. J AOAC Int 91:589–597Google Scholar
  14. 14.
    Van De Riet JM, Gibbs RS, Chou FW, Muggah PM, Rourke WA, Burns G, Thomas K, Quilliam MA (2009) Liquid chromatographic post-column oxidation method for analysis of paralytic shellfish toxins in mussels, clams, scallops, and oysters: single-laboratory validation. J AOAC Int 92:1690–1704Google Scholar
  15. 15.
    Dell'Aversano C, Hess P, Quilliam MA (2005) Hydrophilic interaction liquid chromatography-mass spectrometry for the analysis of paralytic shellfish poisoning (PSP) toxins. J Chromatogr A 1081:190–201CrossRefGoogle Scholar
  16. 16.
    Turrell E, Stobo L, Lacaze JP, Piletsky S, Piletska E (2008) Optimization of hydrophilic interaction liquid chromatography/mass spectrometry and development of solid-phase extraction for the determination of paralytic shellfish poisoning toxins. J AOAC Int 91:1372–1386Google Scholar
  17. 17.
    Diener M, Erler K, Christian B, Luckas B (2007) Application of a new zwitterionic hydrophilic interaction chromatography column for determination of paralytic shellfish poisoning toxins. J Separation Sci 30:1821–1826CrossRefGoogle Scholar
  18. 18.
    Schurmann A, Dvorak V, Cruzer C, Butcher P, Kaufmann A (2009) False-positive liquid chromatography/tandem mass spectrometric confirmation of sebuthylazine residues using the identification points system according to EU directive 2002/657/EC due to a biogenic insecticide in tarragon. Rapid Comm Mass Spectrom 23:1196–1200CrossRefGoogle Scholar
  19. 19.
    Grimalt S, Sancho JV, Pozo OJ, Hernandez F (2010) Quantification, confirmation and screening capability of UHPLC coupled to triple quadrupole and hybrid quadrupole time-of-flight mass spectrometry in pesticide residue analysis. J Mass Spectrom 45:421–436Google Scholar
  20. 20.
    Kellmann M, Muenster H, Zomer P, Mol H (2009) Full scan MS in comprehensive qualitative and quantitative residue analysis in food and feed matrices: how much resolving power is required? J Am Soc Mass Spectrom 20:1464–1476CrossRefGoogle Scholar
  21. 21.
    Zhang NR, Yu S, Tiller P, Yeh S, Mahan E, Emary WB (2009) Quantitation of small molecules using high-resolution accurate mass spectrometers—a different approach for analysis of biological samples. Rapid Commun Mass Spectrom 23:1085–1094CrossRefGoogle Scholar
  22. 22.
    Bateman KP, Kellmann M, Muenster H, Papp R, Taylor L (2009) Quantitative-qualitative data acquisition using a Benchtop Orbitrap mass spectrometer. J Am Soc Mass Spectrom 20:1441–1450CrossRefGoogle Scholar
  23. 23.
    Krauss M, Singer H, Hollender J (2010) LC–high resolution MS in environmental analysis: from target screening to the identification of unknowns. Anal Bioanal Chem 397:943–951CrossRefGoogle Scholar
  24. 24.
    Perez RA, Rehmann N, Crain S, LeBlanc P, Craft C, MacKinnon S, Reeves K, Burton IW, Walter JA, Hess P, Quilliam MA, Melanson JE (2010) The preparation of certified calibration solutions for azaspiracid-1,-2, and-3, potent marine biotoxins found in shellfish. Anal Bioanal Chem 398:2243–2252CrossRefGoogle Scholar
  25. 25.
    McCarron P, Emteborg H, Nulty C, Rundberget T, Loader JI, Miles CO, Emons H, Quilliam MA, Hess P (2011) A mussel tissue certified reference material for multiple toxins: part 1: design and preparation. Anal Bioanal Chem doi:10.1007/s00216-011-4786-9
  26. 26.
    Brenton AG, Godfrey AR (2010) Accurate mass measurement: terminology and treatment of data. J Am Soc Mass Spectrom 21:1821–1835Google Scholar

Copyright information

© Crown copyright in right of Canada 2011

Authors and Affiliations

  • Pearl Blay
    • 1
  • Joseph P. M. Hui
    • 1
  • James Chang
    • 2
  • Jeremy E. Melanson
    • 1
    Email author
  1. 1.National Research Council of Canada, Institute for Marine BiosciencesHalifaxCanada
  2. 2.Thermo Fisher ScientificSan JoseUSA

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