Analytical and Bioanalytical Chemistry

, Volume 409, Issue 24, pp 5675–5687 | Cite as

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for quantitation of paralytic shellfish toxins: validation and application to reference materials

  • Krista M. Thomas
  • Daniel G. Beach
  • Kelley L. Reeves
  • Ryan S. Gibbs
  • Elliott S. Kerrin
  • Pearse McCarron
  • Michael A. QuilliamEmail author
Research Paper


Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by marine dinoflagellates that are responsible for paralytic shellfish poisoning (PSP) in humans. This work highlights our ongoing efforts to develop quantitative methods for PSTs using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). Compared with the commonly used method of liquid chromatography with post-column oxidation and fluorescence detection (LC-ox-FLD), HILIC-MS/MS has the potential of being more robust, sensitive and straightforward to operate, and provides unequivocal confirmation of toxin identity. The main driving force for the present work was the need for a complementary method to LC-ox-FLD to assign values to shellfish tissue matrix reference materials for PSTs. Method parameters that were optimized included LC mobile and stationary phases, electrospray ionization (ESI) conditions, and MS/MS detection parameters. The developed method has been used in the detection and identification of a wide range of PSTs including less common analogues and metabolites in a range of shellfish and algal samples. We have assessed the matrix effects of shellfish samples and have evaluated dilution, standard addition and matrix matched calibration as means of mitigating them. Validation on one LC-MS/MS system for nine common PST analogues (GTX1-4, dcGTX2&3, STX, NEO, and dcSTX) was completed using standard addition. The method was then transferred to a more sensitive LC-MS/MS system, expanded to include five more PSTs (C1&2, dcNEO and GTX5&6) and validated using matrix matched calibration. Limits of detection of the validated method ranged between 6 and 280 nmol/kg tissue using standard addition in extracts of blue mussels, with recoveries between 92 and 108%. Finally, this method was used in combination with the AOAC Official Method based on LC-ox-FLD to measure PSTs in a new mussel tissue matrix reference material.


Hydrophilic interaction liquid chromatography Mass spectrometry Algal toxins Paralytic shellfish poisoning Reference material 



The authors acknowledge funding from the Canadian Food Inspection Agency (CFIA) and the UK Food Standards Agency, technical assistance from Nancy Lewis, Ruth Perez, Stephen Chung, Diane Marciniak, Sheila Crain, Tobias Karakach and Elliott Wright. Emanuel Hignutt from the Alaska Department of Environmental Conservation is acknowledged for his support and early adoption of the described methodology. The authors would also like to thank Sciex for supporting the validation work on the QTRAP 5500.

Compliance with ethical standards

The research did not involve human participants or animals.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_507_MOESM1_ESM.pdf (1 mb)
ESM 1 (DOC 1512 kb)


  1. 1.
    Hall S, Strichartz GR, Moczydlowski E, Ravindran A, Reichardt PB. The saxitoxins: sources, chemistry, and pharmacology. In: Hall S, Strichartz GR, editors. Marine toxins, ACS symposium series 418. Woods Hole: American Chemical Society; 1990. p. 29–65.CrossRefGoogle Scholar
  2. 2.
    Carmichael WW. The toxins of cyanobacteria. Sci Am. 1994;270:78–86.CrossRefGoogle Scholar
  3. 3.
    Wiese M, D'Agostino PM, Mihali TK, Moffitt MC, Neilan BA. Neurotoxic alkaloids: saxitoxin and its analogs. Mar Drugs. 2010;8:2185–211.CrossRefGoogle Scholar
  4. 4.
    Anon. AOAC Official Method 959.08. Paralytic shellfish poison, biological method. Official Methods Analysis, AOAC International. Gaithersberg, MD, USA: AOAC International; 1959.Google Scholar
  5. 5.
    Hess P, Grune B, Anderson DB, Aune T, Botana LM, Caricato P, et al. Three Rs approaches in marine biotoxin testing. The report and recommendations of a joint ECVAM/DG Sanco workshop (ECVAM workshop 55). ATLA Altern Lab Anim. 2006;34:193–224.Google Scholar
  6. 6.
    Campbell K, Vilariño N, Botana LM, Elliott CT. A European perspective on progress in moving away from the mouse bioassay for marine toxin analysis. TrAC Trends Anal Chem. 2011;30:239–53.CrossRefGoogle Scholar
  7. 7.
    Hall S, Reichardt PB. Cryptic paralytic shellfish toxins. In: Ragelis EP, editor. Seafood toxins. Washington DC: Am. Chem. Soc; 1984. p. 113–23.CrossRefGoogle Scholar
  8. 8.
    Lawrence JF, Ménard C. Liquid chromatographic determination of paralytic shellfish poisons in shellfish after prechromatographic oxidation. J AOAC Int. 1991;74:1006–12.Google Scholar
  9. 9.
    Janecek M, Quilliam MA, Lawrence JF. Analysis of paralytic shellfish poisoning toxins by automated pre-column oxidation and microcolumn liquid chromatography with fluorescence detection. J Chromatogr. 1993;644:321–31.CrossRefGoogle Scholar
  10. 10.
    Anon. AOAC Official Method 2005.06. Paralytic shellfish poisoning toxins in shellfish, prechromatographic oxidation and liquid chromatography with fluorescence detection. Official Methods Analysis, AOAC International. Gaithersberg, MD, USA: AOAC International; 2005.Google Scholar
  11. 11.
    Lawrence JF, Menard C, Cleroux C. Evaluation of prechromatographic oxidation for liquid chromatographic determination of paralytic shellfish poisons in shellfish. J AOAC Int. 1995;78:514–20.Google Scholar
  12. 12.
    Rourke WA, Murphy CJ, Pitcher G, van de Riet JM, Burns BG, Thomas KM, et al. Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue. J AOAC Int. 2008;91:589–97.Google Scholar
  13. 13.
    van de Riet J, Gibbs RS, Muggah PM, Rourke WA, MacNeil JD, Quilliam MA. Liquid chromatography post-column oxidation (PCOX) method for the determination of paralytic shellfish toxins in mussels, clams, oysters, and scallops: collaborative study. J AOAC Int. 2011;94:1154–76.Google Scholar
  14. 14.
    Anon. AOAC Official Method 2011.02. Paralytic shellfish toxins in mussels, clams, oysters, and scallops; post-column oxidation (PCOX) method. Official Methods Analysis, AOAC International. Gaithersberg, MD, USA: AOAC International; 2011.Google Scholar
  15. 15.
    Hatfield RG, Punn R, Algoet M, Turner AD. A rapid method for the analysis of paralytic shellfish toxins utilizing standard pressure HPLC: refinement of AOAC 2005.06. J AOAC Int. 2016;99:475–80.CrossRefGoogle Scholar
  16. 16.
    Turner AD, Tarnovius S, Johnson S, Higman WA, Algoet M. Testing and application of a refined rapid detection method for paralytic shellfish poisoning toxins in UK shellfish. Toxicon. 2015;100:32–41.CrossRefGoogle Scholar
  17. 17.
    Turner AD, Lewis AM, Rourke WA, Higman WA. Interlaboratory comparison of two AOAC liquid chromatographic fluorescence detection methods for paralytic shellfish toxin analysis through characterization of an oyster reference material. J AOAC Int. 2014;97:380–90.CrossRefGoogle Scholar
  18. 18.
    Harwood DT, Boundy M, Selwood AI, van Ginkel R, MacKenzie L, McNabb PS. Refinement and implementation of the Lawrence method (AOAC 2005.06) in a commercial laboratory: assay performance during an Alexandrium catenella bloom event. Harmful Algae. 2013;24:20–31.CrossRefGoogle Scholar
  19. 19.
    Turner AD, Hatfield RG. Refinement of AOAC official method 2005.06 liquid chromatography-fluorescence detection method to improve performance characteristics for the determination of paralytic shellfish toxins in king and queen scallops. J AOAC Int. 2012;95:129–42.CrossRefGoogle Scholar
  20. 20.
    Turrell E, Stobo L, Lacaze JP, Piletsky S, Piletska E. 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. 2008;91:1372–86.Google Scholar
  21. 21.
    van den Top HJ, Gerssen A, McCarron P, van Egmond HP. Quantitative determination of marine lipophilic toxins in mussels, oysters and cockles using liquid chromatography-mass spectrometry: inter-laboratory validation study. Food Add Contam Part A. 2011;28:1745–57.Google Scholar
  22. 22.
    McCarron P, Wright E, Quilliam MA. Liquid chromatography/mass spectrometry of domoic acid and lipophilic shellfish toxins with selected reaction monitoring and optional confirmation by library searching of product ion spectra. J AOAC Int. 2014;97(2):316–24.CrossRefGoogle Scholar
  23. 23.
    Quilliam MA, Hess P, Dell’Aversano C. 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, editors. Mycotoxins and phycotoxins in perspective at turn century. Wageningen: W.J. de Koe; 2001. p. 383–91.Google Scholar
  24. 24.
    Dell'Aversano C, Hess P, Quilliam MA. Hydrophilic interaction liquid chromatography-mass spectrometry for the analysis of paralytic shellfish poisoning (PSP) toxins. J Chromatogr A. 2005;1081:190–201.CrossRefGoogle Scholar
  25. 25.
    Dell'Aversano C, Walter JA, Burton IW, Stirling DJ, Fattorusso E, Quilliam MA. Isolation and structure elucidation of new and unusual saxitoxin analogues from mussels. J Nat Prod. 2008;71:1518–23.CrossRefGoogle Scholar
  26. 26.
    Zhuo L, Yin Y, Fu W, Qiu B, Lin Z, Yang Y, et al. Determination of paralytic shellfish poisoning toxins by HILIC-MS/MS coupled with dispersive solid phase extraction. Food Chem. 2013;137:115–21.CrossRefGoogle Scholar
  27. 27.
    Watanabe R, Matsushima R, Harada T, Oikawa H, Murata M, Suzuki T. Quantitative determination of paralytic shellfish toxins in cultured toxic algae by LC-MS/MS. Food Add Contam Part A. 2013;30:1351–7.CrossRefGoogle Scholar
  28. 28.
    Jansson D, Åstot C. Analysis of paralytic shellfish toxins, potential chemical threat agents, in food using hydrophilic interaction liquid chromatography-mass spectrometry. J Chromatogr A. 2015;1417:41–8.CrossRefGoogle Scholar
  29. 29.
    Boundy MJ, Selwood AI, Harwood DT, McNabb PS, Turner AD. Development of a sensitive and selective liquid chromatography-mass spectrometry method for high throughput analysis of paralytic shellfish toxins using graphitised carbon solid phase extraction. J Chromatogr A. 2015;1387:1–12.CrossRefGoogle Scholar
  30. 30.
    Blay P, Hui JPM, Chang J, Melanson JE. Screening for multiple classes of marine biotoxins by liquid chromatography-high-resolution mass spectrometry. Anal Bioanal Chem. 2011;400:577–85.CrossRefGoogle Scholar
  31. 31.
    Turner AD, McNabb PS, Harwood DT, Selwood AI, Boundy MJ. Single-laboratory validation of a multitoxin ultra-performance LC-hydrophilic interaction LC-MS/MS method for quantitation of paralytic shellfish toxins in bivalve shellfish. J AOAC Int. 2015;98:609–21.CrossRefGoogle Scholar
  32. 32.
    Beach DG, Melanson JE, Purves RW. Analysis of paralytic shellfish toxins using high-field asymmetric waveform ion mobility spectrometry with liquid chromatography-mass spectrometry. Anal Bioanal Chem. 2015;407:2473–84.CrossRefGoogle Scholar
  33. 33.
    Beach DG. Differential mobility spectrometry for improved selectivity in hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of paralytic shellfish toxins. J Am Soc Mass Spectrom. 2017; doi: 10.1007/s13361-017-1651-x.
  34. 34.
    Van Den Top HJ, Boenke A, Burdaspal PA, Bustos J, Van Egmond HP, Legarda T, et al. The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: certification study. Food Add Contam. 2001;18:810–24.CrossRefGoogle Scholar
  35. 35.
    Reeves K, Thomas K, Quilliam MA. A mussel tissue certified reference material for paralytic shellfish poisoning toxins. In: Henshilwood K, Deegan B, McMahon T, Cusack C, Keaveney S, Silke J, et al., editors. Molluscan shellfish safety. Galway: The Marine Institute; 2006. p. 116–22.Google Scholar
  36. 36.
    Li A, Ma J, Cao J, Wang Q, Yu R, Thomas K, et al. Analysis of paralytic shellfish toxins and their metabolites in shellfish from the north Yellow Sea of China. Food Add Contam Part A. 2012;29:1455–64.CrossRefGoogle Scholar
  37. 37.
    Thomas K, Wechsler D, Chen YM, Crain S, Quilliam MA. Analysis of natural toxins by liquid chromatography-chemiluminescence nitrogen detection and application to the preparation of certified reference materials. J AOAC Int. 2016;99:1173–85.CrossRefGoogle Scholar
  38. 38.
    Youden WJ, Steiner EH, editors. Statistical manual of the association of analytical chemists. Gaithersburg: Association of Official Analytical Chemists; 1975.Google Scholar
  39. 39.
    Turner AD, Lewis AM, Hatfield RG, Galloway AW, Higman WA. Transformation of paralytic shellfish poisoning toxins in Crassostrea gigas and Pecten maximus reference materials. Toxicon. 2012;60(6):1117–34.CrossRefGoogle Scholar
  40. 40.
    McCarron P, Burrell S, Hess P. 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. 2007;387(7):2495–502.CrossRefGoogle Scholar
  41. 41.
    McCarron P, Giddings SD, Reeves KL, Hess P, Quilliam MA. A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids. Anal Bioanal Chem. 2015;407:2985–96.CrossRefGoogle Scholar
  42. 42.
    McCarron P, Reeves KL, Giddings SD, Beach DG, Quilliam MA. Development of certified reference materials for diarrhetic shellfish poisoning toxins, part 2: shellfish matrix materials. J AOAC Int. 2016;99:1163–72.CrossRefGoogle Scholar
  43. 43.
    Dörr FA, Kovačević B, Maksić ZB, Pinto E, Volmer DA. Intriguing differences in the gas-phase dissociation behavior of protonated and deprotonated gonyautoxin epimers. J Am Soc Mass Spectrom. 2011;22:2011–20.CrossRefGoogle Scholar
  44. 44.
    Costa PR, Robertson A, Quilliam MA. Toxin profile of Gymnodinium catenatum (Dinophyceae) from the Portuguese coast, as determined by liquid chromatography tandem mass spectrometry. Mar Drugs. 2015;13:2046–62.CrossRefGoogle Scholar
  45. 45.
    Hignutt E, Sawasaki V, Knue J. LC/MS/MS determination of paralytic shellfish toxins in Alaskan shellfish. Poster presentation at 124th AOAC Annual Meeting and Exposition; Sept.25–29, 2010; Orlando, FA, USA. 2010.Google Scholar
  46. 46.
    McCarron P, Giddings SD, Quilliam MA. A mussel tissue certified reference material for multiple phycotoxins. Part 2: liquid chromatography-mass spectrometry, sample extraction and quantitation procedures. Anal Bioanal Chem. 2011;400:835–46.CrossRefGoogle Scholar
  47. 47.
    McCarron P, Wright E, Emteborg H, Quilliam MA. A mussel tissue certified reference material for multiple phycotoxins. Part 4: certification. Anal Bioanal Chem. 2017;409:95–106.CrossRefGoogle Scholar
  48. 48.
    Anon. Guidelines for collaborative study procedures to validate characteristics of a method of analysis. Gaithersburg: AOAC International; 2002.Google Scholar
  49. 49.
    Anon. Accuracy (trueness and precision) of measurement methods and results—Part 2: basic method for the determination of repeatability and reproducibility of a standard measurement method. IS0 5725-2:1994. Switzerland: International Organization for Standardization; 1994.Google Scholar
  50. 50.
    Burrell S, Crum S, Foley B, Turner AD. Proficiency testing of laboratories for paralytic shellfish poisoning toxins in shellfish by QUASIMEME: a review. TrAC Trends Anal Chem. 2016;75:10–23.CrossRefGoogle Scholar
  51. 51.
    Authority EFS. Scientific opinion on marine biotoxins in shellfish—saxitoxin group. EFSA J. 2009;1019:1–76.Google Scholar
  52. 52.
    Quilliam MA, Janecek M, Lawrence JF. Characterization of the oxidation products of paralytic shellfish poisoning toxins by liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 1993;7:482–7.CrossRefGoogle Scholar
  53. 53.
    Pauwels J, Van Der Yeen A, Lamberty A, Schimmel H. Evaluation of uncertainty of reference materials. Accred Qual Assur. 2000;5:95–9.CrossRefGoogle Scholar
  54. 54.
    McCarron P, Emteborg H, Giddings SD, Wright E, Quilliam MA. A mussel tissue certified reference material for multiple phycotoxins. Part 3: homogeneity and stability. Anal Bioanal Chem. 2011;400:847–58.CrossRefGoogle Scholar
  55. 55.
    Beach DG, Crain S, Lewis N, LeBlanc P, Hardstaff WR, Perez RA, et al. Development of certified reference materials for diarrhetic shellfish poisoning toxins, part 1: calibration solutions. J AOAC Int. 2016;99:1151–62.CrossRefGoogle Scholar
  56. 56.
    Burton IW, Quilliam MA, Walter JA. Quantitative 1H NMR with external standards: use in preparation of calibration solutions for algal toxins and other natural products. Anal Chem. 2005;77:3123–31.CrossRefGoogle Scholar
  57. 57.
    Bragg WA, Lemire SW, Coleman RM, Hamelin EI, Johnson RC. Detection of human exposure to saxitoxin and neosaxitoxin in urine by online-solid phase extraction-liquid chromatography-tandem mass spectrometry. Toxicon. 2015;99:118–24.CrossRefGoogle Scholar

Copyright information

© Her Majesty the Queen in Right of Canada 2017

Authors and Affiliations

  • Krista M. Thomas
    • 1
  • Daniel G. Beach
    • 1
  • Kelley L. Reeves
    • 1
  • Ryan S. Gibbs
    • 2
  • Elliott S. Kerrin
    • 1
  • Pearse McCarron
    • 1
  • Michael A. Quilliam
    • 1
    Email author
  1. 1.Measurement Science and StandardsNational Research Council CanadaHalifaxCanada
  2. 2.Canadian Food Inspection AgencyDartmouthCanada

Personalised recommendations