Hydrophilic interaction liquid chromatography-tandem mass spectrometry for quantitation of paralytic shellfish toxins: validation and application to reference materials
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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.
KeywordsHydrophilic 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.
- 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.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
- 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
- 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.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.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.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.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
- 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.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
- 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
- 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
- 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.
- 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
- 38.Youden WJ, Steiner EH, editors. Statistical manual of the association of analytical chemists. Gaithersburg: Association of Official Analytical Chemists; 1975.Google Scholar
- 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
- 48.Anon. Guidelines for collaborative study procedures to validate characteristics of a method of analysis. Gaithersburg: AOAC International; 2002.Google Scholar
- 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
- 51.Authority EFS. Scientific opinion on marine biotoxins in shellfish—saxitoxin group. EFSA J. 2009;1019:1–76.Google Scholar