, Volume 81, Issue 2, pp 277–288 | Cite as

Liquid Chromatography–Tandem Mass Spectrometry Method for the Screening of Eight Paralytic Shellfish Poisoning Toxins, Domoic Acid, 13-Desmethyl Spirolide C, Palytoxin and Okadaic Acid in Seawater

  • Carmela Riccardi
  • Francesca Buiarelli
  • Patrizia Di Filippo
  • Sisto Distratis
  • Luigi Giannetti
  • Maura Manganelli
  • Bruno Neri
  • Donatella Pomata
  • Mara Stefanelli


A quick and reproducible screening analytical method for the simultaneous determination of algal toxins, belonging to different chemical classes, was developed to provide a toxin profile in seawater, useful to assess potential risks to environment and human health. The target compounds were: gonyautoxin-1,4, gonyautoxin-2,3, decarbamoylgonyautoxin-2,3, N‐sulfocarbamoyl-gonyautoxin-1,2, neosaxitoxin, decarbamoyl-neosaxitoxin, saxitoxin, decarbamoyl-saxitoxin, domoic acid, 13-desmethyl spirolide C (SPX1), palytoxin and okadaic acid. Extraction and clean-up were carried out with a combination of Bond Elut LRC-C18 and Carbograph4 cartridges connected in series. Analyte separation was performed in gradient elution mode in 12 min with a Gemini C18 column. Compound detection was carried out in multiple reaction monitoring and in positive ionization mode for paralytic shellfish poisoning toxins, domoic acid, SPX1 and palytoxin, and in negative ionization mode for okadaic acid. The toxins were quantified with matrix-matched calibration curves constructed by spiked seawater samples (concentration levels 0.02–2 μg L−1 depending on the compound). The method was reproducible with intra-day and inter-day relative standard deviations ranging from 4 to 9% and from 8 to 16%, respectively. Good recoveries (84–105%) and good accuracy (2–20%) were obtained by spiking experiments. Limits of detection were calculated for each toxin and varied from 0.011 to 0.12 μg L−1 depending on the compound. The developed method was applied to cultured Ostreopsis cf. ovata samples. The proposed procedure may be considered a valuable alternative to existing methods for monitoring toxic microalgae, since it offers a rapid screening of target toxins, reduced organic solvent consumption, and handling of smaller sample volumes while providing good sensitivity and accuracy.

Graphical Abstract


Column liquid chromatography Solid-phase extraction Harmful algal blooms Algal toxins Seawater samples 



The authors wish to thank prof. Honsell (University of Udine) for providing strains of Ostreopsis cf ovata isolated from the Adriatic Sea. This research was funded by INAIL/Project P20L03.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

10337_2017_3440_MOESM1_ESM.docx (735 kb)
Supplementary material 1 (DOCX 735 kb)


  1. 1.
    Davidson K, Gowen RJ, Tett P, Bresnan E, Harrison PJ, McKinney A, Milligan S, Mills DK, Silke J, Crooks AM (2012) Harmful algal blooms: how strong is the evidence that nutrient ratios and forms influence their occurrence? Estuar Coast Shelf Sci 115:399–413CrossRefGoogle Scholar
  2. 2.
    Villacorte LO, Tabatabai SAA, Anderson DM, Amy GL, Schippers JC, Kennedy MD (2015) Seawater reverse osmosis desalination and (harmful) algal blooms. Desalination 360:61–80CrossRefGoogle Scholar
  3. 3.
    Kirkpatrick B, Fleming LE, Squicciarini D, Backer LC, Clark R, Abraham W, Benson J, Cheng YS, Johnson D, Pierce R, Zaias J, Bossart GD, Baden DG (2004) Literature review of Florida red tide: implications for human health effects. Harmful Algae 3:99–115CrossRefGoogle Scholar
  4. 4.
    Dyson K, Huppert DD (2010) Regional economic impacts of razor clam beach closures due to harmful algal blooms (HABs) on the Pacific coast of Washington. Harmful Algae 9:264–271CrossRefGoogle Scholar
  5. 5.
    Boehm AB, Bischel HN (2011) Oceans and human health. In: Nriagu JO (ed) Encyclopedia of environmental health. Elsevier, BurlingtonGoogle Scholar
  6. 6.
    Landsberg JH (2002) The effects of harmful algal blooms on aquatic organisms. Rev Fish Sci 10:113–390CrossRefGoogle Scholar
  7. 7.
    Fire SE, Wang Z, Byrd M, Whitehead HR, Paternoster J, Morton SL (2011) Co-occurrence of multiple classes of harmful algal toxins in bottlenose dolphins (Tursiops truncatus) stranding during an unusual mortality event in Texas, USA. Harmful Algae 10:330–336CrossRefGoogle Scholar
  8. 8.
    Turner AD, Stubbs B, Coates L, Dhanji-Rapkova M, Hatfield RG, Lewis AM, Rowland-Pilgrim S, O’Neil A, Stubbs P, Ross S, Baker C, Algoet M (2014) Variability of paralytic shellfish toxin occurrence and profiles in bivalve mollusks from Great Britain from official control monitoring as determined by pre-column oxidation liquid chromatography and implications for applying immunochemical tests. Harmful Algae 31:87–99CrossRefGoogle Scholar
  9. 9.
    Rúbies A, Munoza E, Gibert D, Cortés-Francisco N, Granados M, Caixach J, Centrich F (2015) New method for the analysis of lipophilic marine biotoxins in fresh and canned bivalves by liquid chromatography coupled to high resolution mass spectrometry: a quick, easy, cheap, efficient, rugged, safe approach. J Chromatogr A 1386:62–73CrossRefGoogle Scholar
  10. 10.
    Ciminiello P, Dell’Aversano C, Fattorusso E, Forino M, Magno GS, Tartaglione L, Grillo C, Melchiorre N (2006) The Genoa 2005 outbreak. Determination of putative palytoxin in Mediterranean Ostreopsis cf. ovata by a new liquid chromatography tandem mass spectrometry method. Anal Chem 78:6153–6159CrossRefGoogle Scholar
  11. 11.
    Wang Z, King KL, Ramsdell JS, Doucette GJ (2007) Determination of domoic acid in seawater and phytoplankton by liquid chromatography–tandem mass spectrometry. J Chromatogr A 1163:169–176CrossRefGoogle Scholar
  12. 12.
    Mafra LL Jr, Léger 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
  13. 13.
    Rundberget T, Sandvik M, Larsen K, Pizarro GM, Reguera B, Castberg T, Gustad E, Loader JI, 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
  14. 14.
    Deeds JR, Schwartz MD (2010) Human risk associated with palytoxin exposure. Toxicon 56:150–162CrossRefGoogle Scholar
  15. 15.
    Ciminiello P, Dell’Aversano C, Dello Iacovo E, Fattorusso E, Forino M, Tartaglione L, Benedettini G, Onorari M, Serena F, Battocchi C, Casabianca S, Penna A (2014) First finding of Ostreopsis cf. ovata toxins in marine aerosols. Environ Sci Technol 48:3532–3540CrossRefGoogle Scholar
  16. 16.
    Funari E, Manganelli M, Testai E (2015) Ostreopsis cf. ovata blooms in coastal water: Italian guidelines to assess and manage the risk associated to bathing waters and recreational activities. Harmful Algae 50:45–56CrossRefGoogle Scholar
  17. 17.
    Regulation (EC) No. 854/2004 of the European Parliament and of the Council laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off J Eur Union L226:83Google Scholar
  18. 18.
    Reguera B, Riobó P, Rodríguez F, Díaz PA, Pizarro G, Paz B et al (2014) Dinophysis toxins: causative organisms, distribution and fate in shellfish. Mar Drugs 12:394–461CrossRefGoogle Scholar
  19. 19.
    Paredes I, Rietjens IMCM, Vieites JM, Cabado AG (2011) Update of risk assessments of main marine biotoxins in the European Union. Toxicon 58:336–354CrossRefGoogle Scholar
  20. 20.
    Commission Regulation (EU) No. 15/2011 of 10 January 2011 amending Regulation (EC) No. 2074/2005 as regards recognised testing methods for detecting marine biotoxins in live bivalve molluscs (2011). Off J Eur Union L6:3Google Scholar
  21. 21.
    Thomas KM, Beach DG, Reeves KL, Gibbs RS, Kerrin ES, McCarron P, Quilliam MA (2017) Hydrophilic interaction liquid chromatography–tandem mass spectrometry for quantitation of paralytic shellfish toxins: validation and application to reference materials. Anal Bioanal Chem 409:5675–5687CrossRefGoogle Scholar
  22. 22.
    Quilliam MA (2003) The role of chromatography in the hunt for red tide toxins. J Chromatogr A 1000:527–548CrossRefGoogle Scholar
  23. 23.
    Stobo LA, Lacaze JPCL, Scott AC, Petrie J, Turrell EA (2008) Surveillance of algal toxins in shellfish from Scottish waters. Toxicon 51:635–648CrossRefGoogle Scholar
  24. 24.
    Gerssen A, McElhinney MA, Mulder PPJ, Bire R, Hess P, de Boer J (2009) Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 394:1213–1226CrossRefGoogle Scholar
  25. 25.
    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
  26. 26.
    Campbell K, Vilarino N, Botana LM, Elliott CT (2011) A European perspective on progress in moving away from the mouse bioassay for marine-toxin analysis. Trends Anal Chem 30:239–253CrossRefGoogle Scholar
  27. 27.
    Wang Z, Maucher-Fuquay J, Fire SE, Mikulski CM, Haynes B, Doucette GJ, Ramsdell JS (2012) Optimization of solid-phase extraction and liquid chromatography–tandem mass spectrometry for the determination of domoic acid in seawater, phytoplankton, and mammalian fluids and tissues. Anal Chim Acta 715:71–79CrossRefGoogle Scholar
  28. 28.
    Chan IOM, Tsang VWH, Chu KK, Leung SK, Lam MHW, Lau TC, Lam PKS, Wu RSS (2007) Solid-phase extraction-fluorimetric high performance liquid chromatographic determination of domoic acid in natural seawater mediated by an amorphous titania sorbent. Anal Chim Acta 583:111–117CrossRefGoogle Scholar
  29. 29.
    McNamee SE, Medlin LK, Kegel J, McCoy GR, Raine R, Barra L, Ruggiero MV, Kooistra WHCF, Montresor M, Hagstrom J, Blanco EP, Graneli E, Rodrıguez F, Escalera L, Reguera B, Dittami S, Edvardsen B, Taylor J, Lewis JM, Pazos Y, Elliott CT, Campbell K (2016) Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: a comparison of methods of analysis. Harmful Algae 55:112–120CrossRefGoogle Scholar
  30. 30.
    Blay P, Hui JPM, Chang J, Melanson JE (2011) Screening for multiple classes of marine biotoxins by liquid chromatography–high-resolution mass spectrometry. Anal Bioanal Chem 400:577–585CrossRefGoogle Scholar
  31. 31.
    Li X, Li Z, Chen J, Shi Q, Zhang R, Wang S, Wang X (2014) Detection, occurrence and monthly variations of typical lipophilic marine toxins associated with diarrhetic shellfish poisoning in the coastal seawater of Qingdao City, China. Chemosphere 111:560–567CrossRefGoogle Scholar
  32. 32.
    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
  33. 33.
    Pietsch J, Fichtner S, Imhof L, Schmidt W, Brauch HJ (2001) Simultaneous determination of cyanobacterial hepato- and neurotoxins in water samples by ion-pair supported enrichment and HPLC–ESI-MS–MS. Chromatographia 54:339–344CrossRefGoogle Scholar
  34. 34.
    Deeds JR, Petitpas CM, Shue V, White KD, Keafer BA, McGillicuddy DJ Jr, Milligan PJ, Anderson DM, Jefferson T, Turner JT (2014) PSP toxin levels and plankton community composition and abundance in size-fractionated vertical profiles during spring/summer blooms of the toxic dinoflagellate Alexandrium fundyense in the Gulf of Maine and on Georges Bank, 2007, 2008, and 2010: 1. Toxin levels. Deep-Sea Res II 103:329–349CrossRefGoogle Scholar
  35. 35.
    Mac Kenzie L, Beuzenberg V, Holland P, McNabb P, Selwood A (2004) Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves. Toxicon 44:901–918CrossRefGoogle Scholar
  36. 36.
    Tartaglione L, Dell’Aversano C, Mazzeo A, Forino M, Wieringa A, Ciminiello P (2016) Determination of palytoxins in soft coral and seawater from a home aquarium. Comparison between palythoa- and ostreopsis-related inhalatory poisonings. Environ Sci Technol 50:1023–1030CrossRefGoogle Scholar
  37. 37.
    McNamee SE, Elliott CT, Delahaut P, Campbell K (2013) Multiplex biotoxin surface plasmon resonance method for marine biotoxins in algal and seawater samples. Environ Sci Pollut Res 20:6794–6807CrossRefGoogle Scholar
  38. 38.
    Reis Costa P, Botelho MJ, Lefebvre KA (2010) Characterization of paralytic shellfish toxins in seawater and sardines (Sardina pilchardus) during blooms of Gymnodinium catenatum. Hydrobiologia 655:89–97CrossRefGoogle Scholar
  39. 39.
    Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can J Microbiol 8:229–239CrossRefGoogle Scholar
  40. 40.
    Honsell G, Bonifacio A, De Bortoli M, Penna A, Battocchi C, Ciminiello P, Dell’Aversano C, Fattorusso E, Sosa S, Yasumoto T, Tubaro A (2013) New insights on cytological and metabolic features of Ostreopsis cf. ovata Fukuyo (Dinophyceae): a multidisciplinary approach. PLoS One 8:e5729CrossRefGoogle Scholar
  41. 41.
    Brissard C, Hervé F, Sibat M, Séchet V, Hess P, Amzil Z, Herrenknecht C (2015) Characterization of ovatoxin-h, a new ovatoxin analog, and evaluation of chromatographic columns for ovatoxin analysis and purification. J Chromatogr A 1388:87–101CrossRefGoogle Scholar
  42. 42.
    Sayfritz SJ, Aasen JAB, Aune T (2008) Determination of paralytic shellfish poisoning toxins in Norwegian shellfish by liquid chromatography with fluorescence and tandem mass spectrometry detection. Toxicon 52:330–340CrossRefGoogle Scholar
  43. 43.
    Halme M, Rapinoja ML, Karjalainen M, Vanninen P (2012) Verification and quantification of saxitoxin from algal samples using fast and validated hydrophilic interaction liquid chromatography–tandem mass spectrometry method. J Chromatogr B 880:50–57CrossRefGoogle Scholar
  44. 44.
    Ciminiello P, Dell’Aversano C, Dello Iacovo E, Fattorusso E, Forino M, Grauso L, Tartaglione L, Guerrini F, Pistocchi R (2010) Complex palytoxin-like profile of Ostreopsis cf. ovata. Identification of four new ovatoxins by high-resolution liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 24:2735–2744CrossRefGoogle Scholar
  45. 45.
    Ciminiello P, Dell’Aversano C, Dello Iacovo E, Fattorusso E, Forino M, Tartaglione L, Yasumoto T, Battocchi C, Giacobbe M, Amorim A, Penna A (2013) Investigation of toxin profile of Mediterranean and Atlantic strains of Ostreopsis cf. siamensis (Dinophyceae) by liquid chromatography–high resolution mass spectrometry. Harmful Algae 23:19–27CrossRefGoogle Scholar
  46. 46.
    Regueiro J, Martín-Morales E, Álvarez G, Blanco J (2011) Sensitive determination of domoic acid in shellfish by on-line coupling of weak anion exchange solid-phase extraction and liquid chromatography–diode array detection–tandem mass spectrometry. Food Chem 129:672–678CrossRefGoogle Scholar
  47. 47.
    Quilliam MA, Hess P, Dell’Aversano C (2001) Recent developments in the analysis of phycotoxins by liquid chromatography–mass spectrometry. In: deKoe WJ, Samson RA, van Egmond HP, Gilbert J, Sabino M (eds) Mycotoxins and phycotoxins in perspective at the turn of the century. Wageningen, The Netherlands, pp 383–391Google Scholar
  48. 48.
    Dell’Aversano C, Eaglesham GK, Quilliam MA (2004) Analysis of cyanobacterial toxins by hydrophilic interaction liquid chromatography–mass spectrometry. J Chromatogr A 1028:155–164CrossRefGoogle Scholar
  49. 49.
    Fux E, Rode D, Bire R, Hess P (2008) Approaches to the evaluation of matrix effects in the liquid chromatography–mass spectrometry (LC–MS) analysis of three regulated lipophilic toxin groups in mussel matrix (Mytilus edulis). Food Addit Contam 25:1024–1032CrossRefGoogle Scholar
  50. 50.
    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 Sep Sci 30:1821–1826CrossRefGoogle Scholar
  51. 51.
    Riobó P, Franco JM (2011) Palytoxins: biological and chemical determination. Toxicon 57:368–375CrossRefGoogle Scholar
  52. 52.
    Boundy MJ, Selwood AI, Harwood DT, McNabb PS, Turner AD (2015) 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 1387:1–12CrossRefGoogle Scholar
  53. 53.
    Bogialli S, Bruno M, Curini R, Di Corcia A, Fanali C, Laganà A (2006) Monitoring algal toxins in lake water by liquid chromatography tandem mass spectrometry. Environ Sci Technol 40:2917–2923CrossRefGoogle Scholar
  54. 54.
    Ciminiello P, Fattorusso E, Magno S, Oshima Y, Poletti R, Vivian R, Yasumoto T (1995) Determination of PSP toxins in mussels from the Adriatic Sea. Mar Pollut Bull 30:733–735CrossRefGoogle Scholar
  55. 55.
    Suzuki T, Ichimi K, Oshima Y, Kamiyama T (2003) Paralytic shellfish poisoning (PSP) toxin profiles and short-term detoxification kinetics in mussels Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense. Harmful Algae 2:201–206CrossRefGoogle Scholar
  56. 56.
    Guerrini F, Pezzolesi L, Feller A, Riccardi M, Ciminiello P, Dell’Aversano C, Tartaglione L, Dello Iacovo E, Fattorusso E, Forino M, Pistocchi R (2010) Comparative growth and toxin profile of cultured Ostreopsis ovata from the Tyrrhenian and Adriatic Seas. Toxicon 55:211–220CrossRefGoogle Scholar
  57. 57.
    Zhang C, Zhang J (2015) Current techniques for detecting and monitoring algal toxins and causative harmful algal blooms. J Environ Anal Chem 2:1–12Google Scholar
  58. 58.
    Ito S, Tsukada K (2001) Matrix effect and correction by standard addition in quantitative liquid chromatographic–mass spectrometric analysis of diarrhetic shellfish poisoning toxins. J Chromatogr A 943:39–46CrossRefGoogle Scholar
  59. 59.
    Kilcoyne J, Fux E (2010) Strategies for the elimination of matrix effects in the liquid chromatography tandem mass spectrometry analysis of the lipophilic toxins okadaic acid and azaspiracid-1 in molluscan shellfish. J Chromatogr A 1217:7123–7130CrossRefGoogle Scholar
  60. 60.
    Ciminiello P, Dell’Aversano C, Dello Iacovo E, Fattorusso E, Forino M, Tartaglione L, Rossi R, Soprano V, Capozzo D, Serpe L (2011) Palytoxin in seafood by liquid chromatography tandem mass spectrometry: investigation of extraction efficiency and matrix effect. Anal Bioanal Chem 401:1043–1050CrossRefGoogle Scholar
  61. 61.
    ICH Harmonised Tripartite Guideline (2005) Validation of analytical procedures: text and methodology Q2(R1). International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use, Geneva, pp 1–13Google Scholar
  62. 62.
    Pelin M, Sosa S, Tubaro A (2014) Palytoxins: toxicological profile. In: Gopalakrishnakone P Jr, Haddad V Jr, Kem WR, Tubaro A, Kim E (eds) Marine and freshwater toxins. Springer, DordrechtGoogle Scholar
  63. 63.
    Pelin M, Forino M, Brovedani V, Tartaglione L, Dell’Aversano C, Pistocchi R et al (2016) Ovatoxin-a, A palytoxin analogue isolated from Ostreopsis cf. ovata Fukuyo: cytotoxic activity and ELISA detection. Environ Sci Technol 50:1544–1551CrossRefGoogle Scholar
  64. 64.
    Ciminiello P, Dell’Aversano C, Fattorusso E, Forino M, Tartaglione L, Grillo C et al (2008) Putative palytoxin and its new analogue, ovatoxin-a, in Ostreopsis ovata collected along the Ligurian coasts during the 2006 toxic outbreak. J Am Soc Mass Spectrom 19:111–120CrossRefGoogle Scholar
  65. 65.
    Pezzolesi L, Guerrini F, Ciminiello P, Dell’Aversano C, Iacovo ED, Fattorusso E et al (2012) Influence of temperature and salinity on Ostreopsis cf. ovata growth and evaluation of toxin content through HR LC–MS and biological assays. Water Res 46:82–92CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Carmela Riccardi
    • 1
    • 2
  • Francesca Buiarelli
    • 2
  • Patrizia Di Filippo
    • 1
    • 2
  • Sisto Distratis
    • 2
  • Luigi Giannetti
    • 3
  • Maura Manganelli
    • 4
  • Bruno Neri
    • 3
  • Donatella Pomata
    • 1
    • 2
  • Mara Stefanelli
    • 1
    • 4
  1. 1.INAIL, DITRomeItaly
  2. 2.Department of ChemistryUniversity of Rome “Sapienza”RomeItaly
  3. 3.Istituto Zooprofilattico Sperimentale Regioni Lazio e ToscanaRomeItaly
  4. 4.Department of Environment and Primary PreventionISSRomeItaly

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