Abstract
Azaspiracids (AZAs) are lipophilic biotoxins produced by marine algae that can contaminate shellfish and cause human illness. The European Union (EU) regulates the level of AZAs in shellfish destined for the commercial market, with liquid chromatography-mass spectrometry (LC-MS) being used as the official reference method for regulatory analysis. Certified reference materials (CRMs) are essential tools for the development, validation, and quality control of LC-MS methods. This paper describes the work that went into the planning, preparation, characterization, and certification of CRM-AZA-Mus, a tissue matrix CRM, which was prepared as a wet homogenate from mussels (Mytilus edulis) naturally contaminated with AZAs. The homogeneity and stability of CRM-AZA-Mus were evaluated, and the CRM was found to be fit for purpose. Extraction and LC-MS/MS methods were developed to accurately certify the concentrations of AZA1 (1.16 mg/kg), AZA2 (0.27 mg/kg), and AZA3 (0.21 mg/kg) in the CRM. Quantitation methods based on standard addition and matrix-matched calibration were used to compensate for the matrix effects in LC-MS/MS. Other toxins present in this CRM at lower levels were also measured with information values reported for okadaic acid, dinophysistoxin-2, yessotoxin, and several spirolides.
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References
McMahon T, Silke J (1996) Winter toxicity of unknown aetiology in mussels. Harmful Algae News 14:2
Satake M, Ofuji K, Naoki H, James KJ, Furey A, McMahon T, Silke J, Yasumoto T (1998) Azaspiracid, a new marine toxin having unique spiro ring assemblies, isolated from Irish mussels, Mytilus edulis. J Am Chem Soc 120:9967–9968
Twiner MJ, Rehmann N, Hess P, Doucette G (2008) Azaspiracid shellfish poisoning: a review on the chemistry, ecology, and toxicology with an emphasis on human health impacts. Mar Drugs 6:39–72
Nicolaou KC, Vyskocil S, Koftis TV, Yamada YMA, Ling T, Chen DYK, Tang W, Petrovic G, Frederick MO, Li Y, Satake M (2004) Structural revision and total synthesis of azaspiracid-1, part 1: intelligence gathering and tentative proposal. Angewandte Chem Int Ed 43:4312–4318
Brombacher S, Edmonds S, Volmer DA (2002) Studies on azaspiracid biotoxins. II. Mass spectral behavior and structural elucidation of azaspiracid analogs. Rapid Commun Mass Spectrom 16:2306–2316
James KJ, Sierra MD, Lehane M, Magdalena AB, Furey A (2003) Detection of five new hydroxyl analogues of azaspiracids in shellfish using multiple tandem mass spectrometry. Toxicon 41:277–283
Rehmann N, Hess P, Quilliam MA (2008) Discovery of new analogs of the marine biotoxin azaspiracid in blue mussels (Mytilus edulis) by ultra-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 22:549–558
Ofuji K, Satake M, McMahon T, Silke J, James KJ, Naoki H, Oshima Y, Yasumoto T (1999) Two analogs of azaspiracids isolated from mussels, Mytilus edulis, involved in human intoxication in Ireland. Nat Toxins 7:99–102
Ofuji K, Satake M, McMahon T, James KJ, Naoki H, Oshima Y, Yasumoto T (2001) Structures of azaspiracid analogs, azaspiracid-4 and azaspiracid-5, causative toxins of azaspiracid poisoning in Europe. Biosci Biotechnol Biochem 65:740–742
Furey A, Moroney C, Magdalena AB, Saez MJF, Lehane M, James KJ (2003) Geographical, temporal, and species variation of the polyether toxins, azaspiracids, in shellfish. Environ Sci Technol 37:3078–3084
James KJ, Furey A, Lehane M, Ramstad H, Aune T, Hovgaard P, Morris S, Higman W, Satake M, Yasumoto T (2002) First evidence of an extensive northern European distribution of azaspiracid poisoning (AZP) toxins in shellfish. Toxicon 40:909–915
Magdalena A, Lehane M, Krys S, Fernandez M, Furey A, James K (2003) The first identification of azaspiracids in shellfish from France and Spain. Toxicon 42:105–108
Torgersen T, Bremnes NB, Rundberget T, Aune T (2008) Structural confirmation and occurrence of azaspiracids in Scandinavian brown crabs (Cancer pagurus). Toxicon 51:93–101
Taleb H, Vale P, Amanhir R, Benhadouch A, Sagou R, Chafik A (2006) First detection of azaspiracids in mussels in north west Africa. J Shellfish Res 25:1067–1070
Trainer V, Moore L, Bill B, Adams N, Harrington N, Borchert J, da Silva D, Eberhart BT (2013) Diarrhetic shellfish toxins and other lipophilic toxins of human health concern in Washington State. Mar Drugs 11:1815–1835
Ueoka R, Ito A, Izumikawa M, Maeda S, Takagi M, Shin-ya K, Yoshida M, van Soest RWM, Matsunaga S (2009) Isolation of azaspiracid-2 from a marine sponge Echinoclathria sp. as a potent cytotoxin. Toxicon 53:680–684
Krock B, Tillmann U, John U, Cembella A (2008) LC-MS-MS aboard ship: tandem mass spectrometry in the search for phycotoxins and novel toxigenic plankton from the North Sea. Anal Bioanal Chem 392:797–803
Tillmann U, Elbrachter M, Krock B, John U, Cembella A (2009) Azadinium spinosum gen. et sp. nov. (Dinophyceae) identified as a primary producer of azaspiracid toxins. Eur J Phycol 44:63–79
Salas R, Tillmann U, John U, Kilcoyne J, Burson A, Cantwell C, Hess P, Jauffrais T, Silke J (2011) The role of Azadinium spinosum (Dinophyceae) in the production of azaspiracid shellfish poisoning in mussels. Harmful Algae 10:774–783
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 57:160–169
Jauffrais T, Marcaillou C, Herrenknecht C, Truquet P, Sechet V, Nicolau E, Tillmann U, Hess P (2012) Azaspiracid accumulation, detoxification and biotransformation in blue mussels (Mytilus edulis) experimentally fed Azadinium spinosum. Toxicon 60:582–595
Hess P, Nguyen L, Aasen J, Keogh M, Kilcoyne J, McCarron P, Aune T (2005) Tissue distribution, effects of cooking and parameters affecting the extraction of azaspiracids from mussels (Mytilus edulis) prior to analysis by liquid chromatography coupled to mass spectrometry. Toxicon 46:62–71
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. Off J Eur Union L 139 of 30 April 2004
Anonymous (2008) Marine biotoxins in shellfish—azaspiracid group. Scientific opinion of the panel on contaminants in the food chain. Eur Food Safety Authority, vol 723
Hess P, Butter T, Petersen A, Silke J, McMahon T (2009) Performance of the EU-harmonised mouse bioassay for lipophilic toxins for the detection of azaspiracids in naturally contaminated mussel (Mytilus edulis) hepatopancreas tissue homogenates characterised by liquid chromatography coupled to tandem mass spectrometry. Toxicon 53:713–722
Anonymous (2011) 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. Off J European Union L 006 of 11 January 2011: 3–6
Forsyth CJ, Xu JY, Nguyen ST, Samdal IA, Briggs LR, Rundberget T, Sandvik M, Miles CO (2006) Antibodies with broad specificity to azaspiracids by use of synthetic haptens. J Am Chem Soc 128:15114–15116
McCarron P, Giddings SD, Miles CO, Quilliam MA (2011) Derivatization of azaspiracid biotoxins for analysis by liquid chromatography with fluorescence detection. J Chrom A 1218:8089–8096
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 398:2243–2252
Quilliam MA, Reeves K, MacKinnon S, Craft C, Whyte H, Walter J, Stobo L, Gallacher S (2006) Preparation of reference materials for azaspiracids. In: Proceedings of the 5th International Conference of Molluscan Shellfish Safety, 14–18 June 2004, Galway, Ireland, Molluscan Shellfish Safety. ISBN: 1 902895-33-9. pp 111–115
Turner AD, Lewis AM, Hatfield RG, Higman WA, Burrell S (2013) A feasibility study into the production of a freeze-dried oyster reference material for paralytic shellfish poisoning toxins. Anal Bioanal Chem 405:8621–8632
McCarron P, Kotterman M, de 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 387:2487–2493
Bogialli S, Dicorcia A (2007) Matrix solid-phase dispersion as a valuable tool for extracting contaminants from foodstuffs. J Biochem Biophys Methods 70:163–179
Mountfort DO, Suzuki T, Truman P (2001) Protein phosphatase inhibition adapted for determination of total DSP in contaminated mussel. Toxicon 39:383–390
McCarron P, Giddings SD, Quilliam MA (2011) A mussel tissue certified reference material for multiple phycotoxins. Part 2: liquid chromatography-mass spectrometry, sample extraction and quantitation procedures. Anal Bioanal Chem 400:835–846
Hardstaff WR, Jamieson WD, Milley JE, Quilliam MA, Sim PG (1990) Reference materials for domoic acid, a marine neurotoxin. Fres J Anal Chem 338:520–525
Hess P, McCarron P, Quilliam MA (2007) Fit-for-purpose shellfish reference materials for internal and external quality control in the analysis of phycotoxins. Anal Bioanal Chem 387:2463–2474
McCarron P, Burrell S, Hess P (2007) 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 387:2495–2502
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 387:2475–2486
McCarron P, Emteborg H, Giddings SD, Wright E, Quilliam MA (2011) A mussel tissue certified reference material for multiple phycotoxins. Part 3: homogeneity and stability. Anal Bioanal Chem 400:847–858
van der Veen AMH, Linsinger T, Pauwels J (2001) Uncertainty calculations in the certification of reference materials. 2. Homogeneity study. Accred Qual Assur 6:26–30
Nzoughet K, Hamilton J, Floyd S, Douglas A, Nelson J, Devine L, Elliott C (2008) Azaspiracid: first evidence of protein binding in shellfish. Toxicon 51:1255–1263
Alfonso C, Rehmann N, Hess P, Alfonso A, Wandscheer CB, Abuin M, Vale C, Otero P, Vieytes MR, Botana LM (2008) Evaluation of various pH and temperature conditions on the stability of azaspiracids and their importance in preparative isolation and toxicological studies. Anal Chem 80:9672–9680
Barker S (2007) Matrix solid phase dispersion (MSPD). J Biochem Biophys Methods 70:151–162
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 Chrom A 1217:7123–7130
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 25:1024–1032
Ito S, Tsukada K (2002) Matrix effect and correction by standard addition in quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic shellfish poisoning toxins. J Chromatogr A 943:39–46
Gerssen A, Olst EHW, Mulder PPJ, 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 397:3079–3088
Kilcoyne J, McCarron P, Twiner MJ, Nulty C, Crain S, Quilliam MA, Rise F, Wilkins AL, Miles CO (2014) Epimers of azaspiracids: isolation, structural elucidation, relative LC-MS response, and in vitro toxicity of 37-epi-azaspiracid-1. Chem Res Toxicol 27:587–600
Ihnat M (1998) A synopsis of different approaches to the certification of reference materials. Fresenius J Anal Chem 360:308–311
Burton IW, Quilliam MA, Walter JA (2005) Quantitative 1H NMR with external standards: use in preparation of calibration solutions for algal toxins and other natural products. Anal Chem 77:3123–3131
ISO-guide-35 (2006) Reference materials—general statistics and principles for certification. REMCO.
BIPM (1995) Guide to the expression of uncertainty in measurement, 1st edition. International Organisation for Standardisation (ISO). Geneva
Acknowledgments
The following staff members at the NRCC (1), the Canadian Institute of Fisheries and Technology (2), and Agriculture and Agrifood Canada (3) contributed to the production and certification of CRM-AZA-Mus: Dian Marciniak (1), Sheila Crain (1), Elliott Wright (1), Ruth A. Perez (1), Douglas Singer (2), Anne Timmins (2), John Thompson (2), and Bruno Laventure (3). This project was supported by the ASTOX project (ST/02/02, 2003–2006) funded through the Irish National Development Plan (NDP) and the Irish Marine Institute.
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Published in the topical collection Reference Materials for Chemical Analysis with guest editors Hendrik Emons and Stephen A. Wise.
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McCarron, P., Giddings, S.D., Reeves, K.L. et al. A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids. Anal Bioanal Chem 407, 2985–2996 (2015). https://doi.org/10.1007/s00216-014-8250-5
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DOI: https://doi.org/10.1007/s00216-014-8250-5