Liquid chromatography—multiple tandem mass spectrometry for the determination of ten azaspiracids, including hydroxyl analogues in shellfish
Introduction
Azaspiracid Poisoning (AZP) is a recently identified human toxic syndrome that is caused by the consumption of shellfish that are contaminated by natural toxins. Azaspiracids (AZAs) were first identified in 1995 [1] following intoxications in The Netherlands, and the human symptoms, included nausea, vomiting, severe diarrhoea and stomach cramps [2], [3]. Acute and chronic toxicological studies using mice have shown that AZAs caused widespread organ damage and induced tumours [4], [5] but the mechanisms of toxicity remain to be elucidated [6]. Although AZAs were first identified in mussels that were cultivated in Ireland, a widespread European distribution of these toxins has recently been confirmed [7]. The EU regulatory limit for AZAs has recently been set at 0.16 μg/g total shellfish tissue.
Several major classes of polyether marine toxins, such as dinophysistoxins, pectenotoxins, yessotoxins and brevetoxins, are produced by marine dinoflagellates [8]. A common phytoplankton, belonging to the genus, Protoperidinium, has recently been discovered as the progenitor of AZAs [9]. These toxins accumulate in filter-feeding bivalve molluscs, including mussels (Mytilus edulis) [7] and scallops (Pecten maximus) [10] which can lead to the poisoning of human consumers.
Structurally, azaspiracids are polyether amino acids that have a 6,5,6-trispiroacetal moiety, rings A, B and C, together with a 2,9-dioxabicyclo[3.3.1]nonane ring that is fused with an azaspiro ring system, rings F, G, H, and I (Fig. 1A, Table 1) [1]. Three azaspiracids, AZA1–3, have been identified in phytoplankton and they are the predominant toxins in shellfish. AZA2 and AZA3 are the 8-methyl and 22-demethyl analogues of AZA1, respectively [2]. Toxins that have been found in low abundance in shellfish include AZA4 and AZA5, which are the 3- and 23-hydroxy analogues, respectively, of AZA3 [11] and AZA6, which is an isomer of AZA1 [12], [13]. AZA7–10 [14] are hydroxy analogues of AZA1 and AZA6 (Fig. 1, Table 1). The hydroxy analogues, AZA4, AZA5 and AZA7–10, are most likely the products of bioconversion in shellfish as they are not found in phytoplankton.
The determination of AZA1–3 in shellfish is possible using a single quadrupole mass spectrometer provided that a solid phase extraction (SPE) clean-up is used [11]. However, the development of a SPE protocol, that can be successfully applied to all 10 AZAs, may be problematic [14]. The high selectivity of multiple tandem MS is required for determining the minor azaspiracid contaminants in shellfish. LC-MS/MS, using triple quadrupole instruments [15], [16], [17] and LC-MS3, using an ion-trap instrument, have been developed for the determination of AZAs [12], [18], [19]. The aim of this study was to develop multiple tandem MS methods for the simultaneous analysis of the predominant azaspiracids, as well as their minor analogues, in biological tissues. These methods employed both chromatographic resolution and mass selection of fragments ions in multiple tandem MS to permit the resolution of isomers.
Section snippets
Chemicals and toxin standards
HPLC-grade acetonitrile and water were purchased from Labscan (Dublin, Ireland) and trifluoroacetic acid (TFA), deuterated methanol (CD3OD) and water (D2O) were obtained from Sigma-Aldrich (Dorset, UK). Azaspiracid standards, AZA1–3, were isolated from toxic mussels (Mytilus edulis) as described previously [2]. Contaminated mussels were collected from various locations on the west coast of Ireland and extraction for analysis of azaspiracids was carried out as described previously [19].
Liquid chromatography conditions
The LC
Results and discussion
LC-tandem MS is undoubtedly the method of choice for the determination of trace quantities of analytes in complex biological matrices [20]. A number of LC-MS approaches were examined with the aim of developing a method for the simultaneous determination of the predominant azaspiracids and their bioconversion analogues. There are two main challenges in the development of a rapid LC-MS method for the simultaneous analysis of azaspiracids and their hydroxyl analogues in biological tissues.
Acknowledgements
We acknowledge funding from EU sponsored programmes; Higher Education Authority of Ireland (PRTLI-2), Irish Research Council for Science (Engineering and Technology), under the National Development Plan, Enterprise Ireland (Applied Research Programme, Strand 1) and a post-doctoral fellowship (to M.D.S) from FICYT, Spain.
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Cited by (42)
Phycotoxins and Food Safety
2017, Chemical Contaminants and Residues in Food: Second EditionLC/MS Analysis of Marine Toxins
2017, Comprehensive Analytical ChemistryCitation Excerpt :Due to the positively charged amine nitrogen at the end of the AZA molecule, fragmentation occurs by elimination of parts of the molecule with heterogenic oxygen atom from its charged end (Fig. 21). In addition to the key ions shown in the fragmentation diagram assigned by previous reports [134,136,138,140,141], series of several losses of H2O from the key ions were observed (Fig. 20). Although qTOF or quadruple tandem MS/MS fragmentation of AZA analogues on the negative mode were not reported, FAB MS/MS fragmentation of AZA on the negative mode gave excellent structural confirmation [130,142].
Confirmation of extensive natural distribution of azaspiracids in the tissue compartments of mussels (Mytilus edulis)
2014, ToxiconCitation Excerpt :This was a consequence of poor sensitivity of the assay and the fact that azaspiracids are not exclusively found in the shellfish digestive glands that were initially used for toxin testing (James et al. 2002a). Soon after the discovery of azaspiracids, liquid chromatography multiple tandem mass spectrometry (LC–MS/MS, LC–MSn), using triple quadrupole and ion-trap instruments, were developed for the determination of azaspiracids in shellfish (Draisci et al. 2000; James et al. 2001; Furey et al. 2002; Lehane et al. 2004). These analytical tools permitted a detailed evaluation of the impacts of azaspiracids on the marine environment, including their geographical distribution, and proved to be essential for the regulatory control of shellfish destined for human consumption, as outlined in two reviews (James et al. 2008a, 2008b).
Phycotoxins and food safety
2012, Chemical Contaminants and Residues in Food