Liquid chromatography—multiple tandem mass spectrometry for the determination of ten azaspiracids, including hydroxyl analogues in shellfish

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Abstract

Azaspiracids (AZAs) are a group of polyether toxins that cause food poisoning in humans. These toxins, produced by marine dinoflagellates, accumulate in filter-feeding shellfish, especially mussels. Sensitive liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MSn) methods have been developed for the determination of the major AZAs and their hydroxyl analogues. These methods, utilising both chromatographic and mass resolution, were applied for the determination of 10 AZAs in mussels (Mytilus edulis). An optimised isocratic reversed phase method (3 μm Luna-2 C18 column) separated 10 azaspiracids using acetonitrile/water (46:54, v/v) containing 0.05% trifluoroacetic acid (TFA) and 0.004% ammonium acetate in 55 min. Analyte determination using MS3 involved trapping and fragmentation of the [M+H]+ and [M+H−H2O]+ ions with detection of the [M+H−2H2O]+ ion for each AZA. Linear calibrations were obtained for AZA1, using spiked shellfish extracts, in the range 0.05–1.00 μg/ml (r2=0.997) with a detection limit of 5 pg (signal:noise=3). The major fragmentation pathways in hydroxylated azaspiracids were elucidated using hydrogen/deuterium (H/D) exchange experiments. An LC-MS3 method was developed using unique parent ions and product ions, [M+H−H2O−C9H10O2R1R3]+, that involved fragmentation of the A-ring. This facilitated the discrimination between 10 azapiracids, AZA1–10. Thus, this rapid LC-MS3 method did not require complete chromatographic resolution and the run-time of 7 min had detection limits better than 20 pg for each toxin.

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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