Brett Hamilton

First evidence of azaspiracids (AZAs): A family of lipophilic polyether marine toxins in scallops (Argopecten purpuratus) and mussels (Mytilus chilensis) collected in two regions of Chile.

Azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 following the consumption by consumers of intoxicated shellfish (Mytilus edulis). These azaspiracids have now been identified in mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) from two Chilean locations. This is the first report of the occurrence of azaspiracid toxins in these species (Mytilus chilensis and Argopecten purpuratus) from Chile. The areas studied were Bahía Inglesa (III Region, 27 degrees SL) and Chiloé Archipelago, both important scallop and mussels farming areas. Separation of azaspiracid (AZA1), azaspiracid isomer (AZA6) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and toxins were identified using a turbo electrospray ionisation (ESI) source, to a triple quadrupole mass spectrometer. In mussels, AZA1 was the predominant toxin in mussel hepatopancreas with AZA2, AZA3 and AZA6 present in approximate equivalent amounts in the remaining tissues, 20-30% of the AZA1 level. AZA2 predominated in the scallop samples with the toxin almost entirely present in the hepatopancreas (digestive gland). AZA1 was only observed in some of the scallop samples and was present at 12-15% of the AZA2 levels. Whilst the levels of AZAs in Chilean samples are below the EU regulatory limit of 160mug/kg, it is significant that this toxin is present in Pacific Ocean species. Consequently measures should be taken by regulatory authorities to implement regular seafood monitoring to ensure safety of harvested product.

The fragmentation pathways of azaspiracids elucidated using positive nanospray hybrid quadrupole time-of-flight (QqTOF) mass spectrometry

The azaspiracids, AZA1, AZA2 and AZA3, are the predominant shellfish toxins responsible for the human toxic syndrome, azaspiracid poisoning. Collision induced dissociation (CID) mass spectra were generated for azaspiracids using nano-electrospray ionisation (ESI) with a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer in positive mode. Six main backbone fragmentations of the polyether skeleton of azaspiracids were observed as well as multiple neutral losses of water molecules from the parent and product ions. The characteristic charge-remote fragmentation of the carbon skeleton of azaspiracids produced nitrogenous ions. The three azaspiracids differ from one another by 14 Da due to methylation in the A- and E-rings. Three fragmentation pathways, involving cleavage of the E-ring, C27–C28 and G-ring, gave ions that were common to all azaspiracids. Another three fragmentations involving the A-ring, C-ring and C19–C20, were useful for distinguishing between azaspiracid analogues. Multiple tandem ion‒trap mass spectrometry (MSn) was used to confirm the fragmentation pathways.

Elucidation of the mass fragmentation pathways of the polyether marine toxins, dinophysistoxins, and identification of isomer discrimination processes

RATIONALE Most of the liquid chromatography/mass spectrometry (LC/MS) methods that have been developed for the analysis of Diarrhetic Shellfish Poisoning (DSP) toxins in shellfish and algae samples have been unable to differentiate the isomers okadaic acid (OA) and dinophysistoxin-2 (DTX2), unless separated by chromatography. Since there are many bioconversion products of these compounds it is imperative to determine characteristic product ions, which can provide unequivocal identification of OA and DTX2 and their analogs. METHODS Using electrospray ionization, the fragmentation processes for two types of precursor ions, [M+Na](+) and [M-H](-), of the polyether marine toxins, dinophysistoxins (DTXs), were studied using a hybrid linear ion trap Orbitrap mass spectrometer which provided high mass accuracy data in combination with multiple tandem mass (MS(n)) spectra. Three structurally related toxins were compared; okadaic acid (OA), dinophysistoxin-2 (DTX2) and dinophysistoxin-1 (DTX1). A quick multiple reaction monitoring (MRM) LC/MS/MS method was developed utilizing the characteristic precursor/product ion mass transitions. RESULTS Comparison of the high-resolution product ion, [M-H](-), spectra of these toxins featured dominant signals that resulted from two six-centered rearrangements and previously proposed fragmentation pathways for the ion of m/z 321 and 293 have been corrected and identified. By contrast, the [M+Na](+) product ion spectra only revealed distinctive ions for the isomers, OA (m/z 595, 443 and 151) and DTX2 (m/z 581, 429 and 165). To illustrate the benefits of this study, a mass selective LC/MS/MS method was developed in which the isomers OA and DTX2 co-eluted but were distinguished using the mass transitions, m/z 827/595, 827/443 (OA) and m/z 827/581, 827/429 (DTX2). CONCLUSIONS Comparison of OA, DTX2 and DTX1 led to the correction of proposed negative ion mode fragmentation pathways. Through extensive study and comparison of the [M+Na](+) product ion spectra, distinctive product ions were identified which allowed for these compounds to be identified and distinguished without separation for the first time.