A Review of In Situ Methods-Solid Phase Adsorption Toxin Tracking (SPATT) and Polar Organic Chemical Integrative Sampler (POCIS) for the Collection and Concentration of Marine Biotoxins and Pharmaceuticals in Environmental Waters.

Solid Phase Adsorption Toxin Tracking (SPATT) and Polar Organic Chemical Integrative Sampler (POCIS) are in situ methods that have been applied to pre-concentrate a range of marine toxins, pesticides and pharmaceutical compounds that occur at low levels in marine and environmental waters. Recent research has identified the widespread distribution of biotoxins and pharmaceuticals in environmental waters (marine, brackish and freshwater) highlighting the need for the development of effective techniques to generate accurate quantitative water system profiles. In this manuscript, we reviewed in situ methods known as Solid Phase Adsorption Toxin Tracking (SPATT) and Polar Organic Chemical Integrative Sampler (POCIS) for the collection and concentration of marine biotoxins, freshwater cyanotoxins and pharmaceuticals in environmental waters since the 1980s to present. Twelve different adsorption substrates in SPATT and 18 different sorbents in POCIS were reviewed for their ability to absorb a range of lipophilic and hydrophilic marine biotoxins, pharmaceuticals, pesticides, antibiotics and microcystins in marine water, freshwater and wastewater. This review suggests the gaps in reported studies, outlines future research possibilities and guides researchers who wish to work on water contaminates using Solid Phase Adsorption Toxin Tracking (SPATT) and Polar Organic Chemical Integrative Sampler (POCIS) technologies.

Development, Validation and Application of an ICP-SFMS Method for the Determination of Metals in Protein Powder Samples, Sourced in Ireland, with Risk Assessment for Irish Consumers.

A method has been developed, optimised and validated to analyse protein powder supplements on an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS), with reference to ICH Guideline Q2 Validation of Analytical Procedures: Text and Methodology. This method was used in the assessment of twenty-one (n = 21) elements (Al, Au, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Pb, Pt, Sn, Ti, Tl, V) to evaluate the safety of thirty-six (n = 36) protein powder samples that were commercially available in the Irish marketplace in 2016/2017. Using the determined concentrations of elements in samples (µg·kg-1), a human health risk assessment was carried out to evaluate the potential carcinogenic and other risks to consumers of these products. While the concentrations of potentially toxic elements were found to be at acceptable levels, the results suggest that excessive and prolonged use of some of these products may place consumers at a slightly elevated risk for developing cancer or other negative health impacts throughout their lifetimes. Thus, the excessive use of these products is to be cautioned, and consumers are encouraged to follow manufacturer serving recommendations.

High-resolution mass spectrometry analysis of tetrodotoxin (TTX) and its analogues in puffer fish and shellfish.

Tetrodotoxin (TTX) is an emerging toxin in the European marine environment. It has various known structural analogues. It acts as a sodium channel blocker; the ability of each analogue to bind to the sodium channel varies with the particular structure of each analogue. Thus, each analogue will vary in its toxic potential. TTX analogues co-occur in food samples at variable concentrations. An LC-MS method was developed for the identification and quantitation of several analogues of TTX using an LTQ-Orbitrap XL mass spectrometer. The LTQ-Orbitrap XL mass spectrometer facilitates high mass accuracy measurement up to 100,000 full width at half maximum (FWHM). Using high resolution at 100,000 FWHM allows for the identification of TTX and its analogues in various matrices, including puffer fish and molluscan shellfish samples (Δ ppm = 0.28-3.38). The confirmation of characteristic fragment ions of TTX and its analogues was achieved by determining their elemental formulae via high mass accuracy. A quantitative method was then developed and optimised using these characteristic fragment ions. The limit of quantitation (LOQ) of the method was 0.136 µg g(-1) (S/N = 10) and the limit of detection (LOD) was 0.041 µg g(-1) (S/N = 3) spiking TTX standard into TTX-free mackerel fish extracts. The method was applied to naturally contaminated puffer fish and molluscan shellfish samples to confirm the presence of TTX and its analogues.

LC-MS/MS method for the determination of tetrodotoxin (TTX) on a triple quadruple mass spectrometer.

Tetrodotoxin (TTX), often referred to as the 'puffer fish' poison, is a marine toxin and it has been identified as the agent responsible for many food poisoning incidents around the world. It is a neurotoxin that blocks voltage-gated sodium channels, resulting in respiratory paralysis and even death in severe cases. It is known to occur in many different species of fish and other organisms. The toxin is mainly found in the Southeast Asia region. Worryingly, TTX is starting to appear in European waters. It is suspected that this is a consequence of Lessepsian migration, also known as the Erythrean invasion. Therefore, straightforward and reliable extraction and analytical methods are now urgently required to monitor seafood of European origin for TTX. This paper provides a versatile, dependable and robust method for the analysis of TTX in puffer fish and trumpet shellfish using LC-MS/MS. A three-stage approach was implemented involving: (1) the screening of samples using fast multiple reaction monitoring (MRM) mass spectral analysis to identify quickly positive samples on a triple quadrupole mass spectrometer (QqQMS/MS), the API 3000; (2) a Fourier-transform (FT)-MS full-scan analysis of positive samples to collect qualitative data; and (3) a method with a longer chromatography run to identify and quantitate the positive samples using the QqQMS. The quantitative LC-QqQMS method delivered excellent linearity for solvent-based standards (0.01-7.5 µg ml-1; R2 ≥ 0.9968) as well as for matrix-matched standards (0.05-37.50 µg g-1; R2 ≥ 0.9869). Good inter-day repeatability was achieved for all the relevant analytes with %RSD values (n = 9) ranging from 1.11% to 4.97% over a concentration range of 0.01-7.5 µg ml-1. A sample clean-up procedure for the puffer fish and trumpet shellfish was developed to ensure acceptable and reproducible recoveries to enable accurate and precise determination of TTX in a myriad of tissues types. Blank mackerel matrix was used for the TTX standard spiking studies in order to calculate the recoveries of the toxin during the extraction procedure. The recovery was 61.17% ± 5.42% for the extraction protocol. MS/MS studies were performed on a linear-trap quadruple-Orbitrap mass spectrometer (LTQ-Orbitrap) to obtain high-mass-accuracy data of the target analytes and their characteristic fragment ions in the puffer fish and trumpet shellfish samples. This facilitated identification of TTX and its associated analogues. These high-mass-accuracy studies facilitated the development of a rapid MRM-based quantitative method for TTX determination on the LC-QqQMS.

Tetrodotoxin: chemistry, toxicity, source, distribution and detection.

Tetrodotoxin (TTX) is a naturally occurring toxin that has been responsible for human intoxications and fatalities. Its usual route of toxicity is via the ingestion of contaminated puffer fish which are a culinary delicacy, especially in Japan. TTX was believed to be confined to regions of South East Asia, but recent studies have demonstrated that the toxin has spread to regions in the Pacific and the Mediterranean. There is no known antidote to TTX which is a powerful sodium channel inhibitor. This review aims to collect pertinent information available to date on TTX and its analogues with a special emphasis on the structure, aetiology, distribution, effects and the analytical methods employed for its detection.

Ion suppression; a critical review on causes, evaluation, prevention and applications.

The consequences of matrix effects in mass spectrometry analysis are a major issue of concern to analytical chemists. The identification of any ion suppressing (or enhancing) agents caused by sample matrix, solvent or LC-MS system components should be quantified and measures should be taken to eliminate or reduce the problem. Taking account of ion suppression should form part of the optimisation and validation of any quantitative LC-MS method. For example the US Food and Drug Administration has included the evaluation of matrix effects in its "Guidance for Industry on Bioanalytical Method Validation" (F.D.A. Department of Health and Human Services, Guidance for industry on bioanalytical method validation, Fed. Regist. 66 (100) 2001). If ion suppression is not assessed and corrected in an analytical method, the sensitivity of the LC-MS method can be seriously undermined, and it is possible that the target analyte may be undetected even when using very sensitive instrumentation. Sample analysis may be further complicated in cases where there are large sample-to-sample matrix variations (e.g. blood samples from different people can sometimes vary in certain matrix components, shellfish tissue samples sourced from different regions where different phytoplankton food sources are present, etc) and therefore exhibit varying ion-suppression effects. Although it is widely agreed that there is no generic method to overcome ion suppression, the purpose of this review is to: provide an overview of how ion suppression occurs, outline the methodologies used to assess and quantify the impact of ion suppression, discuss the various corrective actions that have been used to eliminate ion suppression in sample analysis, that is to say the deployment of techniques that eliminate or reduce the components in the sample matrix that cause ion suppression. This review article aims to collect together the latest information on the causes of ion suppression in LC-MS analysis and to consider the efficacy of common approaches to eliminate or reduce the problem using relevant examples published in the literature.

Development of a nano-electrospray MSn method for the analysis of serotonin and related compounds in urine using a LTQ-orbitrap mass spectrometer.

Serotonin, its key metabolite hydroxyindole acetic acid (5-HIAA) and dopamine have been shown to be potential biomarkers whose levels in serum and urine can be correlated with certain psychiatric and physiological disorders and illness, including depression, schizophrenia, anxiety and dementia. Recently we have published elsewhere that 5-HIAA has been identified as a potential biomarker for Attention Deficit Hyperactivity/Hyperkinetic Disorder (AD-HKD). This study describes a versatile and validated method for the analysis of these three compounds in urine using a nanoelectrospray-MS(n) method interfaced with an LTQ Orbitrap mass spectrometer. No chromatographic separation is required prior to nanoelectrospray infusion. Good linear calibrations were obtained for analytes in urine (with serotonin and dopamine giving R(2)=0.9999 and 5-HIAA having a lower R(2) value of 0.9955). Acceptable intraday repeatability was achieved for all analytes with RSD values (n=5) ranging from 4.4% to 6.2% (57, 65 and 52 nmol/L for serotonin, dopamine and 5-HIAA respectively) to 2.1-8.1% (2837, 3268, 2618 nmol/L for serotonin, dopamine and 5-HIAA respectively). Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with spiked samples for all compounds; with LODs of 9-12.9 nmol/L and LOQs of 27.2-57.7 nmol/L for analytes in urine. An appropriate sample clean-up procedure for urine was developed to ensure highest recovery and reproducibility on analysis.

Hawthorn (Crataegus spp.) in the treatment of cardiovascular disease.

The medicinal properties of hawthorn (Crataegus spp., a genus comprising approximately 300 species) have been utilized by many cultures for a variety of therapeutic purposes for many centuries. In the Western world cardiovascular disease (CVD) has become one of the single most significant causes of premature death. Echoing this situation, more recent research into the therapeutic benefits of hawthorn preparations has focused primarily upon its cardiovascular effects. This review covers research into the various mechanisms of action proposed for Crataegus preparations, clinical trials involving Crataegus preparations, and the herb's safety profile.Clinical trials reviewed have been inconsistent in terms of criteria used (sample size, preparation, dosage, etc) but have been largely consistent with regard to positive outcomes. An investigation into data available to date regarding hawthorn preparations and herb/drug interactions reveals that theoretical adverse interactions have not been experienced in practice. Further, adverse reactions relating to the use of hawthorn preparations are infrequent and mild, even at higher dosage ranges. A recent retrospective study by Zick et al. has suggested a negative outcome for the long-term use of hawthorn in the prognosis of heart failure. These findings are examined in this paper.Although further research is needed in certain areas, current research to date suggests that hawthorn may potentially represent a safe, effective, nontoxic agent in the treatment of CVD and ischemic heart disease (IHD).

Azaspiracid poisoning (AZP) toxins in shellfish: toxicological and health considerations.

It has been almost a decade since a previously unknown human toxic syndrome, azaspiracid poisoning (AZP), emerged as the cause of severe gastrointestinal illness in humans after the consumption of mussels (Mytilus edulis). Structural studies indicated that these toxins, azaspiracids, were of a new unprecedented class containing novel structural features. It is now known that the prevalent azaspiracids in mussels are AZA1, AZA2 and AZA3, which differ from each other in their degree of methylation. Several hydroxylated and carboxylated analogues of the main azaspiracids have also been identified, presumed to be metabolites of the main toxins. Since its first discovery in Irish mussels, the development of facile sensitive and selective LC-MS/MS methods has resulted in the discovery of AZA in other countries and in other species. Mice studies indicate that this toxin class can cause serious tissue injury, especially to the small intestine, and chronic exposure may increase the likelihood of the development of lung tumours. Studies also show that tissue recovery is very slow following exposure. These observations suggest that AZA is more dangerous than the other known classes of shellfish toxins. Consequently, in order to protect human consumers, proper risk assessment and regulatory control of shellfish and other affected species is of the utmost importance.

The occurrence of domoic acid linked to a toxic diatom bloom in a new potential vector: the tunicate Pyura chilensis (piure).

The tunicate Pyura chilensis (Molina, 1782); Phylum Chordata; Subphylum Urochordata; Class Ascidiacea, common local name "piure" or sea squirt; a filter-feeder (plankton and suspended particles) sessile species; may play an important role in monitoring domoic acid (DA) the principal toxic component of Amnesic Shellfish Poisoning (ASP). Significant DA concentrations have been determined in tunicate samples, collected during a recent ASP outbreak in Bahía Inglesa, an important scallop (Argopecten purpuratus) farming area. Several infaunal species were tested for the presence of DA, in addition to the usual scallop monitoring programme. DA was found at sub-toxic levels in filtering bivalves such as mussels (Mytilus chilensis), large mussels (Aulacomya ater) and clams (Protothaca thaca) (6.4, 5.4 and 4.7 microg DA/g tissue respectively). Of particular interest was the observation of significant accumulations of toxic Pseudo-nitzschia sp. diatoms in the internal siphon and atrium spaces of the tunicate. Toxin distribution within major tunicate organs was heterogeneous with 8.7-15.5 microg DA/g in edible tissues, 14.9-17.9 microg DA/g in the fecal material and 13.6-32.7 microg DA/g in the gut content. DA was determined by HPLC-UV and confirmed by diode-array detection and LC-MS/MS analysis. This is the first report of the presence of DA in a tunicate that is regularly consumed by coastal populations. These results confirm the need to include these organisms in sanitation programs for marine toxins.

Liquid chromatography-tandem mass spectrometry application, for the determination of extracellular hepatotoxins in Irish lake and drinking waters.

A novel method for the determination of hepatotoxins; microcystins (MCs), and nodularin (Nod) in lake water and domestic chlorinated tap water has been developed using liquid chromatography hyphenated with electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS). Optimization of the mass spectrometer parameters and mobile-phase composition was performed to maximize the sensitivity and reproducibility of the method. Detection of the hepatotoxins was carried out using multiple reaction monitoring experiments, thus improving the selectivity of the method. A total ion chromatogram and a precursor ion scan on ion m/z 135 was also applied to all samples to detect unknown microcystins or microcystins for which there are no standards available. A comprehensive validation of the LC-ESI-MS/MS method was completed that took into account matrix effects, specificity, linearity, accuracy, and precision. Good linear calibrations were obtained for MC-LR (1-200 microg/L; R2=0.9994) in spiked lake and tap water samples (1-50 microg/L; R2=0.9974). Acceptable interday repeatability was achieved for MC-LR in lake water with RSD values (n=9) ranging from 9.9 (10 microg/L) to 5.1% (100 microg/L). Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with spiked MCs and Nod samples; LOD=0.27 microg/L and LOQ=0.90 microg/L for MC-LR in the "normal linear range" and LOD=0.08 microg/L and LOQ=0.25 microg/L in the "low linear range" in both lake and chlorinated tap water. Similar results were obtained for a suite of microcystins and nodularin. This sensitive and rapid method does not require any sample preconcentration, including the elimination of solid-phase extraction (SPE) for the effective screening of hepatotoxins in water below the 1 microg/L WHO provisional guideline limit for MC-LR. Furthermore, SPE techniques are time-consuming, nonreproducible at trace levels, and offer poor recoveries with chlorinated water. The application of this LC-ESI-MS/MS method for routine screening of hepatotoxins in lake and chlorinated tap water (average Cl2=0.23 mg/L) is achieved and this study represents the first direct method for the screening of hepatotoxins in chlorinated tap water.

Amnesic shellfish poisoning toxins in bivalve molluscs in Ireland.

In December 1999, domoic acid (DA) a potent neurotoxin, responsible for the syndrome Amnesic Shellfish Poisoning (ASP) was detected for the first time in shellfish harvested in Ireland. Two liquid chromatography (LC) methods were applied to quantify DA in shellfish after sample clean-up using solid-phase extraction (SPE) with strong anion exchange (SAX) cartridges. Toxin detection was achieved using photodiode array ultraviolet (LC-UV) and multiple tandem mass spectrometry (LC-MS(n)). DA was identified in four species of bivalve shellfish collected along the west and south coastal regions of the Republic of Ireland. The amount of DA that was present in three species was within EU guideline limits for sale of shellfish (20 microg DA/g); mussels (Mytilus edulis), <1.0 microg DA/g; oysters (Crassostrea edulis), <5.0 microg DA/g and razor clams (Ensis siliqua), <0.3 microg DA/g. However, king scallops (Pecten maximus) posed a significant human health hazard with levels up to 240 microg DA/g total tissues. Most scallop samples (55%) contained DA at levels greater than the regulatory limit. The DA levels in the digestive glands of some samples of scallops were among the highest that have ever been recorded (2,820 microg DA/g).

Strategies to avoid the mis-identification of anatoxin-a using mass spectrometry in the forensic investigation of acute neurotoxic poisoning.

Anatoxin-a (AN) is a potent neurotoxin, produced by a number of cyanobacterial species, and consumption of freshwater contaminated with this toxin has led to animal deaths. Forensic investigations of suspected AN poisonings are frequently hampered by difficulties in detecting this toxin in biological matrices due to its rapid decay. In addition, detection of AN using single quadrupole mass spectrometry (MS) is suspect due to the presence of the amino acid, phenylalanine (Phe), since these compounds are isobaric and elute similarly in reversed phase liquid chromatography (LC). Approaches to prevent the misidentification of AN that have been explored in these studies included: (a) fluorimetric LC following derivatisation using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F); (b) methylation using diazomethane prior to LC-MS determination; (c) multiple tandem MS using a quadrupole ion-trap (LC-MS3); and (d) hybrid quadrupole time-of-flight (QqTOF). Interference from Phe was not observed in any of procedures, (a)-(c), and the high mass accuracy obtained in method (d), readily distinguished between AN (165.11536) and Phe (165.07898). LC-MSn was also employed to study the fragmentation pathway of Phe and multi-stage MS spectra provided characteristic fragmentation information that clearly distinguished between AN and Phe. The difficulties associated with the over reliance on low resolution MS without MS/MS data in forensic toxicology are discussed.

Anatoxins and degradation products, determined using hybrid quadrupole time-of-flight and quadrupole ion-trap mass spectrometry: forensic investigations of cyanobacterial neurotoxin poisoning.

The potent neurotoxins from cyanobacteria, anatoxin-a (AN), its methyl analogue, homoanatoxin-a (HMAN), and their degradation products, have been studied using nano-electrospray hybrid quadrupole time-of-flight mass spectrometry (QqTOF-MS). The anatoxin degradation products, which are readily produced in vivo by either reduction or epoxidation, were also examined in this study. The high mass accuracy QqTOF-MS data was used to confirm formula assignments for major product ions and quadrupole ion-trap (QIT)-MS was used to construct fragmentation pathways for anatoxins. Significant differences between these fragmentation pathways were observed. Comparisons between the spectra of compounds that differ in side-chain length (the AN and HMAN series) were used to identify ions that are characteristic of the homologues. The application to forensic samples in which the principal neurotoxin had undergone rapid biodegradation has been demonstrated and used to confirm anatoxin poisoning of dogs.

Rapid determination of polyether marine toxins using liquid chromatography-multiple tandem mass spectrometry.

The diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA), dinophysistoxins (DTX); pectenotoxin-2 (PTX2) and pectenotoxin-2 seco acids, were determined in marine phytoplankton, Dinophysis acuta, and mussels (Mytilus edulis) collected along the southwest coast of Ireland. Liquid chromatography-multiple tandem mass spectrometry (LC-MS/MS) was employed for the simultaneous determination of a series of marine toxins with large polarity differences. Separation of five DSP toxins was achieved on a C18 column (Luna-2, 150 mm x 2.1 mm, 5 microm) using an acetonitrile-water gradient with ammonium acetate as an eluent modifier. Electrospray ionisation (ESI) in negative mode, was used to generate the molecule related ion, [M-H]-, for each toxin. To develop a multiple reaction monitoring (MRM) method, fragmentation studies were performed to determine the optimum precursor-product ion combinations: OA (803/255), DTX2 (803/255), DTX1 (817/255), PTX2SAs (875/137) and PTX2 (857/137). This highly sensitive method had detection limits better than 1 pg (on-column). Linear calibrations were obtained for shellfish extracts that were spiked with toxins, OA, 0.007-1.00 microg/ml (r2 = 0.9993, N = 3) and DTX2, 0.054-8.5 microg/ml (r2 = 0.9992, N = 3). Good reproducibility data were also achieved with %RSD values (N = 3) ranging from 3.15% (0.56 microg DTX2/ml) to 5.71% (0.14 microg DTX2/ml), for shellfish extracts. The method was sufficiently sensitive to permit the determination of DSP toxins in small numbers of picked phytoplankton cells (N = 12-40). In one sample of D. acuta the average toxin composition per cell was: OA (7.0 pg), DTX2 (11 pg) and PTX2 (7.2 pg).

Determination of toxic cyclic heptapeptides by liquid chromatography with detection using ultra-violet, protein phosphatase assay and tandem mass spectrometry.

Microcystins, toxic cyclic heptapeptides and nodularin-R, a toxic cyclic pentapeptide, were determined using liquid chromatography (LC) with detection using photo-diode array ultra-violet (PDA-UV) and protein phosphatase (PP) assay. Positive fractions were analysed for toxins using collision-induced dissociation (CID) and tandem MS/MS experiments which were carried out simultaneously using electrospray ion-trap instrumentation. Reversed-phase liquid chromatography (LC) using an acetonitrile/water gradient was used for the LC-MS(2) determination of six microcystins standards and nodularin. The molecular related ion species, [M+H](+)([M+2H](2+) in the case of MC-RR), were used as the precursor ions for MS(2) experiments. Optimum calibration and reproducibility data were obtained for MC-LR using LC-MS(2); 0.1-5.0 microg/ml, r2 = 0.992 (n = 3); % RSD < or =7.3 at 0.25 microg MC-LR/ml (n = 3). The detection limit (S/N = 3) was better than 0.1 ng. Water samples for microcystin analysis were first screened using protein phosphatase (PP) assays and positives were concentrated using C-18 solid-phase extraction. The developed method was applied to examine a lake in Ireland contaminated by Microcystis sp. and MC-LR and MC-LA were identified.

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

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-MS(n)) 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 microm 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 microg/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 - CgH10O2R1R3]+, 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.

Elucidation of the fragmentation pathways of azaspiracids, using electrospray ionisation, hydrogen/deuterium exchange, and multiple-stage mass spectrometry.

Collision-induced dissociation (CID) mass spectra were generated for azaspiracids using electrospray ionisation (ESI), and hydrogen/deuterium (H/D) exchange was used to ascertain the number and type of replaceable hydrogens in the three predominant azaspiracid toxins. H/D exchange was conveniently achieved using deuterated solvents for liquid chromatography (LC). Using ion-trap mass spectrometry, multiple-stage CID experiments (MS(n)) on the protonated and fully exchanged ions were performed to decipher characteristic fragmentation pathways. The precursor and product ions from azaspiracids lost up to five water molecules from different regions during MS(n) experiments and it was possible to distinguish between the water losses from different molecular regions. These studies confirmed that the first water-loss ion in the spectra of azaspiracids resulted from dehydration at the vicinal diol at C20-C21. Five MS dissociation pathways were identified that resulted from fragmentation of the carbon skeleton of azaspiracids producing nitrogen-containing ions. Two pathways, involving cleavage of the E-ring and C27-C28, gave ions that were found in all azaspiracids. Three pathways, A-ring, C-ring and C19-C20 cleavages, were useful for distinguishing between azaspiracid analogues. The same product ions from backbone fragmentation were also observed using hybrid quadrupole time-of-flight mass spectrometry (QqTOFMS). The fragmentation of the A-ring was the most facile and was exploited in the development of LC/MS(n) methods for the analysis of azaspiracids.

The first identification of azaspiracids in shellfish from France and Spain.

Incidents of human intoxications throughout Europe, following the consumption of mussels have been attributed to Azaspiracid Poisoning (AZP). Although first discovered in Ireland, the search for the causative toxins, named azaspiracids, in other European countries has now led to the first discovery of these toxins in shellfish from France and Spain. Separation of the toxins, azaspiracid (AZA1) and analogues, AZA2 and AZA3, was achieved using isocratic reversed-phase liquid chromatography coupled, via an electrospray ionisation source, to an ion-trap mass spectrometer. Azaspiracids were identified in mussels (Mytilus galloprovincialis), 0.24 microg/g, from Galicia, Spain, and scallops (Pecten maximus), 0.32 microg/g, from Brittany, France. Toxin profiles were similar to those found in the equivalent shellfish in Ireland in which AZA1 was the predominant toxin.

Geographical, temporal, and species variation of the polyether toxins, azaspiracids, in shellfish.

Azaspiracid Poisoning (AZP) is a new toxic syndrome that has caused human intoxications throughout Europe following the consumption of mussels (Mytilus edulis), harvested in Ireland. Shellfish intoxication is a consequence of toxin-bearing microalgae in the shellfish food chain, and these studies demonstrated a wide geographic distribution of toxic mussels along the entire western coastal region of Ireland. The first identification of azaspiracids in other bivalve mollusks including oysters (Crassostrea gigas), scallops (Pecten maximus), clams (Tapes phillipinarium), and cockles (Cardium edule) is reported. Importantly, oysters were the only shellfish that accumulated azaspiracids at levels that were comparable with mussels. The highest levels of total azaspiracids (microg/g) recorded to-date were mussels (4.2), oysters (2.45), scallops (0.40), cockles (0.20), and clams (0.61). An examination of the temporal variation of azaspiracid contamination of mussels in a major shellfish production area revealed that, although maximum toxin levels were recorded during the late summer period, significant intoxications were observed at periods when marine dinoflagellate populations were low. Although human intoxications have so far only been associated with mussel consumption, the discovery of significant azaspiracid accumulation in other bivalve mollusks could pose a threat to human health.