Isolation of Caribbean Ciguatoxin-5 (C-CTX5) and confirmation of its structure by NMR spectroscopy.

Ciguatera poisoning occurs throughout subtropical and tropical regions globally. The Virgin Islands in the Caribbean Sea is a known hyperendemic region for ciguatera and has been associated with Caribbean ciguatoxin (C-CTX) contamination in fish. An algal C-CTX (C-CTX5) was identified in Gambierdiscus silvae and G. caribeaus isolated from benthic algal samples collected in waters south St. Thomas, US Virgin Islands. The highest CTX-producing isolate, G. silvae 1602 SH-6, was grown at large-scale to isolate sufficient C-CTX5 for structural confirmation by NMR spectroscopy. A series of orthogonal extraction and fractionation procedures resulted in purification of approximately 40 µg of C-CTX5, as estimated by quantitative NMR. A suite of 1D and 2D NMR experiments were acquired that verified the structure originally proposed for C-CTX5. The structural confirmation and successful isolation of C-CTX5 opens the way for work on the stability, toxicology and biotransformation of C-CTXs, as well as for the production of quantitative reference materials for analytical method development and validation. The strategies developed for purification of C-CTX5 may also apply to isolation and purification of CTXs from the Pacific Ocean and other regions.

Identification of 24-O-ß-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Dinophysis Blooms, Dinophysis and Prorocentrum Cultures, and Shellfish in Europe, North America and Australasia.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-ß-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1-5, the 24-O-ß-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4-6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

Selective Extraction and Purification of Azaspiracids from Blue Mussels ( Mytilus edulis) Using Boric Acid Gel.

Azaspiracids belong to a family of more than 50 polyether toxins originating from marine dinoflagellates such as Azadinium spinosum. All of the azaspiracids reported thus far contain a 21,22-dihydroxy group. Boric acid gel can bind selectively to compounds containing vic-diols or α-hydroxycarboxylic acids via formation of reversible boronate complexes. Here we report use of the gel to selectively capture and release azaspiracids from extracts of blue mussels. Analysis of the extracts and fractions by liquid chromatography-tandem mass spectrometry (LC-MS) showed that this procedure resulted in an excellent cleanup of the azaspiracids in the extract. Analysis by enzyme-linked immunoasorbent assay (ELISA) and LC-MS indicated that most azaspiracid analogues were recovered in good yield by this procedure. The capacity of boric acid gel for azaspiracids was at least 50 µg/g, making this procedure suitable for use in the early stages of preparative purification of azaspiracids. In addition to its potential for concentration of dilute samples, the extensive cleanup provided by boric acid gel fractionation of azaspiracids in mussel samples almost eliminated matrix effects during subsequent LC-MS and could be expected to reduce matrix effects during ELISA analysis. The method may therefore prove useful for quantitative analysis of azaspiracids as part of monitoring programs. Although LC-MS data showed that okadaic acid analogues also bound to the gel, this was much less efficient than for azaspiracids under the conditions used. The boric acid gel methodology is potentially applicable to other important groups of natural toxins containing diols including ciguatoxins, palytoxins, pectenotoxins, tetrodotoxin, trichothecenes, and toxin glycosides.

Differential Mobility-Mass Spectrometry Double Spike Isotope Dilution Study of Release of ß-Methylaminoalanine and Proteinogenic Amino Acids during Biological Sample Hydrolysis.

The non-protein amino acid ß-methylamino-L-alanine (BMAA) has been linked to neurodegenerative disease and reported throughout the environment. Proposed mechanisms of bioaccumulation, trophic transfer and chronic toxicity of BMAA rely on the hypothesis of protein misincorporation. Poorly selective methods for BMAA analysis have led to controversy. Here, a recently reported highly selective method for BMAA quantitation using hydrophilic interaction liquid chromatography-differential mobility spectrometry-tandem mass spectrometry (HILIC-DMS-MS/MS) is expanded to include proteinogenic amino acids from hydrolyzed biological samples. For BMAA quantitation, we present a double spiking isotope dilution approach using D3-BMAA and 13C15N2-BMAA. These methods were applied to study release of BMAA during acid hydrolysis under a variety of conditions, revealing that the majority of BMAA can be extracted along with only a small proportion of protein. A time course hydrolysis of BMAA from mussel tissue was carried out to assess the recovery of BMAA during sample preparation. The majority of BMAA measured by typical methods was released before a significant proportion of protein was hydrolyzed. Little change was observed in protein hydrolysis beyond typical hydrolysis times but the concentration of BMAA increased linearly. These findings demonstrate protein misincorporation is not the predominant form of BMAA in cycad and shellfish.

Development of Certified Reference Materials for Diarrhetic Shellfish Poisoning Toxins, Part 1: Calibration Solutions.

Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties.

Development of Certified Reference Materials for Diarrhetic Shellfish Poisoning Toxins, Part 2: Shellfish Matrix Materials.

Okadaic acid (OA) and its analogs, dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic biotoxins produced by marine algae that can accumulate in shellfish and cause the human illness known as diarrhetic shellfish poisoning (DSP). Regulatory testing of shellfish is required to protect consumers and the seafood industry. Certified reference materials (CRMs) are essential for the development, validation, and quality control of analytical methods, and thus play an important role in toxin monitoring. This paper summarizes work on research and development of shellfish tissue reference materials for OA and DTXs. Preliminary work established the appropriate conditions for production of shellfish tissue CRMs for OA and DTXs. Source materials, including naturally incurred shellfish tissue and cultured algae, were screened for their DSP toxins. This preliminary work informed planning and production of a wet mussel (Mytilus edulis) tissue homogenate matrix CRM. The homogeneity and stability of the CRM were evaluated and found to be fit-for-purpose. Extraction and LC-tandem MS methods were developed to accurately certify the concentrations of OA, DTX1, and DTX2 using a combination of standard addition and matrix-matched calibration to compensate for matrix effects in electrospray ionization. The concentration of domoic acid was also certified. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization. The presence of other toxins in the CRM was also assessed and information values are reported for OA and DTX acyl esters.

Structure Elucidation, Relative LC-MS Response and In Vitro Toxicity of Azaspiracids 7-10 Isolated from Mussels (Mytilus edulis).

Azaspiracids (AZAs) are marine biotoxins produced by dinoflagellates that can accumulate in shellfish, which if consumed can lead to poisoning events. AZA7-10, 7-10, were isolated from shellfish and their structures, previously proposed on the basis of only LC-MS/MS data, were confirmed by NMR spectroscopy. Purified AZA4-6, 4-6, and 7-10 were accurately quantitated by qNMR and used to assay cytotoxicity with Jurkat T lymphocyte cells for the first time. LC-MS(MS) molar response studies performed using isocratic and gradient elution in both selected ion monitoring and selected reaction monitoring modes showed that responses for the analogues ranged from 0.3 to 1.2 relative to AZA1, 1. All AZA analogues tested were cytotoxic to Jurkat T lymphocyte cells in a time- and concentration-dependent manner; however, there were distinct differences in their EC50 values, with the potencies for each analogue being: AZA6 > AZA8 > AZA1 > AZA4 ≈ AZA9 > AZA5 ≈ AZA10. This data contributes to the understanding of the structure-activity relationships of AZAs.

A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids.

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.

Analysis of ß-N-methylamino-L-alanine (BMAA) in spirulina-containing supplements by liquid chromatography-tandem mass spectrometry.

Over the last decade the amino acid beta-N-methylamino-L-alanine (BMAA) has come under intense scrutiny. International laboratory and epidemiological research continues to support the hypothesis that environmental exposure to BMAA (e.g., through dietary practices, water supply) can promote the risk of various neurodegenerative diseases. A wide variety of cyanobacteria spp. have previously been reported to produce BMAA, with production levels dependent upon species, strain and environmental conditions. Since spirulina (Arthrospira spp.) is a member of the cyanobacteria phylum frequently consumed via dietary supplements, the presence of BMAA in such products may have public health implications. In the current work, we have analyzed ten spirulina-containing samples for the presence of BMAA; six pure spirulina samples from two separate raw materials suppliers, and four commercially-available multi-ingredient products containing 1.45 g of spirulina per 8.5 g serving. Because of controversy surrounding the measurement of BMAA, we have used two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: one based on reversed phase LC (RPLC) with derivatization and the other based on hydrophilic interaction LC (HILIC). Potential matrix effects were corrected for by internal standardization using a stable isotope labeled BMAA standard. BMAA was not detected at low limits of detection (80 ng/g dry weight) in any of these product samples. Although these results are reassuring, BMAA analyses should be conducted on a wider sample selection and, perhaps, as part of ongoing spirulina production quality control testing and specifications.

Derivatization of azaspiracid biotoxins for analysis by liquid chromatography with fluorescence detection.

Azaspiracids (AZAs) are an important group of regulated lipophilic biotoxins that cause shellfish poisoning. Currently, the only widely available analytical method for quantitation of AZAs is liquid chromatography-mass spectrometry (LC-MS). Alternative methods for AZA analysis are needed for detailed characterization work required in the preparation of certified reference materials (CRMs) and by laboratories not equipped with LC-MS. Chemical derivatization of the amine and carboxyl groups on AZAs was investigated for the purpose of facilitating analysis by LC with fluorescence detection (FLD). Experiments towards chemical modification of AZA1 at the amine achieved only limited success. Derivatization of the carboxyl group, on the other hand, proved successful using the 9-anthryldiazomethane (ADAM) method previously applied to the okadaic acid (OA) group toxins. Extraction and clean-up methods were investigated for shellfish tissue samples and a post-reaction solid phase extraction procedure was developed for the AZA ADAM derivatives. Chromatographic separations were developed for the LC-FLD analysis of derivatized AZAs alone or in the presence of other derivatized toxins. This new analytical method for analysis of AZAs enabled verification of AZA1-3 concentrations in recently certified reference materials. The method demonstrated good linearity, repeatability and accuracy showing its potential as an alternative to LC-MS for measurement of AZAs.

A mussel tissue certified reference material for multiple phycotoxins. Part 2: liquid chromatography-mass spectrometry, sample extraction and quantitation procedures.

A freeze-dried mussel tissue certified reference material (CRM-FDMT1) containing multiple groups of shellfish toxins has been prepared. Toxin groups present in the material include okadaic acid and the dinophysistoxins, azaspiracids, yessotoxins, pectenotoxins, spirolides and domoic acid. In this work, analytical methods have been examined for the characterisation of the candidate CRM. A comprehensive extraction procedure was developed, which gave good recovery (>98%) for all lipophilic toxins studied. A fast liquid chromatography-mass spectrometry (LC-MS) method was developed that separates the major toxins according to the MS ionisation mode of optimum sensitivity. Matrix effects associated with analysis of these extracts using the developed LC-MS method were assessed. Standard addition and matrix-matched calibration procedures were evaluated to compensate for matrix effects. The methods and approaches will be used for the precise characterisation of the homogeneity and stability of the various toxins in CRM-FDMT1 and for the accurate assignment of certified values. The developed methods also have excellent potential for application in routine regulatory monitoring of shellfish toxins.

A mussel tissue certified reference material for multiple phycotoxins. Part 3: homogeneity and stability.

A candidate certified reference material (CRM) for multiple shellfish toxins (domoic acid, okadaic acid and dinophysistoxins, pectenotoxins, yessotoxin, azaspiracids and spirolides) has been prepared as a freeze-dried powder from mussel tissues (Mytilus edulis). Along with the certified values, the most important characteristics for a reference material to be fit-for-purpose are homogeneity and stability. Acceptable between-bottle homogeneity was found for this CRM. Within-bottle homogeneity was assessed using domoic acid, and it was shown that repeated subsampling of the CRM can be performed precisely down to 0.35 g. Both short- and long-term stability studies carried out under isochronous conditions demonstrated excellent stability of the various toxins present in the material. While degradation of some analytes was observed at +60°C in short-term studies, it was determined that shipping at ambient temperature is adequate. No instability was detected in long-term stability studies, and it was shown that the material can be held at +18°C safely for up to 1 year. To guarantee stability of the CRM over its lifetime the stock will be maintained at -20°C. The results of the homogeneity and stability testing show that CRM-FDMT1 is appropriate for its intended use in quality assurance and quality control of shellfish toxin analysis methods.