Publications by authors named "Pearse McCarron"

58 Publications

Production and stability of Oxygen-18 labeled Caribbean ciguatoxins and gambierones.

Toxicon 2022 May 14;211:11-20. Epub 2022 Mar 14.

Biotoxin Metrology, National Research Council, 1411 Oxford Street, Halifax, NS, B3H 2Z1, Canada.

Ciguatoxins (CTXs) and gambierones are ladder-shaped polyethers associated with ciguatera poisoning and Gambierdiscus spp. Several of these compounds contain carbonyl or hemiketal groups, which have the potential to exchange with O-labeled water under acidic conditions. The effects of solvent composition and acid on the rate of exchange and on the stability of the labels at various pH values were assessed to optimize the incorporation of O into Caribbean ciguatoxin-1 and -2 (C-CTX1/2), gambierone, and 44-methylgambierone. LC-HRMS results showed that O-labeling occurred at the hydroxy group of the hemiketal at C-56 in C-CTX1/2, and at the hydroxy group of the hemiketal at C-4 and the ketone at C-40 in gambierones. Labeling occurred very rapidly (complete in <30 min) for C-CTX1/2, and more slowly (complete in ca. 16 h) for both gambierones. Labeled C-CTX1/2 was reduced with sodium borohydride to produce O-labeled C-CTX3/4. The incorporated O labels in the gambierones and C-CTXs were retained in aqueous solvent mixtures under neutral conditions in a short-term stability study, demonstrating that these O-labeled toxins have the potential to be used in isotope dilution and metabolism studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxicon.2022.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9161730PMC
May 2022

Non-target analysis and stability assessment of reference materials using liquid chromatography‒high-resolution mass spectrometry.

Anal Chim Acta 2022 Apr 17;1201:339622. Epub 2022 Feb 17.

Biotoxin Metrology, National Research Council of Canada, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada.

Development and characterization of biological and environmental matrix certified reference materials (CRMs) for organic analytes typically relies heavily on targeted analytical methods, such as liquid chromatography (LC) with triple-quadrupole mass spectrometry detection. LC with high-resolution mass spectrometry (LC‒HRMS) can also provide high quality data for both targeted and non-targeted analytes, with the potential for retrospective data analysis. Here, we demonstrate the utility of non-target analysis (NTA) using LC‒HRMS for profiling and stability assessment of a mussel tissue matrix CRM certified for several classes of marine algal toxins (CRM-FDMT1). First, the NTA method was developed using data-dependent MS/MS acquisition and commercial metabolomics software for data processing. Of 128 toxin analogues previously reported in CRM-FDMT1, 125 were detected by LC-HRMS, with 97 triggered for MS/MS by data dependant acquisition. Automated data processing detected 119 of these compounds and 109 were retained after automated filtering of results for putative toxin analogues. Those analogues not detected were low abundance ions, or poorly resolved isomers. The method was then used to demonstrate new strategies for CRM stability assessment considering the stability of certified analytes, related toxin analogues, and unrelated matrix compounds. Several analogues from each toxin class in CRM-FDMT1 as well as other unrelated matrix compounds were observed to be significantly less stable than the certified toxins. Using this method, no instability was measured for any compounds at conditions ≤4 °C, providing a greater degree of confidence in CRM stability than could be achieved using conventional approaches to stability assessment targeting only the certified analytes. The NTA method and stability assessment approach presented are applicable to future CRM development with other matrices and organic analyte classes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.aca.2022.339622DOI Listing
April 2022

Acute microcystin exposure induces reversible histopathological changes in Chinook Salmon (Oncorhynchus tshawytscha) and Atlantic Salmon (Salmo salar).

J Fish Dis 2022 May 2;45(5):729-742. Epub 2022 Mar 2.

Pacific Biological Station, Fisheries and Oceans Canada, Nanaimo, British Columbia, Canada.

Atlantic Salmon (Salmo salar) and Chinook Salmon (Oncorhynchus tshawytscha) develop a severe liver disease called net-pen liver disease (NPLD), which is characterized by hepatic lesions that include megalocytosis and loss of gross liver structure. Based on studies where salmonids have been exposed to microcystin (MC) via intraperitoneal injection, NPLD is believed to be caused by MC exposure, a hepatotoxin produced by cyanobacteria. Despite the link between MC and NPLD, it remains uncertain if environmentally relevant MC exposure is responsible for NPLD. To determine if we could produce histopathology consistent with NPLD, we compared the response of Atlantic and Chinook Salmon sub-lethal MC exposure. Salmon were orally gavaged with saline or MC containing algal paste and sampled over 2 weeks post-exposure. Liver lesions appeared by 6 h but were resolved 2-weeks post-exposure; histopathological changes observed in other tissues were not as widespread, nor was their severity as great as those in the liver. There was no evidence for NPLD due to the absence of hepatic megalocytosis. These results indicate that the development of NPLD is not due to acute MC exposure but may be associated with higher MC concentration occurring in food, long-term exposure through drinking of contaminated seawater and/or interactions with other marine toxins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jfd.13599DOI Listing
May 2022

Selective extraction of gambierone and related metabolites in Gambierdiscus silvae using m-aminophenylboronic acid-agarose gel and liquid chromatography-high-resolution mass spectrometric detection.

J Chromatogr B Analyt Technol Biomed Life Sci 2022 Jan 2;1188:123014. Epub 2021 Nov 2.

Biotoxin Metrology, National Research Council Canada, 1411 Oxford Street, Halifax, NS, B3H 3Z1, Canada.

Gambierdiscus spp. are epi-benthic dinoflagellates that have been associated with ciguatera poisoning. These microalgae can have complex secondary metabolite profiles including ciguatoxins, maitotoxins, and gambierones, with varying compositions and toxicities across species and strains. Given this chemical diversity there is a need to develop selective and sensitive methods for secondary metabolite profiling. In this study, we used a cultured Caribbean strain of Gambierdiscus silvae to develop sample preparation and analysis strategies for characterizing vic-diol containing secondary metabolites. A pooled cellular extract was first screened by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for ciguatoxin-related compounds, which resulted in the confirmation of gambierone (1) and a novel isomer of 44-methylgambierone (3). Treatment of the extract with periodate confirmed that the gambierones each contained one reactive vic-diol, which was exploited for the development of a selective extraction procedure using m-aminophenylboronic acid gel and the non-aqueous binding solvent chloroform. Using this non-traditional boronate affinity procedure, LC-HRMS also revealed the presence of additional sulfated polycyclic ethers in the gambierone-containing vic-diol fraction, while pigments and other contaminants were removed. The developed tools could be applied to screen collections of Gambierdiscus and other benthic algae to provide additional chemical characterization of gambierone-related compounds. The selective extraction procedure may also prove useful as a step in the isolation of these sulfated polyethers for structural, toxicological and biotransformation studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jchromb.2021.123014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9175239PMC
January 2022

CyanoMetDB, a comprehensive public database of secondary metabolites from cyanobacteria.

Water Res 2021 May 8;196:117017. Epub 2021 Mar 8.

Department of Environmental Chemistry, Swiss Federal Institute of Aquatic Science and Technology (Eawag), 8600 Duebendorf, Switzerland. Electronic address:

Harmful cyanobacterial blooms, which frequently contain toxic secondary metabolites, are reported in aquatic environments around the world. More than two thousand cyanobacterial secondary metabolites have been reported from diverse sources over the past fifty years. A comprehensive, publically-accessible database detailing these secondary metabolites would facilitate research into their occurrence, functions and toxicological risks. To address this need we created CyanoMetDB, a highly curated, flat-file, openly-accessible database of cyanobacterial secondary metabolites collated from 850 peer-reviewed articles published between 1967 and 2020. CyanoMetDB contains 2010 cyanobacterial metabolites and 99 structurally related compounds. This has nearly doubled the number of entries with complete literature metadata and structural composition information compared to previously available open access databases. The dataset includes microcytsins, cyanopeptolins, other depsipeptides, anabaenopeptins, microginins, aeruginosins, cyclamides, cryptophycins, saxitoxins, spumigins, microviridins, and anatoxins among other metabolite classes. A comprehensive database dedicated to cyanobacterial secondary metabolites facilitates: (1) the detection and dereplication of known cyanobacterial toxins and secondary metabolites; (2) the identification of novel natural products from cyanobacteria; (3) research on biosynthesis of cyanobacterial secondary metabolites, including substructure searches; and (4) the investigation of their abundance, persistence, and toxicity in natural environments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.watres.2021.117017DOI Listing
May 2021

A mussel tissue certified reference material for multiple phycotoxins. Part 5: profiling by liquid chromatography-high-resolution mass spectrometry.

Anal Bioanal Chem 2021 Mar 4;413(8):2055-2069. Epub 2021 Mar 4.

Biotoxin Metrology, National Research Council Canada, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada.

A freeze-dried mussel tissue-certified reference material (CRM-FDMT1) was prepared containing the marine algal toxin classes azaspiracids, okadaic acid and dinophysistoxins, yessotoxins, pectenotoxins, cyclic imines, and domoic acid. Thus far, only a limited number of analogues in CRM-FDMT1 have been assigned certified values; however, the complete toxin profile is significantly more complex. Liquid chromatography-high-resolution mass spectrometry was used to profile CRM-FDMT1. Full-scan data was searched against a list of previously reported toxin analogues, and characteristic product ions extracted from all-ion-fragmentation data were used to guide the extent of toxin profiling. A series of targeted and untargeted acquisition MS/MS experiments were then used to collect spectra for analogues. A number of toxins previously reported in the literature but not readily available as standards were tentatively identified including dihydroxy and carboxyhydroxyyessotoxin, azaspiracids-33 and -39, sulfonated pectenotoxin analogues, spirolide variants, and fatty acid acyl esters of okadaic acid and pectenotoxins. Previously unreported toxins were also observed including compounds from the pectenotoxin, azaspiracid, yessotoxin, and spirolide classes. More than one hundred toxin analogues present in CRM-FDMT1 are summarized along with a demonstration of the major acyl ester conjugates of several toxins. Retention index values were assigned for all confirmed or tentatively identified analogues to help with qualitative identification of the broad range of lipophilic toxins present in the material.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00216-020-03133-2DOI Listing
March 2021

Fatty acid esters of azaspiracids identified in mussels () using liquid chromatography-high resolution mass spectrometry.

Toxicon X 2020 Dec 28;8:100059. Epub 2020 Sep 28.

Biotoxin Metrology, National Research Council Canada, 1411 Oxford St., Halifax, NS, B3H 3Z1, Canada.

Azaspiracids (AZAs) are lipophilic polyether toxins produced by and species of marine microalgae. The main dinoflagellate precursors AZA1 and AZA2 are metabolized by shellfish to produce an array of AZA analogues. Many marine toxins undergo fatty acid esterification in shellfish, therefore mussel tissues contaminated with AZAs were screened for intact fatty acid esters of AZAs using liquid chromatography-high resolution mass spectrometry. Acyl esters were primarily observed for AZAs containing hydroxy groups at C-3 with 3--palmitoylAZA4 identified as the most abundant acyl ester, while other fatty acid esters including 18:1, 16:1, 17:0, 20:2 and 18:0 acyl esters were detected. The structures of these acyl derivatives were determined through LC-MS/MS experiments, and supported by periodate cleavage reactions and semi-synthesis of palmitate esters of the AZAs. Esters of the hydroxy groups at C-20 or C-21 were not observed in mussel tissue. The relative proportion of the most abundant AZA ester was less than 3% of the sum of the major free AZA analogues. These findings reveal an additional metabolic pathway for AZAs in shellfish.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxcx.2020.100059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549145PMC
December 2020

Rapid quantitative screening of cyanobacteria for production of anatoxins using direct analysis in real time high-resolution mass spectrometry.

Rapid Commun Mass Spectrom 2021 Jan;35(1):e8940

Biotoxin Metrology, National Research Council Canada, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada.

Rationale: Anatoxins (ATXs) are a potent class of cyanobacterial neurotoxins that are increasingly problematic in drinking water reservoirs and recreational water bodies worldwide. Because of their high polarity and low molecular weight, analysis of ATXs is challenging and they can be considered underreported compared with other classes of cyanobacterial toxins. Improved screening methods are therefore needed to effectively assess their occurrence and concentrations in the environment.

Methods: A rapid screening method was developed for ATXs in cyanobacteria using direct analysis in real time combined with high-resolution mass spectrometry (DART-HRMS), requiring less than 2 min per sample for triplicate analysis. The developed method was evaluated for its quantitative capabilities, applied to the screening of 30 cyanobacterial culture samples for the presence of anatoxin-a, homoanatoxin-a and dihydroanatoxin-a, and compared with a more typical liquid chromatography (LC)/HRMS method.

Results: Excellent linearity was observed in the analysis of a matrix-matched calibration curve using DART-HRMS, with ionization suppression of about 50% and relative standard deviations between replicate analyses of approximately 30%. Limits of detection for both anatoxin-a and homoanatoxin-a were estimated as 1 ng/mL. Excellent agreement was observed between DART-HRMS and LC/HRMS with all ATX-producing cultures correctly identified and only one false positive culture by DART-HRMS.

Conclusions: DART-HRMS shows excellent promise for the rapid, quantitative screening of ATXs in cyanobacteria and could be expanded in the future to include the analysis of field samples and drinking water, as well as additional ATX analogues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rcm.8940DOI Listing
January 2021

Dihydrodinophysistoxin-1 Produced by in the Gulf of Maine, USA and Its Accumulation in Shellfish.

Toxins (Basel) 2020 08 20;12(9). Epub 2020 Aug 20.

Washington State Department of Health Public Health Laboratories, Shoreline, WA 98155, USA.

Dihydrodinophysistoxin-1 (dihydro-DTX1, (M-H) 819.5), described previously from a marine sponge but never identified as to its biological source or described in shellfish, was detected in multiple species of commercial shellfish collected from the central coast of the Gulf of Maine, USA in 2016 and in 2018 during blooms of the dinoflagellate . Toxin screening by protein phosphatase inhibition (PPIA) first detected the presence of diarrhetic shellfish poisoning-like bioactivity; however, confirmatory analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) failed to detect okadaic acid (OA, (M-H) 803.5), dinophysistoxin-1 (DTX1, (M-H) 817.5), or dinophysistoxin-2 (DTX2, (M-H) 803.5) in samples collected during the bloom. Bioactivity-guided fractionation followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) tentatively identified dihydro-DTX1 in the PPIA active fraction. LC-MS/MS measurements showed an absence of OA, DTX1, and DTX2, but confirmed the presence of dihydro-DTX1 in shellfish during blooms of in both years, with results correlating well with PPIA testing. Two laboratory cultures of isolated from the 2018 bloom were found to produce dihydro-DTX1 as the sole DSP toxin, confirming the source of this compound in shellfish. Estimated concentrations of dihydro-DTX1 were >0.16 ppm in multiple shellfish species (max. 1.1 ppm) during the blooms in 2016 and 2018. Assuming an equivalent potency and molar response to DTX1, the authority initiated precautionary shellfish harvesting closures in both years. To date, no illnesses have been associated with the presence of dihydro-DTX1 in shellfish in the Gulf of Maine region and studies are underway to determine the potency of this new toxin relative to the currently regulated DSP toxins in order to develop appropriate management guidance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/toxins12090533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551465PMC
August 2020

Semiquantitation of Paralytic Shellfish Toxins by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry Using Relative Molar Response Factors.

Toxins (Basel) 2020 06 16;12(6). Epub 2020 Jun 16.

Biotoxin Metrology, National Research Council Canada, 1411 Oxford St, Halifax, NS B3H 3Z1, Canada.

Paralytic shellfish toxins (PSTs) are a complex class of analogs of the potent neurotoxin saxitoxin (STX). Since calibration standards are not available for many PSTs, including -11 hydroxyl analogs called M-toxins, accurate quantitation by liquid chromatography-mass spectrometry (LC-MS) can be challenging. In the absence of standards, PSTs are often semiquantitated using standards of a different analog (e.g., STX), an approach with a high degree of uncertainty due to the highly variable sensitivity between analytes in electrospray ionization. Here, relative molar response factors (RMRs) were investigated for a broad range of PSTs using common LC-MS approaches in order to improve the quantitation of PSTs for which standards are unavailable. First, several M-toxins (M1-M6, M9 and dcM6) were semipurified from shellfish using preparative gel filtration chromatography and quantitated using LC-charged aerosol detection (LC-CAD). The RMRs of PST certified reference materials (CRMs) and M-toxins were then determined using selective reaction monitoring LC-MS/MS and full scan LC-high-resolution MS (LC-HRMS) methods in positive and negative electrospray ionization. In general, RMRs for PSTs with similar chemical structures were comparable, but varied significantly between subclasses, with M-toxins showing the lowest sensitivity. For example, STX showed a greater than 50-fold higher RMR than M4 and M6 by LC-HRMS. The MS instrument, scan mode and polarity also had significant impacts on RMRs and should be carefully considered when semiquantitating PSTs by LC-MS. As a demonstration of their utility, the RMRs determined were applied to the semiquantitation of PSTs in contaminated mussels, showing good agreement with results from calibration with CRMs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/toxins12060398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354571PMC
June 2020

Fatty acid ester metabolites of gymnodimine in shellfish collected from China and in mussels (Mytilus galloprovincialis) exposed to Karenia selliformis.

Harmful Algae 2020 02 20;92:101774. Epub 2020 Feb 20.

College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China; Key Laboratory of Marine Environment and Ecology, Ocean University of China, Ministry of Education, Qingdao 266100, China. Electronic address:

Marine shellfish exposed to the microalgae Karenia selliformis can accumulate gymnodimines (GYM). Shellfish samples collected from Beihai City in Guangxi Autonomous Region, and Ningde City in Fujian Province, in the South China Sea, as well as mussels Mytilus galloprovincialis fed on K. selliformis under laboratory conditions were analyzed. Gymnodimines and various fatty acid ester metabolites were detected in the clam Antigona lamellaris and pen shell Atrina pectinata, while no esters were found in the oyster Crassostrea sp. and the gastropod Batillaria zonalis despite positive detection of free GYM in both species. When present, the predominant acyl esters observed were 18:0-GYM-A and 20:1-GYM-A. Under laboratory conditions GYM-A was accumulated and metabolized to fatty acid esters in mussels exposed to K. selliformis, with 16:0-GYM-A and 20:1-GYM-A as the major variants. A novel compound with the same accurate mass as GYM-A and its 16:0 fatty acid ester were observed in the experimental mussels but was not present in the microalgal strain to which mussels were exposed. No significant differences of reactive oxygen species (ROS) levels and antioxidant enzymes were found between mussels fed on K. selliformis or GYM-free microalgae Isochrysis galbana. This suggests the accumulation of GYM and its metabolites does not significantly impact the physiological status of mussels. While it is currently not proven that GYM affects human health, risk assessments should consider the presence of GYM esters in naturally contaminated shellfish as part of exposure analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.hal.2020.101774DOI Listing
February 2020

Isolation and Characterization of [DLeu]microcystin-LY from CPCC-464.

Toxins (Basel) 2020 01 23;12(2). Epub 2020 Jan 23.

Biotoxin Metrology, National Research Council, 1411 Oxford Street, Halifax NS B3H 3Z1, Canada.

[D-Leu]MC-LY () ([M + H] / 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from strain CPCC464. The compound was characterized by H and C NMR spectroscopy, liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and UV spectroscopy. A calibration reference material was produced after quantitation by H NMR spectroscopy and LC with chemiluminescence nitrogen detection. The potency of in a protein phosphatase 2A inhibition assay was essentially the same as for MCLR (). Related microcystins, [D-Leu]MC-LR () ([M + H] / 1037.6041, Δ 1.0 ppm), [D-Leu]MC-M(O)R () ([M + H] / 1071.5565, Δ 2.0 ppm) and [D-Leu]MC-MR () ([M + H] / 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu]MC-M(O)R () ([M + H] / 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC-HRMS/MS experiments. The relative abundances of , , , and in a freshly extracted culture in the positive ionization mode LC-HRMS were ca. 84, 100, 3.0, 11 and 0.05, respectively. These and other results indicate that [D-Leu]-containing MCs may be more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC-MS/MS screening methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/toxins12020077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076857PMC
January 2020

Phylogenetic analysis and yessotoxin profiles of Gonyaulax spinifera cultures from the Benguela Current upwelling system.

Harmful Algae 2019 05 9;85:101626. Epub 2019 Jul 9.

Leibniz Institute for Baltic Sea Research Warnemünde (IOW), Seestrasse 15, D-18119 Rostock, Germany.

The Benguela Current in the Atlantic is one of the four major upwelling systems on the Eastern boundary of the world ocean. Thus the coastal regions off Namibia are prone to high primary productivity that can lead to Harmful Algae Blooms as this nutrient rich water reaches the euphotic zone. Yessotoxins (YTXs) produced by G. spinifera were detected in Namibian phytoplankton field samples in 2011. Isolation of G. spinifera cultures from this location in 2012 enabled molecular genetics work and further liquid chromatography-mass spectrometry assessment of toxin profiles. The molecular work grouped the Benguela G. spinifera with other toxic G. spinifera strains originating from Italy and New Zealand. The main YTX analogs present in the Benguela G. spinifera are homo-YTX, YTX and a hydroxylated analogue. This work adds important knowledge on the occurrence of Harmful Algae Blooms in this region and is of relevance for safety.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.hal.2019.101626DOI Listing
May 2019

Structure Elucidation and Relative Toxicity of (24)-24-Hydroxyyessotoxin from a Namibian Isolate of .

J Nat Prod 2019 07 8;82(7):1945-1952. Epub 2019 Jul 8.

Biotoxin Metrology , National Research Council Canada , 1411 Oxford Street , Halifax , Nova Scotia B3H 3Z1 , Canada.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of a Namibian strain of showed the presence of a number of yessotoxins (YTXs). Principal among these were YTX (), homoYTX (), and a tentative hydroxylated analogue that did not correspond to any previously confirmed YTX structures. Culturing the strain afforded sufficient biomass for purification of the new analogue through a series of solvent partitioning and chromatographic steps, yielding ∼0.9 mg as a solid. NMR spectroscopy, ion-trap mass spectrometry, and HRMS identified the new analogue as 24-hydroxyYTX (). Purified 24-hydroxyYTX was quantitated by NMR, and its relative toxicity evaluated using two embryonic zebrafish toxicity assays. 24-HydroxyYTX demonstrated reduced toxicity compared to YTX.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jnatprod.9b00318DOI Listing
July 2019

Screening of cyclic imine and paralytic shellfish toxins in isolates of the genus Alexandrium (Dinophyceae) from Atlantic Canada.

Harmful Algae 2018 07 6;77:108-118. Epub 2018 Jul 6.

National Research Council Canada, 1411 Oxford St, Halifax, NS, B3H 3Z1, Canada. Electronic address:

The dinoflagellate genus Alexandrium Halim has frequently been associated with harmful algal blooms. Although a number of species from this genus are known to produce paralytic shellfish toxins (PST) and/or cyclic imines (CI), studies on comprehensive toxin profiling using techniques capable of detecting the full range of PST and CI analogues are limited. Isolates of Alexandrium spp. from Atlantic Canada were analyzed by targeted and untargeted liquid chromatography-tandem mass spectrometry (LC-MS). Results showed a number of distinct profiles and wide ranging cell quotas of PST and spirolides (SPX) in both A. catenella (Whedon & Kofoid) Balech and A. ostenfedii (Paulsen) Balech & Tangen. The concentration of PST in A. catenella ranged from 0.0029 to 54 fmol cell with the major components being C2 and GTX4. In addition, putative PST metabolites were confirmed for the first time in A. catenella by high resolution MS/MS. By comparison, A. ostenfeldii isolates showed much lower concentrations of PST (
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.hal.2018.05.005DOI Listing
July 2018

Dynamics of paralytic shellfish toxins and their metabolites during timecourse exposure of scallops Chlamys farreri and mussels Mytilus galloprovincialis to Alexandrium pacificum.

Aquat Toxicol 2018 Jul 16;200:233-240. Epub 2018 May 16.

College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China; Key Laboratory of Marine Environment and Ecology, Ocean University of China, Ministry of Education, Qingdao 266100, China. Electronic address:

New C-11 hydroxyl metabolites of paralytic shellfish toxins (PSTs) have been reported in shellfish. To gain further information on these metabolites, as well as the potential for formation of phase-II metabolites and acyl esters of PSTs, bivalves were fed with the PSTs-producing dinoflagellate Alexandrium pacificum (strain ATHK). Through independent experiments, scallops (Chlamys farreri) were fed for 9 days and mussels (Mytilus galloprovincialis) for 5 days plus an additional 5 days of depuration, with representative samples taken throughout. Several common PSTs (C1-4, GTX1-6 and NEO) and metabolites including M1, M3, M5, M7, M9, M2 and M8 were detected in the hepatopancreas of scallops during toxin accumulation and in the hepatopancreas of mussels during both toxin accumulation and elimination periods. The relative molar ratio of metabolites to precursor molecules was used to estimate relative metabolic conversion rates. Conversion rates of C1/2 and GTX2/3 were higher than those of C3/4 and GTX1/4, in scallops and mussels. The first metabolites observed in both bivalve species investigated were M1/3, which are formed from C1/2. However, the conversion of GTX2/3 to M2 was more complete than other biotransformation reactions in both mussels and scallops. In general, metabolic conversion of PSTs was observed after a shorter time and to a greater extent in mussels than in scallops in the exposure period. No acyl esters or conjugation products of PSTs with glucuronic acid, glutathione, cysteine and taurine were detected by liquid chromatography with high resolution tandem mass spectrometry in the samples investigated. Additionally, only GTX1/4 and GTX2/3 were detected in the kidney of scallops, which demonstrates that PSTs are mainly metabolized through the hepatic metabolism pathway in bivalves. This work improves the understanding of PST metabolism during toxin accumulation and depuration in commercially harvested shellfish.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.aquatox.2018.05.003DOI Listing
July 2018

Identification, growth and toxicity assessment of Coolia Meunier (Dinophyceae) from Nova Scotia, Canada.

Harmful Algae 2018 05 27;75:45-56. Epub 2018 Apr 27.

Measurement Science and Standards, National Research Council Canada, 1411 Oxford St., Halifax, NS, B3H 3Z1, Canada. Electronic address:

Benthic dinoflagellates of the toxigenic genus Coolia Meunier (Dinophyceae) are known to have a global distribution in both tropical and temperate waters. The type species, C. monotis, has been reported from the Mediterranean Sea, the NE Atlantic and from Rhode Island, USA in the NW Atlantic, whereas other species in the genus have been reported from tropical locations. Coolia cells were observed in algal drift samples collected at seven sites in Nova Scotia, Canada. Clonal isolates were established from four of these locations and identified with light and scanning electron microscopy, then confirmed with genetic sequencing to be C. monotis. This is the first record of this species in Nova Scotia. The isolates were established and incubated at 18 °C under a 14:10 L:D photoperiod with an approximate photon flux density of 50-60 μmol m s. Growth experiments using an isolate from Johnston Harbour (CMJH) were carried out at temperatures ranging from 5 to 30 °C under the same photoperiod with an approximate photon flux density of 45-50 μmol m s. Cells tolerated temperatures from 5 to 25 °C with optimum growth and mucilage aggregate production between 15 and 20 °C. Methanol extracts of this isolate examined by Liquid Chromatography-Mass Spectrometry (LC-MS) did not show the presence of the previously reported cooliatoxin. Toxic effects were assayed using two zebrafish bioassays, the Fish Embryo Toxicity (FET) assay and the General Behaviour and Toxicity (GBT) assay. The results of this study demonstrate a lack of toxicity in C. monotis from Nova Scotia, as has been reported for other genetically-confirmed isolates of this species. Conditions in which cell growth that could potentially degrade water quality and provide substrate and dispersal mechanisms for other harmful microorganisms via mucilage production are indicated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.hal.2018.04.001DOI Listing
May 2018

Capillary electrophoresis-tandem mass spectrometry for multiclass analysis of polar marine toxins.

Anal Bioanal Chem 2018 Sep 16;410(22):5405-5420. Epub 2018 May 16.

Measurement Science and Standards, National Research Council Canada, 1411 Oxford St, Halifax, NS, B3H 3Z1, Canada.

Polar marine toxins are more challenging to analyze by mass spectrometry-based methods than lipophilic marine toxins, which are now routinely measured in shellfish by multiclass reversed-phase liquid chromatography-tandem mass spectrometry (MS/MS) methods. Capillary electrophoresis (CE)-MS/MS is a technique that is well suited for the analysis of polar marine toxins, and has the potential of providing very high resolution separation. Here, we present a CE-MS/MS method developed, with use of a custom-built interface, for the sensitive multiclass analysis of paralytic shellfish toxins, tetrodotoxins, and domoic acid in seafood. A novel, highly acidic background electrolyte (5 M formic acid) was designed to maximize protonation of analytes and to allow a high degree of sample stacking to improve the limits of detection. The method was applied to a wide range of regulated and less common toxin analogues, and exhibited a high degree of selectivity between toxin isomers and matrix interference. The limits of detection in mussel tissue were 0.0052 mg/kg for tetrodotoxins, 0.160 mg/kg for domoic acid, and between 0.0018 and 0.120 mg/kg for paralytic shellfish toxins, all of which showed good linearity. Minimal ionization suppression was observed when the response from neat and mussel-matrix-matched standards was corrected with multiple internal standards. Analysis of shellfish matrix reference materials and spiked samples demonstrated good accuracy and precision. Finally, the method was transferred to a commercial CE-MS/MS system to demonstrate its widespread applicability for use in both R & D and routine regulatory settings. The approach of using a highly acidic background electrolyte is of broad interest, and can be considered generally applicable to simultaneous analysis of other classes of small, polar molecules with differing pK values. Graphical abstract ᅟ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00216-018-1089-4DOI Listing
September 2018

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

J Agric Food Chem 2018 Mar 12;66(11):2962-2969. Epub 2018 Mar 12.

Norwegian Veterinary Institute, P.O. Box 750 Sentrum , N-0106 Oslo , Norway.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jafc.8b00346DOI Listing
March 2018

Identification of 21,22-Dehydroazaspiracids in Mussels ( Mytilus edulis) and in Vitro Toxicity of Azaspiracid-26.

J Nat Prod 2018 04 28;81(4):885-893. Epub 2018 Feb 28.

Measurement Science and Standards , National Research Council Canada , Halifax , NS B3H 3Z1 , Canada.

Azaspiracids (AZAs) are marine biotoxins produced by the genera Azadinium and Amphidoma, pelagic marine dinoflagellates that may accumulate in shellfish resulting in human illness following consumption. The complexity of these toxins has been well documented, with more than 40 structural variants reported that are produced by dinoflagellates, result from metabolism in shellfish, or are extraction artifacts. Approximately 34 μg of a new AZA with MW 823 Da (AZA26 (3)) was isolated from blue mussels ( Mytilus edulis), and its structure determined by MS and NMR spectroscopy. AZA26, possibly a bioconversion product of AZA5, lacked the C-20-C-21 diol present in all AZAs reported thus far and had a 21,22-olefin and a keto group at C-23. Toxicological assessment of 3 using an in vitro model system based on Jurkat T lymphocyte cells showed the potency to be ∼30-fold lower than that of AZA1. The corresponding 21,22-dehydro-23-oxo-analogue of AZA10 (AZA28) and 21,22-dehydro analogues of AZA3, -4, -5, -6, -9, and -10 (AZA25, -48 (4), -60, -27, -49, and -61, respectively) were also identified by HRMS/MS, periodate cleavage reactivity, conversion from known analogues, and NMR (for 4 that was present in a partially purified sample of AZA7).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jnatprod.7b00973DOI Listing
April 2018

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

Sci Rep 2018 01 8;8(1):117. Epub 2018 Jan 8.

Measurement Science and Standards, National Research Council Canada, 1411 Oxford St., Halifax, NS, B3H 3Z1, Canada.

The non-protein amino acid β-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 D-BMAA and CN-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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-18392-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758758PMC
January 2018

Accumulation and transformation of azaspiracids in scallops (Chlamys farreri) and mussels (Mytilus galloprovincialis) fed with Azadinium poporum, and response of antioxidant enzymes.

Toxicon 2018 Mar 8;143:20-28. Epub 2017 Dec 8.

College of Environmental Science and Engineering, Ocean University of China, Qingdao, 266100, China; Key Laboratory of Marine Environment and Ecology, Ocean University of China, Ministry of Education, Qingdao, 266100, China. Electronic address:

Azaspiracid (AZA) producing microalgae have been reported internationally and could potentially impact a variety of seafood. Scallops (Chlamys farreri) and mussels (Mytilus galloprovincialis) from China were fed with the AZA2 producer, Azadinium poporum, to study uptake, metabolism and oxidative stress in the shellfish. LC-MS/MS showed significant accumulation and differential metabolism of AZA2 in the scallops and mussels. In mussels AZA2 was metabolized to AZA19, with subsequent decarboxylation to AZA6. In scallops no AZA19 or AZA6 was observed, however, a novel AZA metabolite was formed that is isobaric with AZA19 ([M+H], m/z 886), but elutes at a different retention time. In addition it was noted that the scallop metabolite was stable during heating, while AZA19 has been shown to decarboxylate. Concentrations of reactive oxygen species (ROS) and activities of antioxidant enzymes were monitored. ROS levels increased slightly in the meat of scallops and mussels due to starvation in the acclimation and depuration periods, but reduced in the feeding periods with non-toxic Isochrysis galbana or toxic A. poporum. No obvious variations were found in activities for a range of antioxidant enzymes. These results provide new insights on the potential for accumulation and metabolism of AZAs in bivalve species relevant to this area of China, which is of importance considering the recent finding of AZA producing microalgae in the region.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxicon.2017.12.040DOI Listing
March 2018

Total Synthesis of (6R,10R,13R,14R,16R,17R,19S,20R,21R,24S, 25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 Reveals Non-Identity with the Natural Product.

Angew Chem Int Ed Engl 2018 01 13;57(3):805-809. Epub 2017 Dec 13.

Department of Chemistry and Biochemistry, The Ohio State University, 151 W. Woodruff Ave, Columbus, OH, 43210, USA.

A convergent and stereoselective total synthesis of the previously assigned structure of azaspiracid-3 has been achieved by a late-stage Nozaki-Hiyama-Kishi coupling to form the C21-C22 bond with the C20 configuration unambiguously established from l-(+)-tartaric acid. Postcoupling steps involved oxidation to an ynone, modified Stryker reduction of the alkyne, global deprotection, and oxidation of the resulting C1 primary alcohol to the carboxylic acid. The synthetic product matched naturally occurring azaspiracid-3 by mass spectrometry, but differed both chromatographically and spectroscopically.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201711006DOI Listing
January 2018

Stereochemical Definition of the Natural Product (6R,10R,13R, 14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 by Total Synthesis and Comparative Analyses.

Angew Chem Int Ed Engl 2018 01 15;57(3):810-813. Epub 2017 Dec 15.

Department of Chemistry and Biochemistry, The Ohio State University, 151 W. Woodruff Ave, Columbus, OH, 43210, USA.

The previously accepted structure of the marine toxin azaspiracid-3 is revised based upon an original convergent and stereoselective total synthesis of the natural product. The development of a structural revision hypothesis, its testing, and corroboration are reported. Synthetic (6R,10R,13R,14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R, 33R,34R,36S,37S,39R)-azaspiracid-3 chromatographically and spectroscopically matched naturally occurring azaspiracid-3, whereas the previously assigned 20R epimer did not.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201711008DOI Listing
January 2018

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for quantitation of paralytic shellfish toxins: validation and application to reference materials.

Anal Bioanal Chem 2017 Sep 20;409(24):5675-5687. Epub 2017 Jul 20.

Measurement Science and Standards, National Research Council Canada, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada.

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by marine dinoflagellates that are responsible for paralytic shellfish poisoning (PSP) in humans. This work highlights our ongoing efforts to develop quantitative methods for PSTs using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). Compared with the commonly used method of liquid chromatography with post-column oxidation and fluorescence detection (LC-ox-FLD), HILIC-MS/MS has the potential of being more robust, sensitive and straightforward to operate, and provides unequivocal confirmation of toxin identity. The main driving force for the present work was the need for a complementary method to LC-ox-FLD to assign values to shellfish tissue matrix reference materials for PSTs. Method parameters that were optimized included LC mobile and stationary phases, electrospray ionization (ESI) conditions, and MS/MS detection parameters. The developed method has been used in the detection and identification of a wide range of PSTs including less common analogues and metabolites in a range of shellfish and algal samples. We have assessed the matrix effects of shellfish samples and have evaluated dilution, standard addition and matrix matched calibration as means of mitigating them. Validation on one LC-MS/MS system for nine common PST analogues (GTX1-4, dcGTX2&3, STX, NEO, and dcSTX) was completed using standard addition. The method was then transferred to a more sensitive LC-MS/MS system, expanded to include five more PSTs (C1&2, dcNEO and GTX5&6) and validated using matrix matched calibration. Limits of detection of the validated method ranged between 6 and 280 nmol/kg tissue using standard addition in extracts of blue mussels, with recoveries between 92 and 108%. Finally, this method was used in combination with the AOAC Official Method based on LC-ox-FLD to measure PSTs in a new mussel tissue matrix reference material.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00216-017-0507-3DOI Listing
September 2017

Relative molar response of lipophilic marine algal toxins in liquid chromatography/electrospray ionization mass spectrometry.

Rapid Commun Mass Spectrom 2017 Sep;31(17):1453-1461

Measurement Science and Standards, National Research Council Canada, Halifax, NS, Canada.

Rationale: Accurate quantitative analysis of lipophilic toxins by liquid chromatography/mass spectrometry (LC/MS) requires calibration solution reference materials (RMs) for individual toxin analogs. Untargeted analysis is aimed at identifying a vast number of compounds and thus validation of fully quantitative untargeted methods is not feasible. However, a semi-quantitative approach allowing for profiling is still required and will be strengthened by knowledge of the relative molar response (RMR) of analogs in LC/MS with electrospray ionization (ESI).

Methods: RMR factors were evaluated for toxins from the okadaic acid (OA/DTXs), yessotoxin (YTX), pectenotoxin (PTX), azaspiracid (AZA) and cyclic imine (CI) toxin groups, in both solvent standards and environmental sample extracts. Since compound ionization and fragmentation influences the MS response of toxins, RMRs were assessed under different chromatographic conditions (gradient, isocratic) and MS acquisition modes (SIM, SRM, All-ion, target MS/MS) on low- and high-resolution mass spectrometers.

Results: In general, RMRs were not significantly impacted by chromatographic conditions (isocratic vs gradient), with the exception of DTX1. MS acquisition modes had a more significant impact, with PnTX-G and SPX differing notably. For a given toxin group, response factors were generally in the range of 0.5 to 2. The cyclic imines were an exception.

Conclusions: Differences in RMRs between toxins of a same chemical base structure were not significant enough to indicate major issues for non-targeted semi-quantitative analysis, where there is limited or no availability of standards for many compounds, and where high degrees of accuracy are not required. Differences in RMRs should be considered when developing methods that use a standard of a single analogue to quantitate other toxins from the same group.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rcm.7918DOI Listing
September 2017

Sulfated diesters of okadaic acid and DTX-1: Self-protective precursors of diarrhetic shellfish poisoning (DSP) toxins.

Harmful Algae 2017 03 11;63:85-93. Epub 2017 Feb 11.

Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, NS B3H 3Z1, Canada; UNC Wilmington Center for Marine Science, Marvin K. Moss Lane, Wilmington, NC 28409, United States. Electronic address:

Many toxic secondary metabolites used for defense are also toxic to the producing organism. One important way to circumvent toxicity is to store the toxin as an inactive precursor. Several sulfated diesters of the diarrhetic shellfish poisoning (DSP) toxin okadaic acid have been reported from cultures of various dinoflagellate species belonging to the genus Prorocentrum. It has been proposed that these sulfated diesters are a means of toxin storage within the dinoflagellate cell, and that a putative enzyme mediated two-step hydrolysis of sulfated diesters such as DTX-4 and DTX-5 initially leads to the formation of diol esters and ultimately to the release of free okadaic acid. However, only one diol ester and no sulfated diesters of DTX-1, a closely related DSP toxin, have been isolated leading some to speculate that this toxin is not stored as a sulfated diester and is processed by some other means. DSP components in organic extracts of two large scale Prorocentrum lima laboratory cultures have been investigated. In addition to the usual suite of okadaic acid esters, as well as the free acids okadaic acid and DTX-1, a group of corresponding diol- and sulfated diesters of both okadaic acid and DTX-1 have now been isolated and structurally characterized, confirming that both okadaic acid and DTX-1 are initially formed in the dinoflagellate cell as the non-toxic sulfated diesters.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.hal.2017.01.012DOI Listing
March 2017

A mussel tissue certified reference material for multiple phycotoxins. Part 4: certification.

Anal Bioanal Chem 2017 Jan 9;409(1):95-106. Epub 2016 Nov 9.

Measurement Science and Standards, National Research Council Canada, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada.

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00216-016-0004-0DOI Listing
January 2017

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

J AOAC Int 2016 Sep 20;99(5):1163-72. Epub 2016 Aug 20.

National Research Council Canada, Measurement Science and Standards, 1411 Oxford St, Halifax, NS, Canada B3H 3Z1.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5740/jaoacint.16-0152DOI Listing
September 2016
-->