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PubMedDate not supplied

Amnesic shellfish poisoning toxins in shellfish: estimation of uncertainty of measurement for a liquid chromatography/tandem mass spectrometry method.

A liquid chromatography/mass spectrometry (LC/MS) method for amnesic shellfish poisoning toxins in shellfish was developed and validated. Tissue homogenate (4 g) was extracted with 16 mL methanol-water (1 + 1, v/v). Dilution into acetonitrile-water (1 + 9, v/v) was followed by C18 solid-phase extraction cleanup. Domoic acid (DA) and epi-domoic acid were determined by LC/MS/MS with electrospray ionization and multiple reaction monitoring. External calibration was performed with dilutions of a certified reference standard. Advantages of this method include speed, lower detection limits, and a very high degree of specificity. The LC/MS response was highly linear, and there were no significant interferences to the determination of DA. Formal method validation was performed on 4 shellfish species. Fortification studies gave recoveries (mean +/- SD; n = 24) of 93 +/- 14% at 1 mg/kg, and 93.3 +/- 7.6% at 20 mg/kg over all the species. Analysis of a mussel certified reference material showed the bias as < 5%. The limits of detection and quantitation were 0.15 and 0.5 mg/kg, respectively. Routine application of the method over 4 months gave a recovery for the QC sample (1 mg/kg fortified blank mussel homogenate) run with each batch of 88.9 +/- 5.5% (mean +/- SD; n = 37). The total uncertainty of measurement results were estimated as 0.12 (12%) at 0.25-5 mg/kg and 0.079 (7.9%) at 5-50 mg/kg. The major contribution to the uncertainty was the repeatability of the LC/MS determination, probably arising from subtle matrix effects.

Amnesia↗
PubMedDate not supplied

Liquid chromatographic determination of paralytic shellfish poisons in shellfish after prechromatographic oxidation.

A liquid chromatographic method for quantitating paralytic shellfish poison toxins in shellfish has been developed in which the toxins are converted to fluorescent purines by prechromatographic oxidation under mildly basic conditions with hydrogen peroxide or periodate. The addition of ammonium formate to the periodate oxidation reaction greatly improved the yield of fluorescent derivatives for neosaxitoxin, gonyautoxin-1, B-2, and C-3 compared to the same reaction without ammonium formate. As little as 3-6 ng of each of the nonhydroxylated toxins and 7-12 ng of the hydroxylated compounds per gram of shellfish could be detected. Reversed-phase chromatography using ammonium formate in the mobile phase improved the chromatography of neosaxitoxin and B-2 compared to results obtained earlier. Because the oxidation products of neosaxitoxin and B-2 could not be separated, parent compounds were separated before oxidation by using an SPE-COOH ion exchange cartridge. The repeatability coefficient of variation for the oxidation reactions ranged from 3 to 8% for the peroxide reaction, and from 4 to 11% for the periodate reaction, depending upon the individual toxin determined and its concentration in the extract (0.04-0.55 micrograms/g). The method was compared to the mouse bioassay and the postcolumn oxidation method. In most cases, results were comparable.

Chromatography, Liquid↗
PubMed2002

Azaspiracid shellfish poisoning: unusual toxin dynamics in shellfish and the increased risk of acute human intoxications.

A number of recent acute human intoxications in Europe from the consumption of Irish mussels have been attributed to the presence of a new class of toxins named azaspiracids. The study demonstrates that azaspiracids behave differently from other polyether toxins, and this accounts for most false-negative results in the mouse bioassay employed by regulatory agencies to detect azaspiracids. Typically, polyether toxins are concentrated in the digestive glands of shellfish, but this is not always the situation with azaspiracids. Liquid chromatography-mass spectrometry (LC-MS), especially multiple tandem MS methods, have been applied to demonstrate that azaspiracid (AZA1) and its methyl- and demethyl- analogues, AZA2 and AZA3 respectively, are distributed throughout shellfish tissues. Using conventional mouse bioassay protocols, only 0-40% of the total azaspiracid content of shellfish was used in the assay, which could directly account for false-negative results. It was also observed that the toxin profiles differed significantly in various mussel tissues with AZA1 as the predominant toxin in the digestive glands and AZA3 predominant in the remaining tissues.

Acute Disease↗
PubMedDate not supplied

Analysis of diarrhetic shellfish poisoning toxins in shellfish tissue by liquid chromatography with fluorometric and mass spectrometric detection.

Diarrhetic shellfish poisoning (DSP) is a severe gastrointestinal illness caused by consumption of shellfish contaminated with toxigenic dinoflagellates. The main toxins responsible for DSP are okadaic acid (OA), DTX-1, DTX-2, and DTX-3, the latter being a complex mixture of 7-O-acyl derivatives of the first 3. In this study, existing methods based on liquid chromatography (LC) combined with mass spectrometry (LC-MS) and LC with fluorometric detection (LC-FLD) of anthryldiazomethane (ADAM) derivatives were improved upon to achieve a high degree of accuracy and precision for the determination of DSP toxins in a new mussel tissue reference material (MUS-2). All experimental parameters were examined comprehensively, and a new internal standard and a new solid-phase extraction cleanup method were introduced. Quantitative extraction of DSP toxins from shellfish tissue was achieved by exhaustive extraction with aqueous 80% methanol. Cleanup was accomplished by partitioning the crude aqueous methanol extract with hexane to remove lipids and then with chloroform to isolate the toxins. A further cleanup based on an aminopropylsilica column was useful for LC-MS and looks promising for the ADAM/LC-FLD method. The internal standard, 7-O-acetylokadaic acid, synthesized by partial acetylation of OA, improved accuracy and precision by correcting for incomplete recoveries in extraction, cleanup, and derivatization steps and for volumetric errors and instrumental drift. An improved silica cleanup after ADAM derivatization also was developed by controlling the activities of both sorbent and solvents. The methods were tested with various mussel tissue samples. The resulting improved methods will be useful to analysts involved in routine monitoring of DSP toxins.

Animals↗
PubMed1989

Serological cross-reactions between crab saxitoxin-induced protein and paralytic shellfish poison-contaminated shellfish.

A polyclonal antiserum generated against crab saxitoxin-induced protein was tested against paralytic shellfish poison (PSP)-contaminated crabs and shellfish. Antibody-reactive proteins in PSP-contaminated bivalve mollusc extracts were localized using SDS-PAGE and immunoblotting. PSP-contaminated clams and oysters possessed a higher degree of immunoreactivity to the saxitoxin-induced protein found in PSP-resistant crabs than their respective non-contaminated controls.

Animals↗
PubMedDate not supplied

Comparison of enzyme immunoassay and mouse bioassay for determining paralytic shellfish poisoning toxins in shellfish.

Sixty shellfish samples (mussel [Mytilus edulis], queen scallops [Chlamys opercularis] and king scallops [Pecten maximus]), collected during routine surveillance for paralytic shellfish poisoning (PSP) toxins conducted by the UK Ministry of Agriculture, Fisheries and Food and the Scottish Office Agriculture, Environmental Fisheries Department, were analysed for contamination with PSP toxins by mouse bioassay (MBA), and by a competitive direct enzyme immunoassay (Ridascreen Saxitoxin ELISA). Using the MBA as the reference method (detection limit: 350 micrograms kg-1), no false negative results out of a total of 45 MBA-positive samples and one false positive result out of a total of 15 MBA-negative samples were obtained by ELISA. The correlation coefficient for MBA-positive samples (n = 45) was 0.78. At the regulatory level for PSP toxins (800 micrograms kg-1), 39 of the samples were correctly determined by ELISA to be above or below this level, toxicity was overestimated in two samples, and underestimated in four samples.

Animals↗
PubMedDate not supplied

Quantitative determination of paralytic shellfish poisoning toxins in shellfish by using prechromatographic oxidation and liquid chromatography with fluorescence detection.

The prechromatographic oxidation LC method developed by Lawrence [J. Assoc. Off. Anal. Chem. 74, 404-409(1991)] for the determination of paralytic shellfish poisoning (PSP) toxins has been tested for the quantitative determination of PSP toxins in shellfish. All aspects of the method were studied and modified as necessary to improve its performance for routine regulatory purposes. The chromatographic conditions were changed to shorten analysis time. The oxidation reaction was tested for repeatability and the influence of the sample matrix on quantitation. An important part of the study was to quantitatively evaluate an ion exchange (-COOH) cleanup step using disposable solid-phase extraction cartridges that separated the PSP toxins into 3 distinct groups for quantitation, namely the C toxins, the GTX toxins, and the saxitoxin group. The cleanup step was very simple and used increasing concentrations of aqueous NaCl for elution of the toxins. The C toxins were not retained by the cartridges and thus were eluted unretained with water. The GTX toxins (GTX1 to GTX6 as well as dcGTX2 and dcGTX3) eluted from the cartridges with 0.05M NaCl while the saxitoxin group (saxitoxin, neosaxitoxin, and dcsaxitoxin) required 0.3M NaCl for elution. Each fraction was analyzed by LC after oxidation with periodate or peroxide. All of the compounds could be separated and quantitatively determined in spiked samples of mussels, clams, and oysters. The nonhydroxylated toxins could be quantitated at concentrations as low as about 0.02 microg/g (2 micro/100 g) of tissue while the hydroxylated toxins could be quantitated at concentrations as low as about 0.1 microg/g (10 microg/100 g). Average recoveries of the toxins through the complete cleanup procedure were 85% or greater for spiked extracts of oysters and clams and greater than 73% for mussels.

Animals↗
PubMedDate not supplied

Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: interlaboratory study.

An interlaboratory study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3 together), gonyautoxins 1 and 4 (GTX1,4 together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2 together), and C-3 and C-4 (C3,4 together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Samples of mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with samples of clams, oysters, and scallops. Twenty-one samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries.

Algorithms↗
PubMedDate not supplied

Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: collaborative study.

A collaborative study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3; together), gonyautoxins 1 and 4 (GTX1,4; together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2; together), and C-3 and C-4 (C3,4; together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with clams, oysters, and scallops. Twenty-one test samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries. It is recommended that the method be adopted First Action by AOAC INTERNATIONAL.

Animals↗
PubMed1999

Detection of diarrhetic shellfish poisoning toxins from tropical shellfish using liquid chromatography-selected reaction monitoring mass spectrometry.

A negative mode liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) method was developed to detect low concentrations of the diarrhetic shellfish poisoning (DSP) toxins okadaic acid and dinophysistoxin-1 (DTX-1). Detection relies upon monitoring the transition of negative precursor ions [M - H]- to a common fragment ion of m/z 255. Our limit of detection for okadaic acid with this method is 0.5 pg on column. LC-SRM MS has allowed us to detect persistent, low concentrations of DSP toxins from Singapore shellfish.

Animals↗
PubMed1987

Dinoflagellate Gymnodinium catenatum as the source of paralytic shellfish toxins in Tasmanian shellfish.

Paralytic shellfish toxins in both cultured cells and natural phytoplankton blooms of the dinoflagellate Gymnodinium catenatum from inshore Tasmanian waters (Australia) were analyzed by high performance liquid chromatography, thin layer chromatography and electrophoresis techniques. The dinoflagellate toxins were dominated by low potency sulfocarbamoyl saxitoxin derivatives (98-99 mole% in total), including gonyautoxin VIII (C2) and its epimer (C1) and sulfocarbamoyl gonyautoxins I and IV (C3 and C4). Mussels and oysters contaminated by the dinoflagellate showed similar toxins, but contained larger proportions of C3 (40-57 mole%) and more potent carbamate toxins (7-23 mole% total).

Animals↗
PubMed1990

A toxin profile for shellfish involved in an outbreak of paralytic shellfish poisoning in India.

Toxin profiles of clams and oysters involved in the outbreak of paralytic shellfish poisoning in India in 1983 were studied by a liquid chromatographic technique. Gonyautoxins 1, 2, 3, 4 and 8, and 11-epigonyautoxin 8 appeared to be the major toxins along with small amounts of saxitoxin, neosaxitoxin, decarbamoylsaxitoxin, decarbamoylgonyautoxins 2 and 3, C3 and C4. Toxin profile suggests the involvement of Alexandrium spp. in this outbreak.

Animals↗
PubMedDate not supplied

Evaluation of prechromatographic oxidation for liquid chromatographic determination of paralytic shellfish poisons in shellfish.

A liquid chromatographic (LC) method employing prechromatographic oxidation for the determination of paralytic shellfish poison (PSP) toxins was evaluated. A number of changes to an earlier method resulted in improved separation and quantitation of most PSP analogues. Modification of the periodate oxidation reaction for the N-hydroxy-containing toxins led to improved sensitivity and stability of the products, enabling automated overnight analyses. These changes also enabled quantitation of gonyautoxins 1 and 4 (together) in the presence of gonyautoxins 2 and 3. Decarbamoylsaxitoxin can be identified and quantitated after peroxide oxidation. A cleanup step using a strong-anion-exchange column removed the C toxins and B-2 from the extracts and enabled a more accurate quantitation of gonyautoxins 1 and 4 and neosaxitoxin. Chiral chromatography, employing a reversed-phase column and chiral mobile-phase additives (copper-proline complex), was briefly evaluated for the separation of the oxidation products of the isomer pairs, gonyautoxins 1 and 4 and gonyautoxins 2 and 3. A comparison of the method with the mouse bioassay for the determination of PSP in lobster hepatopancreas (58 samples) showed a reasonable correlation (0.90) over a concentration range of 40-500 micrograms/100 g (saxitoxin equivalents), although the LC results were consistently higher than the mouse bioassay values by about 40%.

Animals↗
PubMed1996

[Epidemiology of toxic and infectious risk related to shellfish consumption].

For feeding purposes shellfish filter large amounts of water but also concentrate infectious agents and toxins that are present in the marine environment either naturally or because of pollution. Thus, the consumption of raw or undercooked shellfish is a substantial source of foodborne poisoning, mostly epidemic and sometimes sporadic. Most of shellfish-borne infectious diseases are linked to fecal contamination of the marine environment; they include: thyphoid fever, salmonellosis, shigellosis, campylobacteriosis, cholera, Norwalk or Norwalk-like gastroenteritis and hepatitis A. In warm climates, shellfish contains naturally occurring halopilic Vibrios and may cause severe sporadic infections (septicemias) among very susceptible consumers (immunocompromised). Shellfish also causes outbreaks of paralytic shellfish poisoning (PSP) and diarrheic shellfish poisoning (DSP) when they are contaminated by toxins produced when Dinophisis, a marine plancton, proliferates. Chemical compounds (heavy metals and organic toxins) that are dumped in the environment (soil, air, and water) also reach shellfish harvesting waters where they are cocentrated. Although acute or chronic effects of the chemical contamination of shellfish have not been clearly documented, the cadmium pollution of some shellfish harvesting waters raises a serious problem. Since it is impossible to prevent completely the contamination of coastal waters by any of the agents cited above, the prevention of shellfish-borne diseases requires monitoring of the marine environment and shellfish flesh (coliform count, Dinophysis toxins, heavy metals...). This surveillance allows the classification of growing areas as suitable or not for harvesting and distribution of shellfish. However, this surveillance is not always sensitive enough. Indicators of fecal pollution are particularly not reliable for shellfish viral contamination. A better knowledge of marine biology, the limitation of coastal waters pollution, improved surveillance, the development of more sensitive indicators, the responsabilisation of the industry and the information of the public on the health hazards associated with shellfish consumption are the key issues for the improvement of shellfish-borne disease prevention.

Climate↗
PubMed1968

Viral depuration by assaying individual shellfish.

A study was carried out to further evaluate the practicability of viral depuration by assaying individual shellfish. The Northern quahaug and a strain of the type 1 attenuated poliovirus were used as the working model. Two types of depuration systems were employed: the small experimental tanks and a pilot-size tank with a capacity of approximately 24 bushels (836 liters) of shellfish. Volumes of the individual shellfish samples were found uniform throughout the experiments when a prior selection for the weight of the shellfish was made. There was also no significant difference in volumes of the individual samples during the course of depuration (24 to 96 hr). Under controlled hydrographic conditions, however, the uptake of virus in individual shellfish varied considerably. In general, the individual variability reached 10- to 100-fold. This wide variation would explain the variability of viral contents obtained in pooled samples during depuration as reported previously. During a later phase of depuration, although a great majority of shellfish were free of the virus, a few still harbored minimal amounts of contaminants. The presence of virus in some of the shellfish after various periods of depuration would, theoretically, be obscured by the pooling of the sampled shellfish. Further examination of the negative samples by assaying larger quantities than those routinely used revealed that a few still contained virus. To simulate naturally polluted shellfish as closely as technically possible, shellfish were polluted with minimal amounts of virus. The shellfish were cleansed more rapidly by the depuration process than were those polluted with more virus. Since the naturally polluted shellfish were shown to contain less virus than those studied in the laboratory, it is anticipated that the former type of shellfish may be cleansed more readily by this process within a reasonable period of time. Justification for a field trial of depuration in this country is presented.

Animals↗
PubMedDate not supplied

Clinical characteristics and pattern of skin test reactivities in shellfish allergy patients in Hong Kong.

Allergens from crustaceans and mollusks exhibit extensive cross-reactivity in vitro. However, the degree and pattern of cross-reactivity between different shellfish species in vivo is still unclear. The objective of this study was to determine the clinical characteristics of shellfish allergic patients in Hong Kong and the pattern of skin test reactivities to the different species. This cohort study involves patients attending the allergy clinic of a large teaching hospital for suspected shellfish allergy. Each subject underwent skin-prick tests to eight species of shellfish and house-dust mites. Eighty-four consecutive patients were tested. Twenty-eight patients reported a history of severe anaphylaxis. Fourteen patients had no positive shellfish skin test and were excluded. There were 183 positive shellfish skin tests, with an average of 2.61 positive tests per subject. Ninety percent of subjects also had positive skin tests to house-dust mites. Overall, 65. 7% of subjects had more than one positive skin test to shellfish. There were strong statistical associations between species belonging to the same order but also between some mollusks and crustaceans. We found a high degree of skin test cross-reactivity between different species of shellfish and between shellfish and house-dust mites. Therefore, patients with a history of shellfish allergy should be cautious with all types of shellfish.

Adolescent↗