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[Analysis of paralytic shellfish poisoning and diarrhetic shellfish poisoning of bivalves in seafood market of Guangzhou].

OBJECTIVE: Paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) in 7 species of economical shellfishes were analyzed for 1 year, which collected from Huangsha seafood market of Guangzhou from Apr, 2004 to Mar, 2005. METHODS: The levels of PSP and DSP in bivalves were determined with mouse bioassay of AOAC. The risk assessment of PSP and DSP in bivalves was conducted according to FAO and Chinese Administration Organization of Fish Culture and Seaport. RESULTS: PSP was detected in 2 species of the shellfishes assayed and DSP was found out in 6 species. The content of PSP was lower than 4MU/g tissue, whereas the level of PSP in glands was higher than in muscles. DSP toxin was detected in 36 samples of 6 species, what is more, DSP level in 10 samples exceeded the safety threshold. The levels in PSP and DSP of bivalves were all higher in spring and winter with some characteristic of season. CONCLUSION: The results suggested that PSP in economical shellfish in Guangzhou market was lower, and shellfish was safe to eat in term of the PSP level if glands were discarded, but DSP contamination in bivalves was severe. It is essential to detect and assess the risk of DSP and PSP in bivalves from seafood market in the future.

Animals↗

Contamination of shellfish from Shanghai seafood markets with paralytic shellfish poisoning and diarrhetic shellfish poisoning toxins determined by mouse bioassay and HPLC.

This paper reports the results of investigations of shellfish toxin contamination of products obtained from Shanghai seafood markets. From May to October 2003, 66 samples were collected from several major seafood markets. Paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) toxins in shellfish samples were monitored primarily by a mouse bioassay, then analysed by HPLC for the chemical contents of the toxins. According to the mouse bioassay, eight samples were detected to be contaminated by PSP toxins and seven samples were contaminated by DSP toxins. Subsequent HPLC analysis indicated that the concentrations of the PSP toxins ranged from 0.2 to 1.9 microg/100 g tissues and the main components were gonyautoxins 2/3 (GTX2/3). As for DSP, okadaic acid was detected in three samples, and its concentration ranged from 3.2 to 17.5 microg/100 g tissues. Beside okadaic acid, its analogues, dinophysistoxins (DTX1), were found in one sample. According to the results, gastropod (Neverita didyma) and scallop (Argopecten irradians) were more likely contaminated with PSP and DSP toxins, and most of the contaminated samples were collected from Tongchuan and Fuxi markets. In addition, the contaminated samples were always found in May, June and July. Therefore, consumers should be cautious about eating the potential toxic shellfish during this specific period.

Animals↗

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↗

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↗

Comparison evaluation of liquid chromatographic and bioassay methods of analysis for determination of paralytic shellfish poisons in shellfish tissues.

A liquid chromatographic (LC) method was compared with the AOAC mouse bioassay method (18.086-18.092) for determination of paralytic shellfish toxins in shellfish tissues. Shellfish samples were collected from Massachusetts coastal waters as part of a state surveillance program, and extracts of shellfish meat were analyzed for toxins by using both analytical methods. Overall correlation of the LC and bioassay methods is good (r = 0.943), but for samples with toxicities less than 100 micrograms saxitoxin/100 g shellfish meat, the correlation is significantly less (r = 0.531). Limits of detection are 10 micrograms saxitoxin/100 g shellfish meat and 40 micrograms saxitoxin/100 g shellfish meat for the LC and bioassay methods, respectively. Analytical capacity of the LC method is limited to 12 samples/person-day compared with 30 samples/person-day for the bioassay. Sampling capacity of the LC method could be increased by using a fluorescence detector with a wider response range, which would eliminate the need for dilution of concentrated samples.

Animals↗

Proficiency studies on the determination of paralytic shellfish poisoning toxins in shellfish.

Paralytic shellfish poisoning toxins are produced by dinoflagellates. Shellfish filtering these unicellular algae will accumulate the toxins and pose a health risk when consumed by man. In the European Union, paralytic shellfish poisoning toxins in bivalve molluscs are regulated at a maximum content of 80 microg/100 g (91/492/EEC). The current reference method in the European Union is the mouse bioassay, but alternative methods including the liquid chromatography methodology are preferred for ethical reasons. Analyses of suspected shellfish batches revealed, however, unacceptable differences in results reported by a small group of Dutch laboratories all using liquid chromatography methods with precolumn derivatization, followed by fluorescence detection. Therefore, a series of proficiency studies were undertaken among these laboratories. In the first three studies, participants were more or less allowed their own choice of method execution details. This approach yielded unsatisfactory results. A fourth study was then initiated in which a standardized method was mandatory. Two types of test material were used in the fourth study: lyophilized Cardium tuberculatum material containing saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX), and lyophilized mussel material containing dc-STX. The latter material was investigated in an interlaboratory study involving 15 participants and was considered as the reference material. Among the four laboratories, coefficients of variation (ANOVA) for C. tuberculatum material were 10% (n = 11) and 9% (n = 12) for STX and dc-STX, respectively, and for the reference material was 8% (n = 12) for dc-STX. The joint efforts showed that variability in analysis results between laboratories that all apply more or less the same method can be drastically improved if the methodology is rigorously standardized.

Chromatography, Liquid↗

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↗

[Progress in detecting methods on paralytic shellfish poisons].

Paralytic shellfish poisons have led to uncountable economic losses, and done potential harm to human health now. In order to overcome the limits of the traditional bioassay with mouse, many novel detecting methods have been developed, which made it possible to detect the paralytic shellfish poisons with sensitive, high effective and rapid methods. This paper systematically introduces the progress in research on paralytic shellfish poisons detection.

Biological Assay↗

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↗

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↗

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↗

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↗

Comparison of mouse bioassay and sodium channel cytotoxicity assay for detecting paralytic shellfish poisoning toxins in shellfish extracts.

A neuroblastoma cell culture assay was used to analyze shellfish extracts for presence of paralytic shellfish poisoning toxins (saxitoxins). Results were compared with mouse bioassays performed as part of a screening program for shellfish toxins in New Zealand. Twenty-nine samples gave negative results in both assays. Fifty-seven samples gave positive results in at least one assay. The correlation between the assays for saxitoxin equivalent levels in shellfish was 0.867. In spiking studies on shellfish extracts, the neuroblastoma assay showed a good response to added saxitoxin. Although these results support use of the neuroblastoma assay as a screening procedure for shellfish toxicity, results close to regulatory limits should be confirmed by mouse bioassay.

Animals↗

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↗

Commentary on AOAC method for paralytic shellfish poisoning.

Paralytic shellfish poisoning (PSP) is caused by ingesting bivalve molluscan shellfish which have fed on the toxigenic marine dinoflagellates Gonyaulax catanella or G. tamarensis. The toxins from these organisms are neurotoxic alkaloids which interfere with nerve conduction and block muscle-action potential. The U.S. Food and Drug Administration cooperates with state shellfish control officials in the National Shellfish Sanitation Program to prevent marketing of toxin-contaminated shellfish. The toxins are quantitated by the standard mouse bioassay method, as found in Official Methods of Analysis of the AOAC. This paper discusses the procedure followed in the standard bioassay method in an attempt to clarify for the PSP analyst the rather complex official methodology, and, thus, promote more uniform results among laboratories.

Animals↗

A fluorimetric microplate assay for detection and quantitation of toxins causing paralytic shellfish poisoning.

Paralytic shellfish poisoning is one of the most severe forms of food poisoning. The toxins responsible for this type of poisoning are metabolic products of dinoflagellates, which block neuronal transmission by binding to the voltage-gated Na(+) channel. Accumulation of paralytic toxins in shellfish is an unpredictable phenomenon that necessitates the implementation of a widespread and thorough monitoring program for mollusk toxicity. All of these programs require periodical collection and analysis of a wide range of shellfish. Therefore, development of accurate analytical protocols for the rapid determination of toxicity levels would streamline this process. Our laboratory has developed a fluorimetric microplate bioassay that rapidly and specifically determines the presence of paralytic shellfish toxins in many seafood samples. This method is based on the pharmacological activity of toxins and involves several steps: (i) Incubation of excitable cells in 96 well microtiter plates with the fluorescent dye, bis-oxonol, the distribution of which across the membrane is potential-dependent. (ii) Cell depolarization with veratridine, a sodium channel-activating toxin. (iii) Dose-dependent inhibition of depolarization with saxitoxin or natural samples containing paralytic shellfish toxins. Measuring toxin-induced changes in membrane potential allowed for quantification and estimation of the toxic potency of the samples. This new approach offers significant advantages over classical methods and can be easily automated.

Animals↗

A combination fluorescence assay and Folin-Ciocalteau phenol reagent assay for the detection of paralytic shellfish poisons.

Paralytic shellfish poison (PSP) profiles of crude shellfish extracts were determined by linear gradient elution from a Bio-Rad AG-50-X4 strong cation-exchange resin mini-column. STX, GTX2 and GTX3 were detected by fluorescence assay. NeoSTX and GTX1/GTX4 were detected with a Folin-Ciocalteau phenol reagent assay. The major toxicity associated with extracts of Mytilus edulis and Mya arenaria collected during a 1972 red tide off Hampton, New Hampshire, was due to the presence of GTX1/GTX4, with some activity associated with neoSTX, GTX2 and GTX3. STX was also present. Correlations to mouse toxicity are provided.

Amino Acids↗