scholarly journals Proficiency Testing of Eight French Laboratories in Using the AOAC Mouse Bioassay for Paralytic Shellfish Poisoning: Interlaboratory Collaborative Study

2000 ◽  
Vol 83 (2) ◽  
pp. 305-310 ◽  
Author(s):  
Martial LeDoux ◽  
Sherwood Hall ◽  
Madeleine Bohec ◽  
Bernard Charron ◽  
Magali Esnault ◽  
...  
Keyword(s):  
Water Solution ◽  
Collaborative Study ◽  
Paralytic Shellfish Poisoning ◽  
Mouse Bioassay ◽  
Shellfish Poisoning ◽  
Paralytic Shellfish ◽  
Relative Standard ◽  
Standard Solution ◽  
Overall Performance

Abstract In an interlaboratory study, 8 French laboratories were tested for their proficiency in using the AOAC mouse bioassay for paralytic shellfish poisoning (PSP). Each laboratory received 1 saxitoxin (STX) standard solution, 1 STX acidified water solution for determination of the titer, 1 noncontaminated shellfish sample, 1 naturally contaminated shellfish sample, and 2 shellfish samples spiked, respectively, at low (152.8 μg STX/100 g meat) and moderate (334.7 μg STX/100 g meat) levels. All samples were analyzed in duplicate. Mean recoveries were 35.1% for the low level and 46.6% for the moderate level. Relative standard deviations (RSD) for within-laboratory variations (repeatability) ranged from 5.4 to 9.8%; RSD for between-laboratory variations (reproducibility) varied from 7.8 to 39.6%, depending on STX level. On the basis of overall performance, all 8 participating laboratories were proficient in their use of the AOAC mouse bioassay.

Development and Application of Tandem LC-MS Method for the Determination of Paralytic Shellfish Poisoning Toxins for Pollution Monitoring

2011 ◽  
Vol 55-57 ◽  
pp. 51-56
Author(s):  
Xiao Ling Zhang ◽  
Bing Feng ◽  
You Qiong Cai ◽  
Hui Juan Yu
Keyword(s):  
Paralytic Shellfish Poisoning ◽  
Pollution Monitoring ◽  
Reaction Monitoring ◽  
Liquid Chromatography Mass Spectrometry ◽  
Shellfish Poisoning ◽  
Factors Affecting ◽  
Selective Determination ◽  
Paralytic Shellfish ◽  
Relative Standard

In the present study, the development of the tandem liquid chromatography-mass spectrometry (LC-MS) method for the sensitive and selective determination of eight paralytic shellfish poisoning (PSP) (GTX1, GTX2, GTX3, GTX4, GTX5, dcGTX2, dcGTX3 and dcNEO-b) toxins was investigated. Various experimental factors affecting the pretreatment and LC-MS procedures were optimzed, respectively. Using slected reaction monitoring (SRM) mode in LC-MS analysis, the acquired calibration curves were strictly linear with the value ofr2more than 0.996 for the eight targeted analyses. The obtained data expressed as relative standard deviations (RSDs), for the within-laboratory reproducibility and repeatability were less than 6.9% and 7.9%, respectively. The feasibility of the proposed method was finally validated by analyzing the eight PSPs in shellfish samples from diverse Chinese sea areas for the quality assurance of the aquatic producs and enviroment protection monitoring.

scholarly journals Quantitative Determination of Paralytic Shellfish Poisoning Toxins in Shellfish Using Prechromatographic Oxidation and Liquid Chromatography with Fluorescence Detection: Collaborative Study

2005 ◽  
Vol 88 (6) ◽  
pp. 1714-1732 ◽  
Author(s):  
James F Lawrence ◽  
Barbara Niedzwiadek ◽  
Cathie Menard ◽  
L Rojas de Astudillo ◽  
R Biré ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Fluorescence Detection ◽  
Collaborative Study ◽  
Paralytic Shellfish Poisoning ◽  
Shellfish Poisoning ◽  
Paralytic Shellfish ◽  
Study Results ◽  
Concentration Levels ◽  
Psp Toxins

Abstract 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.

Assessment of a Semiquantitative Liquid Chromatography- Fluorescence Detection Method for the Determination of Paralytic Shellfish Poisoning Toxin Levels in Bivalve Molluscs from Great Britain

2014 ◽  
Vol 97 (2) ◽  
pp. 492-497 ◽  
Author(s):  
Andrew D Turner ◽  
Monika Dhanji-Rapkova ◽  
Clothilde Baker ◽  
Myriam Algoet
Keyword(s):  
Great Britain ◽  
Fluorescence Detection ◽  
Detection Method ◽  
Reference Method ◽  
Paralytic Shellfish Poisoning ◽  
Official Method ◽  
Shellfish Poisoning ◽  
Bivalve Molluscs ◽  
Paralytic Shellfish

Abstract AOAC Official Method 2005.06 precolumn oxidation LC-fluorescence detection method has been used for many years for the detection and quantitation of paralytic shellfish poisoning (PSP) toxins in bivalve molluscs. After extensive single- and multiple-laboratory validation, the method has been slowly gaining acceptance worldwide as a useful and practical tool for official control testing. In Great Britain, the method has become routine since 2008, with no requirement since then for reverting back to the bioassay reference method. Although the method has been refined to be semiautomated, faster, and more reproducible, the quantitation step can be complex and time-consuming. An alternative approach was developed to utilize the qualitative screening results for generatinga semiquantitative results assessment. Data obtained over 5 years enabled the comparison of semiquantitative and fully quantitative PSP results in over 15 000 shellfish samples comprising eight different species showed that the semiquantitative approach resulted in over-estimated paralytic shellfish toxin levels by an average factor close to two in comparison with the fully quantified levels. No temporal trends were observed in the data or relating to species type, with the exception of surf clams. The comparison suggested a semiquantitative threshold of 800 μg saxitoxin (STX) eq/kg should provide a safe limitfor the determination of samples to be forwarded to full quantitation. However, the decision was taken to halve this limit to include an additional safety factor of 2, resulting in the use of a semiquantitative threshold of 400 μg STX eq/kg. Implementation of the semiquantitative method into routine testing would result in a significant reduction in the numbers of samples requiring quantitation and have a positive impact on the overall turnaround of reported PSP results. The refined method would be appropriate for any monitoring laboratory faced with high throughput requirements.

scholarly journals Immunoassay Methods for Paralytic Shellfish Poisoning Toxins

2001 ◽  
Vol 84 (5) ◽  
pp. 1649-1656 ◽  
Author(s):  
Ewald Usleber ◽  
Richard Dietrich ◽  
Christine Bürk ◽  
Elisabeth Schneider ◽  
Erwin Märtlbauer
Keyword(s):  
Test Procedure ◽  
Microtiter Plate ◽  
Paralytic Shellfish Poisoning ◽  
Current Status ◽  
Mouse Bioassay ◽  
Shellfish Poisoning ◽  
Sample Extract ◽  
Paralytic Shellfish ◽  
Psp Toxins ◽  
Immunochemical Techniques

Abstract The current status of immunochemical techniques for analysis of paralytic shellfish poisoning (PSP) toxins is summarized. Important aspects regarding production of the biological reagents necessary for immunochemical methods, the characteristics of polyclonal and monoclonal antibodies against saxitoxin and neosaxitoxin, and the importance of test sensitivity and specificity are discussed. Applications of immunochemical techniques for PSP toxins include microtiter plate enzyme immunoasays and enzyme-linked immunofiltration assays for toxin detection, and immunoaffinity chromatography (IAC) for sample extract cleanup. A major advantage of enzyme immunoassay (EIA) is simplicity and rapidity of the test procedure, and higher sensitivity than other methods. However, quantitative agreement between EIA and mouse bioassay is dependent on antibody specificity and the toxin profile in the shellfish; thus, both over- and underestimation of total toxicity may occur. For screening purposes, however, EIAs offer major advantages over the mouse bioassay, which is criticized in Europe because of animal welfare. A major application of antibodies against PSP toxins is their use for extract cleanup by IAC, which gives highly purified extracts, thereby enhancing determination of PSP toxins by conventional physicochemical methods such as liquid chromatography. IAC can also be used to isolate PSP toxins for preparation of analytical standard solutions.

scholarly journals Rapid Postcolumn Methodology for Determination of Paralytic Shellfish Toxins in Shellfish Tissue

2008 ◽  
Vol 91 (3) ◽  
pp. 589-597 ◽  
Author(s):  
Wade A Rourke ◽  
Cory J Murphy ◽  
Ginette Pitcher ◽  
Jeffery M van de Riet ◽  
B Garth Burns ◽  
...  
Keyword(s):  
Reversed Phase ◽  
Mouse Bioassay ◽  
Working Standard ◽  
Shellfish Toxins ◽  
Paralytic Shellfish Toxins ◽  
Paralytic Shellfish ◽  
Relative Standard ◽  
Phase Column ◽  
Reversed Phase Column

Abstract A rapid liquid chromatographic (LC) method with postcolumn oxidation and fluorescence detection (excitation 330 nm, emission 390 nm) for the determination of paralytic shellfish toxins (PSTs) in shellfish tissue has been developed. Extracts prepared for mouse bioassay (MBA) were treated with trichloroacetic acid to precipitate protein, centrifuged, and pH-adjusted for LC analysis. Saxitoxin (STX), neoSTX (NEO), decarbamoylSTX (dcSTX), and the gonyautoxins, GTX1, GTX2, GTX3, GTX4, GTX5, dcGTX2, and dcGTX3, were separated on a polar-linked alkyl reversed-phase column using a step gradient elution; the N-sulfocarbamoyl GTXs, C1, C2, C3, and C4, were determined on a C-8 reversed-phase column in the isocratic mode. Relative toxicities were used to determine STX-dihydrochloride salt (diHCl) equivalents (STXeq). Calibration graphs were linear for all toxins studied with STX showing a correlation coefficient of 0.999 and linearity between 0.18 and 5.9 ng STX-diHCl injected (equivalent to 3.9128 g STXeq/100 g in tissue). Detection limits for individual toxins ranged from 0.07 g STXeq/100 g for C1 and C3 to 4.1 g STXeq/100 g for GTX1. Spike recoveries ranged from 76 to 112 in mussel tissue. The relative standard deviation (RSD) of repeated injections of GTX and STX working standard solutions was <4. Uncertainty of measurement at a level of 195 g STXeq/100 g was 9, and within-laboratory reproducibility expressed as RSD was 4.6 using the same material. Repeatability of a 65 g STXeq/100 g sample was 3.0 RSD. Seventy-three samples were analyzed by the new postcolumn method and both AOAC Official Methods for PST determination: the MBA (y = 1.22x + 13.99, r2 = 0.86) and the precolumn LC oxidation method of Lawrence (y = 2.06x + 12.21, r2 = 0.82).

scholarly journals Assessment of MIST Alert™, a Commercial Qualitative Assay for Detection of Paralytic Shellfish Poisoning Toxins in Bivalve Molluscs

2002 ◽  
Vol 85 (3) ◽  
pp. 632-641 ◽  
Author(s):  
Fiona H Mackintosh ◽  
Susan Gallacher ◽  
Aileen M Shanks ◽  
Elizabeth A Smith
Keyword(s):  
Monitoring Program ◽  
Rapid Test ◽  
Paralytic Shellfish Poisoning ◽  
Ease Of Use ◽  
Mouse Bioassay ◽  
Shellfish Poisoning ◽  
Paralytic Shellfish ◽  
Test Kit ◽  
The Uk ◽  
Psp Toxins

Abstract A recently developed commercial rapid test kit (MIST Alert™) was assessed for determination of the presence of paralytic shellfish poisoning (PSP) toxins in shellfish. Several commercially important shellfish species obtained from the UK shellfish toxin monitoring program, containing a range of total PSP toxicities as determined by the mouse bioassay (MBA), were tested. The kit detected toxin in all samples containing the European Community tolerance level of 80 μg saxitoxin (STX) equivalents/100 g shellfish flesh as determined by the MBA. With one exception, the kit detected toxin in all samples that contained >40 μg STX equivalents/100 g according to the MBA. Among samples in which the MBA did not detect toxin, the kit disagreed in 25% of the tests, although further analysis by liquid chromatography (LC) and MBA of some samples confirmed the presence of toxins. These results suggest that MIST Alert may be suitable as an initial screen for PSP toxins as part of routine monitoring programs, thereby greatly reducing the number of MBAs. Trials were also performed by nonscientific personnel to evaluate the ease of use and interpretation of results obtained by MIST Alert. The results indicated that the kits could be readily used and accurately interpreted by individuals with no technical or scientific background.

Determination of Paralytic Shellfish Toxins in Shellfish by Receptor Binding Assay: Collaborative Study

2012 ◽  
Vol 95 (3) ◽  
pp. 795-812 ◽  
Author(s):  
Frances M Van Dolah ◽  
Spencer E Fire ◽  
Tod A Leighfield ◽  
Christina M Mikulski ◽  
Gregory J Doucette ◽  
...  
Keyword(s):  
East Coast ◽  
Collaborative Study ◽  
Hplc Method ◽  
Mouse Bioassay ◽  
Sea Scallop ◽  
Paralytic Shellfish Toxins ◽  
Paralytic Shellfish ◽  
Aoac Method ◽  
The U.S

Abstract A collaborative study was conducted on a microplate format receptor binding assay (RBA) for paralytic shellfish toxins (PST). The assay quantifies the composite PST toxicity in shellfish samples based on the ability of sample extracts to compete with 3H saxitoxin (STX) diHCl for binding to voltage- gated sodium channels in a rat brain membrane preparation. Quantification of binding can be carried out using either a microplate or traditional scintillation counter; both end points were included in this study. Nine laboratories from six countries completed the study. One laboratory analyzed the samples using the precolumn oxidation HPLC method (AOAC Method 2005.06) to determine the STX congener composition. Three laboratories performed the mouse bioassay (AOAC Method 959.08). The study focused on the ability of the assay to measure the PST toxicity of samples below, near, or slightly above the regulatory limit of 800 (μg STX diHCl equiv./kg). A total of 21 shellfish homogenates were extracted in 0.1 M HCl, and the extracts were analyzed by RBA in three assays on separate days. Samples included naturally contaminated shellfish samples of different species collected from several geographic regions, which contained varying STX congener profiles due to their exposure to different PST-producing dinoflagellate species or differences in toxin metabolism: blue mussel (Mytilus edulis) from the U.S. east and west coasts, California mussel (Mytilus californianus) from the U.S. west coast, chorito mussel (Mytilus chiliensis) from Chile, green mussel (Perna canaliculus) from New Zealand, Atlantic surf clam (Spisula solidissima) from the U.S. east coast, butter clam (Saxidomus gigantea) from the west coast of the United States, almeja clam (Venus antiqua) from Chile, and Atlantic sea scallop (Plactopecten magellanicus) from the U.S. east coast. All samples were provided as whole animal homogenates, except Atlantic sea scallop and green mussel, from which only the hepatopancreas was homogenized. Among the naturally contaminated samples, five were blind duplicates used for calculation of RSDr. The interlaboratory RSDR of the assay for 21 samples tested in nine laboratories was 33.1%, yielding a HorRat value of 2.0. Removal of results for one laboratory that reported systematically low values resulted in an average RSDR of 28.7% and average HorRat value of 1.8. Intralaboratory RSDr, based on five blind duplicate samples tested in separate assays, was 25.1%. RSDr obtained by individual laboratories ranged from 11.8 to 34.9%. Laboratories that are routine users of the assay performed better than nonroutine users, with an average RSDr of 17.1%. Recovery of STX from spiked shellfish homogenates was 88.1–93.3%. Correlation with the mouse bioassay yielded a slope of 1.64 and correlation coefficient (r2) of 0.84, while correlation with the precolumn oxidation HPLC method yielded a slope of 1.20 and an r2 of 0.92. When samples were sorted according to increasing toxin concentration (μg STX diHCl equiv./kg) as assessed by the mouse bioassay, the RBA returned no false negatives relative to the 800 μg STX diHCl equiv./kg regulatory limit for shellfish. Currently, no validated methods other than the mouse bioassay directly measure a composite toxic potency for PST in shellfish. The results of this interlaboratory study demonstrate that the RBA is suitable for the routine determination of PST in shellfish in appropriately equipped laboratories.

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