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.

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.

More than 100 samples of blue-green algae products (consisting of Aphanizomenon, Spirulina, and unidentified blue-green algae) in the form of pills, capsules, and powders were collected from retail outlets from across Canada. The samples were extracted with 75% methanol in water and centrifuged to remove solids. Aliquots of the extracts along with spiked blank sample extracts were sent to each participating laboratory and independently analyzed for microcystins by enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay, and by liquid chromatography–tandem mass spectrometry (LC–MS/MS) after sample cleanup using C18 solid–phase extraction. The results obtained by ELISA and LC–MS/MS agreed very well over a concentration range of about 0.5–35 μg/g. The colorimetric phosphatase results generally agreed with the other 2 methods. While the 2 biochemical assays measured total microcystin content compared with a standard of microcystin LR, the LC–MS/MS method measured specific microcystins (LA, LR, RR, YR) using external standards of these for identification and quantitation. Microcystin LR was found in all positive samples by LC–MS/MS. Microcystin LA was the only other microcystin found in the samples analyzed. These 2 microcystins represent essentially all the microcystins that were present in the extracts. Otherwise, the LC–MS/MS results would have been significantly lower than the results of the biochemical assays had other unknown microcystins been present.

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 s ample 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 μg/g (2 μg/100 g) of tissue while the hydroxylated toxins could be quantitated at concentrations as low as about 0.1 μg/g (10 μg/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.

Beta-N-Methylamino-L-alanine (BMAA) is a neurotoxin originally found in cycad seeds and now known to be produced by many species of freshwater and marine cyanobacteria. We developed a method for its determination in blue-green algae (BGA) food supplements, freshwater fish, and bottled water by using a strong cation-exchange, solid-phase extraction column for cleanup after 0.3 M trichloroacetic acid extraction of BGA supplements and fish. Bottled water was applied directly onto the solid-phase extraction column. For analysis of carbonated water, sonication and pH adjustment to 1.5 were needed. To determine protein-bound BMAA, the protein pellet left after extraction of the BGA supplement and fish was hydrolyzed by boiling with 6 M hydrochloric acid; BMAA was cleaned up on a C18 column and a strong cation-exchange, solid-phase extraction column. Determination of BMAA was by liquid chromatography of the fluorescent derivative formed with 9-fluorenylmethyl chloroformate. The method was validated by recovery experiments using spiking levels of 1.0 to 10 microg/g for BGA supplements, 0.5 to 5.0 microg/g for fish, and 0.002 microg/g for bottled water; mean recoveries were in the range of 67 to 89% for BGA supplements and fish, and 59 to 92% for bottled water. Recoveries of BMAA from spiked extracts of hydrolyzed protein from BGA supplements and fish ranged from 66 to 83%. The cleanup developed provides a useful method for surveying foods and supplements for BMAA and protein-bound BMAA.