Barbara Niedzwiadek

Paralytic shellfish poisoning (PSP) toxin binders for optical biosensor technology: problems and possibilities for the future: a review

This review examines the developments in optical biosensor technology, which uses the phenomenon of surface plasmon resonance, for the detection of paralytic shellfish poisoning (PSP) toxins. Optical biosensor technology measures the competitive biomolecular interaction of a specific biological recognition element or binder with a target toxin immobilised onto a sensor chip surface against toxin in a sample. Different binders such as receptors and antibodies previously employed in functional and immunological assays have been assessed. Highlighted are the difficulties in detecting this range of low molecular weight toxins, with analogues differing at four chemical substitution sites, using a single binder. The complications that arise with the toxicity factors of each toxin relative to the parent compound, saxitoxin, for the measurement of total toxicity relative to the mouse bioassay are also considered. For antibodies, the cross-reactivity profile does not always correlate to toxic potency, but rather to the toxin structure to which it was produced. Restrictions and availability of the toxins makes alternative chemical strategies for the synthesis of protein conjugate derivatives for antibody production a difficult task. However, when two antibodies with different cross-reactivity profiles are employed, with a toxin chip surface generic to both antibodies, it was demonstrated that the cross-reactivity profile of each could be combined into a single-assay format. Difficulties with receptors for optical biosensor analysis of low molecular weight compounds are discussed, as are the potential of alternative non-antibody-based binders for future assay development in this area.

Liquid chromatographic determination of the cyanobacterial toxin β-N-methylamino-L-alanine in algae food supplements, freshwater fish, and bottled water.

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.

Effect of pH on the oxidation of paralytic shellfish poisoning toxins for analysis by liquid chromatography

The effect of pH on the oxidation of individual PSP toxins using both periodate and peroxide oxidations was studied. It was found that the optimum pH for individual toxins varied considerably. For periodate oxidations, pH 8.2 produced the maximum yield of fluorescent products for neosaxitoxin and GTX1/GTX4 while the non-hydroxylated toxins (saxitoxin, GTX2/GTX3, decarbamoyl saxitoxin, GTX5) showed optimum pHs from about pH 10-11.5. Neosaxitoxin and GTX1/GTX4 did not produce significant fluorescent oxidation products with peroxide oxidation at any of the pHs studied (pH 8.2-12.8). The non-hydroxylated toxins all showed optimum pHs above pH 12 with peroxide oxidation. Yields of fluorescent products of these toxins decreased substantially at pHs below pH 12. Neosaxitoxin and GTX1/GTX4 each produced three product peaks at pH 8.2 with periodate oxidation. There was no pH where these toxins produced predominantly a single oxidation product. Decarbamoyl saxitoxin always produced two oxidation products with both oxidation reactions at the pHs studied. However, the relative yields of the products changed with pH. At low pH the second eluting product predominated, while at higher pH values the first eluting product predominated. This pattern was observed for both oxidation reactions. The other non-hydroxylated toxins produced mainly single unique products with both oxidation reactions over the pH range studied. No single pH was found optimum for the oxidation of both hydroxylated and non-hydroxylated toxins without a significant compromise in yield of oxidation products. This has implications for the post column oxidation liquid chromatographic methods, since small changes in pH of the post column oxidant can both positively and negatively affect the yields of oxidation products of toxin mixtures leading to increased error in the subsequent quantitation of these compounds.

Evaluation of surface plasmon resonance relative to high pressure liquid chromatography for the determination of paralytic shellfish toxins

A surface plasmon resonance (SPR) method, incorporating monoclonal and polyclonal antibodies, was compared to HPLC fluorescence for the determination of paralytic shellfish toxins (PSTs) in shellfish collected from different regions of Canada (n = 33) and Europe (n = 55). Cross-reactivity between saxitoxin (STX) and its structural analogues was determined for both monoclonal (GT-13A) and polyclonal (R895) antibodies. Method detection limits based on IC(10) values, using the SPR methodology (0.55-71.3 ng/mL), in particular for GT-13A, were somewhat higher than those determined using HPLC (0.16-1.29 ng/mL). SPR analyses generally resulted in higher PST levels relative to those obtained using HPLC, although neither antibody successfully responded to the N-1-hydroxylated analogues (e.g., neosaxitoxin). Five and 10 (R895 and GT-13A, respectively) of the 88 samples tested resulted in PST concentrations above the regulatory limit (80 microg/100 g shellfish tissue as STX equivalents), although HPLC responses indicated that these samples were within acceptable levels. Two and five samples were found to have PST concentrations below the regulatory limit using the GT-13A and R895, respectively, when HPLC results exceeded the limit. SPR may be applicable as a screening technique, although improved antibody response to the N-1-hydroxylated PSTs is required prior to this method being safely used for routine testing.

Anatoxin-a and its metabolites in blue-green algae food supplements from Canada and Portugal.

Blue-green algae and spirulina are marketed in health food stores and over the Internet as food supplements in Canada, the United States, and Europe. The reported benefits of consuming these products include improved digestion, strengthening of the immune system, and relief from the symptoms of attention deficit disorder. Some of these products have been found to contain elevated concentrations of microcystins, which are known hepatotoxins. In addition to producing microcystins, Anabaena sp. and Aphanizomenon sp. also produce the potent neurotoxin anatoxin-a. Samples of food supplements containing blue-green algae and spirulina were collected in Portugal and from urban centers across Canada in 2005. Extracts of these supplements were analyzed to determine the presence and concentrations of anatoxin-a and its two main metabolites, dihydroanatoxin-a and epoxyanatoxin-a. Initial analyses were performed using high-performance liquid chromatography (HPLC) with fluorescence detection, and confirmation required the use of LC with tandem mass spectrometry (LC-MS-MS). The HPLC with fluorescence detection indicated no anatoxin-a, but four samples were suspected to contain either dihydroanatoxin-a or epoxyanatoxin-a at 0.1 to 0.2 microg/g. LC-MS-MS results, however, indicated no trace of either transformation product in any sample analyzed. The detection limits for anatoxin-a, dihydroanatoxin-a, and epoxyanatoxin-a were similar for both fluorescence detection (0.2 to 0.3, 0.4 to 1.4, and 0.2 to 1.5 pg on the column, respectively) and mass spectrometry (0.3 to 1.5, 0.3 to 0.8, and 0.5 to 0.8 pg on the column, respectively). Because of the higher specificity of the LC-MS-MS analysis, all tested food supplement samples were considered free of anatoxin-a and its transformation products.

Monitoring of shrimp and farmed fish sold in Canada for cyanobacterial toxins.

Sixty-one samples of shrimp and 32 samples of farmed fish collected from retail markets across Canada were analyzed for cyanobacterial toxins, including microcystins, paralytic shellfish poisons (saxitoxins), cylindrospermopsin, and β-N-methylamino-L-alanine, using established methods of analysis. None of these toxins were detected in any of the samples. Some shrimp samples screened for paralytic shellfish poisons showed the presence of unknown peaks in the chromatogram after periodate oxidation.