Analysis saxitoxin

Analyses for the Saxitoxins. Early methods for analysis of the saxitoxins evolved from those used for toxin isolation and purification. The principal landmarks in the development of preparative separation techniques for the saxitoxins were 1) the employment of carboxylate cation exchange resins by Schantz et al. (82) 2) the use of the polyacrylamide gel Bio-Gel P2 by Buckley and by Shimizu (5,78) and 3) the development by Buckley of an effective TLC system, including a new solvent mixture and a new visualization technique (83). The solvent mixture, designated by Buckley as "E", remains the best for general resolution of the saxitoxins. The visualization method, oxidation of the saxitoxins on silica gel TLC plates to fluorescent degradation products with hydrogen peroxide and heat, is an adaptation of the Bates and Rapoport fluorescence assay for saxitoxin in solution. Curiously, while peroxide oxidation in solution provides little or no response for the N-l-hydroxy saxitoxins, peroxide spray on TLC plates is a sensitive test for all saxitoxin derivatives with the C-12 gemdiol intact. 

This structure for saxitoxin (15) was presented at the first Conference on Poisonous Dinoflagellates at Boston, Mass, in November 1974 and published in the Journal of the American Chemical Society, March, 1975 (16). On the basis of this structure, Tanino et al. (17) with Kishi at Harvard University synthesized d,l-saxi-toxin and it was found active in blocking sodium channels in the same manner as natural saxitoxin. Later Bordner et al. (18) working with Rapoport confirmed our structure for saxitoxin with an X-ray analysis of saxitoxin ethyl hemiketal. 

When the homogenates of toxic scallops were incubated, other drastic changes in the toxin profile were observed (10) proportionally gonyautoxin-I - IV and neosaxitoxin decreased and saxitoxin increased. In another instance, the analysis of Mytilus exposed to the 1980 red tide at Sonoma County, California, showed the almost exclusive presence of neosaxitoxin in mussels collected just after the red tide and a gradual increase of saxitoxin (Krueger, Meyer and Shimizu, unpublished). These observations suggested the possible... 

L/mole). It cross-reacts at >90% with saxitoxin but at <1% with neosaxitoxin. This antibody, when used in an anti-rabbit IgG "second antibody" radioimmunoassay format, can detect pmole quantities of saxitoxin. This assay has been shown to be a simple and efficient method for the analysis of saxitoxin in clam extracts. The lack of antibody cross-reactivity to the neosaxitoxin sub-group of the paralytic shellfish poisons limits the general utility of the assay to neurophysiology studies and to certain clam species which preferentially accumulate saxitoxin. However, the radioimmunoassay serves as a good precursor in the development of an enzyme immunoassay for the paralytic shellfish poisons. 

This report describes the development of a radioimmunoassay (RIA) for one of the major PSP (saxitoxin, la) which is based on our successful production of antibodies to a stable saxitoxin derivative-bovine serum albumin conjugate. The application of the RIA to the analysis of PSP contaminated clam extracts and the problems which must be addressed in the development of a routine immunoassay for the PSP are discussed. 

Biomedical Sample Analysis. 291 11.3 Saxitoxin and Other Marine ... 

A major application of LC/ESI/MS is the characterization and detection of toxins, ranging from relatively small molecules, such as mycotoxins and some marine toxins, to the large proteinaceous toxins such as ricin and botulinum toxins. The marine toxin saxitoxin and the plant toxin ricin are specifically listed in Schedule 1 of the CWC as examples of toxins. A comprehensive review of LC/MS in toxin analysis would require a major chapter in its own right. Hancock and D Agostino 1711 reviewed approaches to the mass spectrometric identification of selected low molecular mass toxins. This chapter will describe examples of LC/MS in the analysis of marine, fungal, bacterial, and plant toxins, which are of possible relevance to the CWC. 

Saxitoxin (STX) is a potent neurotoxin that can cause paralytic shellfish poisoning (PSP). Produced by certain strains of dinoflagellates, saxitoxin leads to the contamination of commercial shellfish and cause severe outbreaks of seafood poisoning. The public health problems caused by these outbreaks have led to significant interest in the development of analytical methods for the analysis of saxitoxin in environmental and biological samples. Saxitoxin is also one of a series of several closely related... 

Saxitoxin has been labeled with fluorescamine, o-phthaldialdehyde (OPA) and dansyl chloride and detection limits as low as 0.1 attomole were reported for the OPA derivative of saxitoxin (26). Labeling, separation, and analysis of saxitoxin was best accomplished using fluorescamine, which produces ionic derivatives that can be separated from other fluorescently labeled marine toxins, such as tetrodotoxin and microcystin. However, the precolumn labeling methods required xM concentrations of analyte, limiting the utility of the technique for trace analysis. 

CE analysis with direct UV absorbance detection at 200 nm has been described for the separation and detection of underivatized toxins, including saxitoxin, associated with paralytic shellfish poisoning (27). Confirmation of the electrophoretic peaks was made by CE/ESI/MS. Saxitoxin and neosaxi-toxin (NEO) were separated using a 20 mM sodium citrate buffer at pH 2.1 yielding a mass LOD of 15 pg (5 xM) for saxitoxin. 

Saxitoxin, the paralytic shellfish poison, has finally revealed its structure as (87) by X-ray crystallographic analysis of its ethyl hemiketal " and p-bromobenzene-sulphonate derivatives. C n.m.r. studies show that saxitoxin exists primarily as its ketone hydrate in aqueous solution. ... 

The extreme toxic potential of marine metabolites often prevents their application in medicine. However, a number of metabolites proved to be valuable tools in biochemistry, cell and molecular biology. For instance the neurotoxic maitotoxin [109-112] (interaction with extracellular calcium enhancement of calcium influx [113]), the neurotoxic brevetoxin B [114] (interaction with the binding-site-5 of voltage-sensitive sodium channels [115]), tetrodotoxin and saxitoxin (voltage clamp analysis to study sodium channels and excitatory phenomena [116] tetrodotoxin abolishes brevetoxin B activity [117]), okadaic acid [118-120] (analysis of phosphorylation and dephosphorylation processes in eukaryotic cell metabolism [121]), and palytoxin (stimulation of arachidonic acid metabolism synergistically with TPA-type promoters [122]). 

HPLC is the most common chromatographic assay, predominantly utilizing fluorescence or mass spectrometric detection to measure presence of individual PSP toxins with a detection limit for saxitoxin of 20 fg per 100 g of tissue (0.2 ppm). For clinical purposes, serum and urine are the preferred matrices for analysis of saxitoxin. For postmortem analysis other organ tissues have been analyzed (e.g. heart, brain, liver, gastric, spleen). 

Advantages of using HPLC assays to measure saxitoxin include automated and continuous operation, increased sensitivity and increased precision. HPLC analysis of saxitoxin is limited in that it requires a skilled technician to operate the instmmentation, the analytical instmmentation is expensive, it requires extensive calibration and it is still not legally accepted for regulatory purposes. Advantages and limitations of LC-MS are similar to those... 

Micheli, L., Di Stefano, S. Moscone, D., Palleschi, G., Marini, S., Coletta, M., Draisci, R. and delli Quadri, F. Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis. Anal. Bioanal. Chem., JiTi, 678-684 (2002). 

In the above-mentioned work, DeU Aversano et al. [45] also carried out an exhaustive description about the fragmentation pattern of several saxitoxin analogs using tandem mass spectrometry, indicating that for some PSP toxins the mass spectra vary strongly with instrument type. Table 8.3 shows the typical fragment ions monitored for PSP toxin analysis by LC-MS. 

Inununoassay, based on antibodies against saxitoxin, is also used for detection of PSP toxins, and both radioinununoassay and ELISA have been employed for PSP toxins analysis. Although... 

Papageorgiou, J., et al.. Analysis of cyanobacterial-derived saxitoxins using high-performance ion exchange chromatography with chemical oxidation/fluorescence detection. Environ. Toxicol, 20,549, 2005. 

STX is now distributed worldwide by IAEA and is being used in collaborative trials of the PSTs receptor binding assay (RBA). Single laboratory validation of the RBA using new radiolabeled saxitoxins were presented at the Marine and Freshwater Toxins Analysis First Joint Symposium and AO AC Task Force Meeting in Baiona, Spain, in April 2005. The limit of quantitation of the microplate format assay " was found to be 1.2 pg STX equivalent/100 g shellfish (regulatory limit 80 pg/100 g), with an overall repeatability of 17.7% for shellfish extracts run by one analyst on 5 independent days, and a correlation r =. 98 with the mouse bioassay. 

Suarez-lsla, B.A., Cordova, M., and Fonseca, M. RBA. Evaluation of new radiolabeled saxitoxins and intra-laboratory comparison with mouse bioassay and electrophysiological assay for PSP. Marine and Freshwater Toxins Analysis 1st Joint Symposium and AOAC Task Force Meeting Baiona, Spain, April 2005. 

The blooms generally occur in spring, summer, and autumn and the cyanotoxins that were detected are anatoxin-a, saxitoxins, cylindrospermopsin, microcystins, and thionsulolipid. Also, paralytic shellfish poisons (PSPs) have been confirmed by mouse bioassay following analysis of... 

The same methods used for analysis of marine samples can be apply to saxitoxins originating from cyanobacteria. As already described, the most common methods to quantitate saxitoxins are based on LC with postcolumn derivatization and fluorimetric detection. 

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