Analysis anatoxin

HPLC analysis of anatoxin-a was first carried out by Astrachan and Archer, " who extracted the toxin from Anabaenaflos-aquae using chloroform followed by hydrochloric acid. The HPLC analysis was carried out on an ODS column using hypochlorate-methanol. Other systems used since include acetic acid extraction and analysis on a reversed-phase C g column using methanol-water mobile phase, and extraction in water after ultrasonication and analysis on reversed-phase... 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

The developed biosensor was applied to the analysis of cyanobacterial bloom samples from freshwater lakes of Spain, Greece, France, Scotland and Denmark. Two samples from Scotland and one from Denmark irreversibly inhibit the acetylcholinesterase. The estimated concentrations were between 1.5 and 30nmol/g of dry weight, values extremely high when compared to the intraperitoneal 50% lethal dose of anatoxin-a(s) in mice (121 nmol/kg). 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

Anatoxin-a is a toxic alkaloid occurring in the filamentous blue-green alga Anabaena flos-aquae and has been responsible for fatalities amongst livestock and wildlife. Mass and n.m.r. spectroscopy, and X-ray diffraction analysis of its N-acetyl derivative, pointed to structure and absolute stereochemistry (12) for ana-... 

Despite the ease with which AN and HMAN can rapidly degrade, it is surprising that there have been few reports of the presence of these degradation products, and this may be a consequence of both the lack of commercially available standards and the fact that most studies of anatoxins relied on the use of LC with UV detection for analysis. The degradation products are not detectable using this method as they do not possess the unsaturated ketone chromophore. 

Harada, K., Kimura, I., Ogawa, K., Suzuki, M., Dahlem, A.M., Beasley, VR., and Carmichael, W.W. 1989. A new procedure for the analysis and purification of naturally occurring anatoxin-a from the blue-green a ga Anabaena flos-aquae. Toxicon 27, 1289-1296. 

Smith, R.A., and Lewis, D. 1987. A rapid analysis of water for anatoxin-a, the imstable toxic alkaloid from Anabaena flos-aquae, the stable non-toxic alkaloids left after bioreduction and a related amine which may be nature s precursor to... 

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... 

Cyanobacterial Neurotoxins, Anatoxin-A and Analogues Detection and Analysis... 

The present authors have recently pubhshed two reviews, one on the synthetic approaches to anatoxins (Armesto et al 2006) and another on the discovery, distribution and toxicology of these neurotoxins (James et al 2006). The main focus of this review is therefore confined to the methods used for the detection and analysis of anatoxins from cyanobacteria. 

FIGURE 38.3 Total ion chromatogram (TIC) and MS spectrum from the analysis of a cyanobacteria sample (Nostoc carneum) using SPME-GC-MS. The peak in 11.26 min is related to anatoxin-a (Reprinted from Ghassempour, A., Najafi, N. M., Mehdinia, A., Davarani, S. S. H., Fallahi, M., and Nakhshab, M, J. Chro-matogr. A 1078, 120-127, 2005. With permission. Copyright (2005) Elsevier.)... 

Liquid chromatography with ultraviolet detection (LC-UV) is often used for the analysis of fresh-waters suspected to contain anatoxins. Indeed, both AN and HMAN have a strong absorbance at 227... 

FIGURE 38.6 Examples of the application of the SPME method using PDMS/DVB fibre with on-fiber derivatization for the LC-FLD analysis of anatoxin-a, (a) a river water sample spiked with 1000 ng of anatoxin-a (b) a cyanobacteria sample naturally contaminated with anatoxin-a (Reprinted from Rellan, S., Osswald, J., Vasconcelos, V., and Gago-Martinez, A, J. Chromatogr. A 1156,134-140, 2007. With permission. Copyright (2007) Elsevier). 

Dagnino, D., and Schripsema, J. (2005). H-1 NMR quantification in very dilute toxin solutions application to anatoxin-a analysis. Toxicon 46, 236-240. 

Powell, M. W. (1997). Analysis of anatoxin-a in aqueous samples. Chromatographia, 4525 528. 

Takino, M., Daishima, S., and Yamaguchi, K. (1999). Analysis of anatoxin-a in freshwaters by automated online derivatization-liquid chromatography-electrospray mass spectrometry. J. Chromatogr. 862, 191-197. 

G. Vasas, A. Gdspdr, C. Pdger, G Snranyi, C. Mathe, M.M. Hamvas and G. Borbely, Analysis of cyanobacterial toxins (anatoxin-a, cyhndrospermopsin, microcystin-LR) by capillary electrophoresis. Electrophoresis, 25,108-115, 2004. 

Anatoxin A is the fast-acting and highly effective poison of the cyanobacterium Anabaenaflos-aquae, which is ubiquitous in freshwater. Anatoxin A, also known as Very Fast Death Factor , was isolated from Anabaenaflos-aquae in 1977 by Paul Gorham at the National Research CoimcU in Ottawa. [553,554] The structure had already been determined in 1972 by X-ray analysis of its N-acetyl derivative. [555] Later, the presence of anatoxin A was detected in a range of other toxic strains of Oscillatoria, Anabaena circinalis, Aphanizomenonflos-aquae, Cylindorsperum pp. and Raphidiopsis mediterranea. 

Figure 2 An example of the analysis of a water-borne toxin combining several of the procedures outlined in the text. Analysis of culture medium of the freshwater cyanobacterium Anabaena sp. 86 by LC-photodiode array detection following a concentration step on SDB SPE cartridges (inset UV spectrum of anatoxin-a). Experimental conditions CIS column mobile phase acetonitrile (2.5%)/potassium phosphate 5 mmol I pH 3.5 (97.5%).

Because the presence of anatoxin-a and homoanatoxin-a in the environment represents a risk for animals and humans, several analytical methods have been designed to detect these toxins as well as their natural derivatives (Fig. 3.2). GC-MS was first used to detect and quantify anatoxin-a or its A -acetyl derivative [51,52,66]. High Pressure Liquid chromatography (HPLQ coupled to UV detection was also used, although this detectirm method is not veiy sensitive [67]. Thus, to improve the sensitivity, several authors have used pre-derivatization with a fluorophore, which reacts on the amine of anatoxin-a or of homoanatoxin-a, followed by separation by HPLC coupled to a fluorescence detector [56, 68, 69]. However, the derivatization might lead to false positives even if the technique was improved to remove primary amines present in the sample [70] or by crmcentratirm by extraction of anatoxin-a prior to analysis [71,72]. It is now accepted that the best analytical technique relies on the use of HPLC coupled to tandem mass spectrometry (LC-MS, or even LC-MS") without derivatization to avoid... 

LC-MS 50% acetic acid in methanol (cyanobacteria) No derivatization 0.7 ng 1.15 ng Very high sensitivity. Analysis of anatoxin-a and derivatives. [80]... 

James KJ, Sherlock IR (1996) Determination of the cyanobacterial neurotoxin, anatoxin-a, by derivatisation using 7-fluoro-4-nitro-2,l,3-benzoxadiazole (NBD-F) and HPLC analysis with fluorimetric detection. Biomed Chromatogr 10 46-47. doi 10.1002/(SICI)1099-0801(199601)10 l<46 AID-BMC551>3.0.CO 2-7 [pii]... 

Takino M, Daishima S, Yamaguchi K (1999) Analysis of anatoxin-a in fi-eshwaters by automated on-line derivatization-liquid chromatography-electrospray mass spectrometry. J Chromatogr A 862 191-197. doi 10.1016/s0021-9673(99)00943-7... 

Namera A, So A, Pawliszyn J (2002) Analysis of anatoxin-a in aqueous samples by solid-phase microextraction coupled to high-performance liquid chromatography with fluorescence defection and on-fiber derivatization. J Chromatogr A 963 295-302. doi 10.1016/ s0021-9673(02)00648-9... 

Rellan S, Gago-Martinez A (2007) Improved conditions for the application of solid phase microextraction prior to HPLC-FLD analysis of anatoxin-a. J Sep Sci 30 2522-2528. doi 10.1002/jssc.200700103... 

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