Anatoxins analytical methods

The synthetic aspects of anatoxin-a (AN) and analogues are discussed in a separate chapter of this text, and the analytical methods for anatoxins are the subject of a separate review to be published elsewhere. A number of reviews have demonstrated the importance of understanding toxic cyanobacteria as potential environmental and health hazards as well as a resource of bioactive molecules (Harada 1999 Skulberg 2000 Briand et al. 2003). AN was one of the first cyanobacterial toxins to be chemically and functionally characterized and its high neurotoxicity has attracted extensive research activity. 

Microcystis, Nostoc, and Oscillatoria (Planktothrix). Cyanobacteria toxins (cyanotoxins) include cytotoxins and biotoxins (neurotoxins anatoxin-a, anatoxin-a(s) and saxitoxins, and the hepatotoxins microcystins MCs, and nodularins), with biotoxins being responsible for acute lethal, acute chronic, and subchronic poisonings of wild/domestic animals and humans. In most of the reported cases, afflicted animals consumed water from water bodies where there was an obvious presence of cyano-bacterial scum on the water surface. More recent measurements of cyanobacterial toxins using sensitive modem analytical methods have often revealed high frequencies of toxic blooms even when animal poisonings have not been reported. 

The development of analytical methods for anatoxin-a has been facilitated by the fact that the structure of this toxin was already solved in the late 1970s and chemical synthesis yielded the pure... 

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... 

Anatoxin-a(S) can therefore be measured by its inhibition of acetylcholinesterase whose enzyme activity can be measured by several ways. One example is its degradation of the acetylcholine analog, acetylthiocholine, and subsequent measurement of the released thiocholine by the sulfur reacting chemical Ellman s reagent. Acetylcholinesterase has been cloned and its mutation can increase the enzyme s sensitivity for anatoxin-a(S). Combining different acetylcholinesterase mutants with divergent specificity for anatoxin-a(S) and the above-mentioned organophosphate insecticides has enabled better analyte discrimination. This multiple enzyme method has been implemented in a biosensor that carries several of the acetylcholinesterase mutants. 

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