Determination of anatoxin-a and homoanatoxin in blue—green algal extracts by high-performance liquid chromatography and gas chromatography—mass spectrometry

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Abstract
The production of cyanobacterial toxins as anatoxin-a in the UK by blue—green algae such as Oscillatoria and Anabaena flos-aquae is a potential health hazard, especially to animals, birds and fish. A sensitive reversed-phase ion-pair high-performance liquid chromatographic (RP-HPLC) method is described for the measurement of the neurotoxins anatoxin-a (AnTx) and homoanatoxin (HomoAnTx) in the presence of lyophilized algal extract. A base-deactivated C18 column material was used with acetonitrile—phosphate buffer (pH 3) and sodium dodecyl sulfate as ion-pair reagent with ultraviolet detection at 227 nm. Linear calibrations were obtained between 2 and 93 ng on-column for AnTx·HCI and 2–112 ng on-column for HomoAnTx·HCI with limits of detection of 1 and 2 ng on-column, respectively. A sample of Oscillatoria bloom material collected from Loch Insh, Scotland, in 1991 was found by this method to contain approximately 0.8 mg of AnTx·HCl per gram of lyophilized extract. Homoanatoxin was not present. The identity of AnTx was confirmed by the isolation of the AnTx peak by RP-HPLC, its derivatization to N-butylAnTx and its analysis by RP-HPLC and gas chromatography—mass spectrometry.