Determination of alpha-, beta-, and gamma-amanitin by high performance thin-layer chromatography in Amanita phalloides (Vaill. ex Fr.) secr. from various origin

A fast, sensitive high performance thin-layer chromatographic method for the determination of alpha-, beta-, and gamma-amanitin in crude, methanolic extracts of Amanita phalloides is described. The limit of detection is 50 ng of each amanitin. With this method amanitin was determined in 24 pooled samples of Amanita phalloides, collected between 1970 and 1977 in Germany and Switzerland. The total amanitin content varied between 2010 and 7300 mg/kg dry weight and the average value was 4430 mg/kg of which 43% was alpha-amanitin, 49% beta-amanitin and 8% gamma-amanitin. The origin of the fungi hardly influenced their amanitin content: in samples collected during the same year at different sites it fluctuated within a factor of 1.7. The amanitin content of samples from the same site, but collected in different years, maximally varied within a factor of 3.7. The partial decomposition of amanitins during prolonged storage of the lyophilized samples undoubtedly contributed to this variation. Phalloidin, which was determined by conventional thin-layer-chromatography, could not be detected in a sample from 1970, whereas its concentration in material collected during 1977 amounted to 2400 mg/kg dry weight. The toxicity of the samples (LD50 of lyophilized defatted methanolic extracts intravenously for mice) varied within a factor of 2.5.