Identification and quantification of α- and β-amanitin in wild mushrooms by HPLC-UV-EC and HPLC-DAD-MS detection

Amatoxins are a group of highly toxic peptides, which include - and {beta}-amanitin, found in several species of mushrooms (e.g. Amanita phalloides). Due to their high hepatotoxicity, they account for most deaths occurring after mushrooms ingestion. The determination of - and {beta}- amanitin content in wild mushrooms is invaluable for treating cases involving poisoning. In the present study, we have developed and validated an analytical method based on high-performance liquid chromatography, with in-line ultraviolet and electrochemical detection (HPLC-UV-EC), for the rapid quantification of - and {beta}-amanitin in wild mushroom samples collected from the Inner Center of Portugal. A reproducible and simple solid-phase extraction (SPE) using OASIS(R) PRIME HLB cartridges was used for sample pre-treatment, followed by chromatographic separation based on the RP-C18 column. The UV and EC chromatograms of - and {beta}-amanitin were recorded at 305 nm and +0.600 V vs. Ag/AgCl, respectively. The linear quantification for both amanitins was in the range of 0.5-20.0 g{middle dot}mL-1 (R2 > 0.999). The LOD, calculated based on the calibration curve, was similar for UV and EC detection (0.12-0.33 g ml.-1). Intra-day and inter-day precision were less than 13%, and the recovery ratios ranged from 89% to 117%. Nine Amanita species and five edible mushrooms were analysed by HPLC-UV-EC, and HPLC-DAD-MS confirmed the identification of amatoxins. We find high - and {beta}-amanitin content in A. phalloides and not in the other species analysed. In sum, the developed and validated method provides a simple and fast analysis of - and {beta}-amanitins contents in wild mushrooms and is suitable for screening and routine assessment of mushroom intoxication. HighlightsO_LINew validated method using HPLC-UV-EC to determine - and {beta}-amanitin in wild mushrooms. C_LIO_LIReproducible and fast SPE procedure for small samples. C_LIO_LIEffective sample pre-treatment with the OASIS(R) PRIME HLB SPE cartridge. C_LIO_LIIdentification and quantification of - and {beta}-amanitin in wild mushroom samples from Portugal. C_LIO_LIHPLC-DAD-MS confirmation of amatoxins present in mushroom samples. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/483521v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@734f7aorg.highwire.dtl.DTLVardef@66d9fforg.highwire.dtl.DTLVardef@720f39org.highwire.dtl.DTLVardef@458e64_HPS_FORMAT_FIGEXP M_FIG C_FIG