Identification of Four Toxin Peptides from Amanita in Poisonous Mushroom by Mass Spectrum Fingerprint Based on Ultra Performance Liquid Chromatography Electrospray Ionization Quadrupole Time of Flight-Mass Spectrometry

Abstract

An entire method and a mass spectrum fingerprint for identification of α-amanitin,β-amanitin,phalloidin and phallacidin in mushrooms by ultra performance liquid chromatography(UPLC)-electrospray ionization(ESI) quadrupole time-of-flight(Q-TOF)-mass spectrometry(MS) have been established.A HSS T3 column was used for LC separation,which enabled the detection of the four mushroom toxins in mushroom samples.After homogenizing mushroom debris with methanol acidified with trifluoroacetic acid,the extract solution was subjected to solid phase extraction with an Oasis HLB cartridge.The eluate was analyzed by the UPLC-ESI-Q-TOF MS instrument.Identification for the four toxins was achieved by comparing to mass spectrum fingerprint between samples and standards.The results showed that the actual measured accurate mass spectral data of the protonated molecular peak and its isotope peaks for amatoxins and phallotoxins were very close to the theoretically calculated data.The mass spectra were also recorded by quadrupole-TOF MS2 for the standard compound using protonated molecular ion as precursor.The isotope peaks together with each accurate mass number of a protonated molecular ion and the intense product ions obtained by UPLC-TOF MS/MS2 enable the final identification of the four compounds.The calibration curves for the four mushroom toxins showed good linearities with correlation coefficients 0.9974-0.9985 in the range of 50-1000 μg/L.The LOD for each toxin was 10 μg/L and LOQ was 50 μg/L.The recovery rates of the four toxins were in the range of 68.2%-88.2% and RSD were 7.2%-11.8%.The result of identification for more than twenty wild mushroom samples from Dinghushan in Guangdong showed that five of them contained the toxins by comparing to standard mass spectrum fingerprint.The assay was found to be selective,reliable and accurate.The method is suitable for the identification of α-amanitin,β-amanitin,phallacidin and phalloidin in mushroom samples for poison purposes.