| ObjectiveThe number of mushroom poisoning deaths accounted for 35.57% [1] of the total food poisoning deaths.Amanita species containing toxic oligopeptides,such as amatoxins and phallotoxins should be responsible for 90% deaths.Although Amanita toxins is a highly toxic,it is wildly used in medicine,biology and other fields because significant value.At present,the Amanita toxins can not yet be synthesized and only be extracted from the Amanita species,while majority of oligopeptides standards are still unavailable,especially for virotoxins in the domestic.A.subpallidorosea is one of the new lethal Amanita species that our group discovered in recent years contained variety and high content toxins.Therefore,we establish accurate and reliable method to identify and purity the toxins of A.subpallidorosea.Lethal Amanita species in China are mainly A.fuliginea,A.exitialis,A.rimosa and so on.They frequently cause poisonings by mistakenly due to their similar appearance.In consequence,we established the liquid fingerprints chromatographic of 12 Amanita species using prednisone as the internal standard in order to compare the relative content difference.In addition,the analysis by DNA molecular system showed that A.subpallidorosea and A.virosa were clustered on a branch and it implied they were closely related,as well as toxins analysis indicated that they contained the same virotoxins.Currently it is absence of systematic analysis for oligopeptides of A.subpallidorosea and A.virosa,as a result,we generated chromatography fingerprints of A.subpallidorosea and A.virosa which are collected from different regions to identify variety and compare the difference of the contents of toxins.In the treatment of Amanita poisonings,early diagnosis is significant for successfully rescue of patients of who eat Amanita species by mistake.As a result,we establish the qualitative and quantative method to analyse α-Amanitin(α-AMA),β-Amanitin(β-AMA),Phalloidin(PHD),Ala-viroidin and Viroidin in human plasma and urine.MethodsWith the continuous development of liquid chromatography tandem mass spectrum(LC-MS/MS),LC-MS/MS is an important analysis way for oligopeptide toxins,which has the advantages of less sample usaged,high sensitivity and short-time to analyse.In this paper,high-performance liquid chromatography(HPLC)and high-resolution mass spectrometry(HRMS)were used to isolate and identify the components of oligopeptide toxins from the new species of A.subpallidorosea collected from Zunyi and Guizhou Province.The samples were extracted with 50% methanol in water contained 0.5% formic acid using Agilent 300Extend-C18 column,eluting with ammonium acetate(20 mmol/L)aqueous solution contained 0.05% TFA-methanol as the mobile phase.On the basis of the above methods,the differences in toxin types and contents of A.virosa and A.subpallidorosea were further analyzed.We also compare the differences of types and content of cyclopetide in 12 lethal Amanita species.In this paper α-amanitin,β-amanitin,phalloidin,Ala-viroidin and Viroidin were measured by LC-MS/MS in human plasma and urine.Extraction using the solid phase extraction technique.We establish a high-throughput,fast,simple and high recovery rate of the sample purification method,and we has optimized and validated the analysis methods.ResultsThe results of A.subpallidorosea shows that 12 components were detected,including 9 identified Amanita toxic peptides(4 Amatoxins,2 Phallotoxins,3 Virotoxins),1 isoners of phalloidin and 2 new toxic peptides.It is worth mentioning that Ala-viroidin,Viroidin and Viroisin were simutaneous isolated and identified in the Amanita species for the first time,and then 9 milligram level of oligopeptide toxins were puritied by prepative HPLC which the purity were over 95%,The method not only provides new resources for the development and utilization of oligopeptide toxins but also supplies an effective technology for the rapid diagnosis in emergency of mushroom poisoning incidents.By comparing chromatography of 12 kinds of Amanita species,there were significant differences in the types and relative contents of toxins in Amanita species.which showed apparently chromatographic fingerprint information.19 compounds were identified;13 species were belonged to the Amanita toxins;5 species were unkownen and 1 species was an unknown small molecule compound,which was even not peptide toxin.The results showed that the species of toxins and the contents of α-Amanitin in A.subpallidorosea were all more than A.virosa,as well as there is no β-Amanitin in A.virosa of all regions.The compositions and contents of toxin existed in A.virosa were significantly different because of different producing area.In contrast,the compositions of toxin existed in A.subpallidorosea was no obvious difference except the content of toxin.This research estabilished a rapid and accurate method to analyse five oligopeptide toxins that included virotoxins.This method had satisfactory accuracy,high sensitivity and precision,recovery and the matrix effect by methodology validation.It provided technical support for emergency detection of mushroom poisoning in the clinical incidents.In conclusion,our research offered a scientific basis for correctly identifying species of Amanita,preventing and identifying poisoning incidents of Amanita and clinical diagnose and treatments of Amanita toxins.ConclusionsThe research established a method of analysis,preparation and identification of Amanita species,and the reference production of nine Amanita toxins were prepared with high purity,which is over 95%.We also found 2 new kinds of toxins,and it provided that the A.subpallidorosea is a new species containing the most abundant and the highest content.In addition,the liquid fingerprints chromatographic and the contents of Amanita toxins in Amanita species can compare similarity and difference in Amanita species.As well as,a simple,time-consuming and high recovery rate method of LC-MS/MS was compeleted,it can simultaneously quantitative various kinds of Amanita toxins in biological samples. |
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