The Development Of Analytical Methods For Amanita Cyclopeptide Toxins And The Study Of Their Distribution In Amanita Mushrooms

The death caused by mushroom poisoning accounts for 35.6%of the mortalities in food poisoning in China.The genus Amanita was responsible for 90%of fatalities,which produces the toxic cyclopeptides causing severe liver injury and death.The discovery of the poisonous mushroom and conformation of their toxins play crucial roles in searching the cause of the disease,which is also the basis of the treatment of patients and the health education.However,the cause of the intoxication can be found in a very few poisoning incidents.The reasons can be(1)the lacking of accurate and reliable analytical methods for the detection of peptide toxins in Amanita mushrooms,which lead to numbers of contradictory data from different laboratories,(2)the ignorance about the variation regularity about toxin distribution and composition in Amanitas,(3)unable to confirm the toxins in disease diagnosis without the fast,simple and high throughput methods for analysis of toxic cyclopeptides in human biofluids,(4)a large number of new compounds with high risk in Amanita mushrooms remaining to be discovered and studied.Therefore,we took the most fatal Amanita cyclopeptide toxins as the targets,and(1)developed a full validated analytical method for determination of the amatoxin and phallotoxin in mushrooms,which was applied to confirming the toxins in suspected mushrooms and analyzing the distribution of toxins in our research,(2)established a fast,simple and high throughput LC-MS/MS methods for analysis of amatoxins and phallotoxins in human biofluids designed for clinical emergency analysis in mushroom poisoning early diagnosis,(3)analyzed the effect of the Amanita species,tissues and development stages on the amounts,compositions and distribution of the amatoxin and phallotoxin,(4)based on the high resolution mass spectrometric technique,an untargeted screening method and a database were developed for the discovery of unknown toxins in our study.1.Simultaneous determination of amatoxin and phallotoxin in carpophores by UPLC-MS/MSIn the management of mushroom poisoning incidents and the treatment of patients,the detection and conformation of the toxins in mushroom were seldom performed.Besides,the current knowledge of the toxin composition and distribution in lethal amanitas remain limited and contradictory sometimes.Therefore,a full validated LC-MS/MS method with a "dilute and shoot" sample pretreatment tactics to simplify the procedures.The matrix effects was controlled effectively in this method and the validation data showed that our method was accurate,precise and stable,satisfactory in analysis of mushrooms with a large range of amounts.2.Simultaneous determination of amatoxin and phallotoxin in human biofluids by UPLC-MS/MSIn treatment of Amanita mushroom intoxication,an early and definite diagnosis is necessary for a successful outcome.For this purpose,a simple,rapid,high throughput and sensitive UPLC-MS/MS method for simultaneous determination of amatoxin and phallotoxin in human plasma,serum and urine was developed and validated.The biofluids were purified with a novel PRiME technique on a 96 well?Elution plate platform,which allowed high-throughput sample processing and low amounts of reagent consuming.This method fulfilled the requirement of validation test with good sensitivity,accuracy,precision and matrix effects,and suitable for toxicological and clinical emergency analysis.All of the analytes were analyzed in authentic blood and urine samples obtained from intoxication cases from Yunnan province in 2015.Two of the cases turned to be positive.This definite result provided a solid conformation of the mushroom poisoning.3.The distribution of amatoxin and phallotoxin in Amanita carpophoresTo study the compositions and distribution of major amatoxins and phallotoxins in Amanitas,we collected Amanita mushrooms in 2012-2016 from China and analyzed all the mushroom samples with our LC-MS/MS method.The result indicated the great diversity in compositions and contents of cyclopeptides among Amanita species.For Amanita subpallidorosea,a new lethal Amanita mushroom we identified and published for the first time,the composition and distribution of toxins in different tissues and development stages were systematically analyzed.The concentrations of amatoxins and phallotoxins turned out to be markedly varied in different tissues and development stages which revealed the biosynthesis of toxins in Amanita mushrooms is a dynamic process.This insight of the variation regularity enriched knowledge of the Amanitas and their peptide toxins.4.The establishment of a new untargeted screening method and database based on high resolution mass spectrometryIn our study,a new non-targeted strategy based on liquid chromatography-high resolution mass spectrometer(LC-HRMS)was applied to analyze the toxin profiling of the lethal mushrooms and to find new cyclopeptide toxins.With the LC-HRMS based strategy via parallel reaction monitoring(PRM)and all ion fragmentation(AIF)mode,a toxin screening method was developed and an ion fragments database has been formed.Appling this method,11 high risk cyclopeptide toxins were found and 4 of them were identified as amaninamide,phallisacin,Alal-viroidin and viroidin from six lethal amanita samples.Seven of them were discovered as new compounds.This discovery can be the foundation of the researches on toxicity and the biosynthesis pathway of Amanitas with further study on extraction and confirmation of the new toxins.Besides,the ion fragments database was established for peptide toxin screening with an open and extensible mode of the database workflow,the new compounds will be accumulated remarkably.Virotoxin used to be assumed as a class of exclusive toxins in Amanita virosa.However,we found two virotoxins in Amanita subpallidorosea for the first time with much higher contents.This new discovery provided a new clue for the study on phylogenetic relationship between Amanita virosa and Amanita subpallidorosea.