超高效液相色谱-电喷雾离子化-四级杆飞行时间串联质谱指纹图谱检测毒蕈中4种鹅膏肽类毒素

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

建立了基于超高效液相色谱-电喷雾离子化-四级杆飞行时间串联质谱技术(UPLC-ESI-Q-TOF)的4种鹅膏肽类毒素特征信息的色谱和质谱指纹图谱和分析鉴定方法。样品用含0.1%TFA的甲醇溶液提取后,再用Oasis HLB固相柱净化,洗脱液经UPLC HSS T3色谱柱分离后,采用ESI-TOF MS和ESI-Q-TOF MS2进行测定;建立了包含毒素准分子离子准确质量数及其同位素特征,以及二级质谱特征子离子信息的指纹图谱;方法定量测定的线性范围在50～1000μg/L,相关系数在0.9974～0.9985之间;4种毒素的检出限均为10μg/L;定量限为50μg/L;加标回收率在68.2%～88.2%之间;相对标准偏差为7.2%～11.8%。对采集的20余种野生菌样品进行分析,有5种样品检测出含有毒素。本方法快速、准确、选择性好、特异性强,适用于突发毒蕈中毒事件样品中α-鹅膏毒肽、β-鹅膏毒肽、二羟鬼笔毒肽、羧基二羟鬼笔毒肽的确证分析。

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.