高效液相色谱⁃串联质谱法测定野生蘑菇中鹅膏肽类与色胺类毒素  

Determination of amanita peptide and tryptamine toxins in wild mushrooms by high performance liquid chromatography⁃tandem mass spectrometry

作　　者：刘磊琦 陈镜泽 傅武胜 唐翠英[2,3] LIU Leiqi;CHEN Jingze;FU Wusheng;TANG Cuiying(School of Public Health,Fujian Medical University,Fuzhou 350122,China;Fujian Provincial Key Laboratory for Zoonosis Research,Fujian Center for Disease Control and Prevention,Fuzhou 350012,China;School of Pharmacy,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;Food Science College of Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建医科大学公共卫生学院,福建福州350122 [2]福建省人兽共患病研究重点实验室,福建省疾病预防控制中心,福建福州350012 [3]福建中医药大学药学院,福建福州350122 [4]福建农林大学食品科学学院,福建福州350002

出　　处：《色谱》2023年第11期976-985,共10页Chinese Journal of Chromatography

基　　金：福建省科技厅社会发展引导性(重点)项目(2018Y0007)。

摘　　要：蘑菇毒素种类繁多,化学结构差异大,为了实现蘑菇中毒素的准确、高通量分析,本文采用固相萃取净化技术,以高效液相色谱⁃串联质谱法(HPLC⁃MS/MS)为分析手段,建立了蘑菇中5种鹅膏肽类、2种色胺类蘑菇毒素的分析方法。优化了色谱条件、质谱参数和样品提取净化方法,蘑菇干粉以含0.3%甲酸的甲醇提取,强阳离子交换柱(SCX)净化后,待测样液用T3色谱柱分离,以含0.1%甲酸的5 mmol/L乙酸铵水溶液、乙腈为流动相进行梯度洗脱,采用多反应监测模式(MRM)扫描,以基质匹配标准曲线法对鹅膏肽毒素定量,以同位素内标法对色胺类毒素定量。结果显示,7种毒素在一定的浓度范围内与峰面积(或峰面积比)均呈良好的线性关系(r^(2)>0.99)。蟾蜍色胺、脱磷裸盖菇素、鹅膏肽类毒素的检出限(LOD)分别为2.0、5.0、10μg/kg,定量限(LOQ)分别为5.0、10、20μg/kg;以香菇干粉为基质,在3个水平下加标,5种鹅膏肽类毒素的回收率为71.8%~115%,相对标准偏差(RSD)为2.14%~9.92%;2种色胺类毒素的回收率为80.6%~117%,RSD为1.73%~5.98%;与国家市场监管总局食品补充检验方法BJS 202008进行比对,结果表明鹅膏毒素含量具有可比性,无显著性差异(p>0.05)。该方法简便、快速,准确度和精密度较高,符合要求,适用于野生蘑菇中7种蘑菇毒素的检测。采用该法开展了福建省野生蘑菇中蘑菇毒素分布情况的调查,在全省9个设区市采集了59份野生蘑菇样品,采用核糖体DNA内转录间隔区(rDNA⁃ITS)分子条形码技术进行了种属鉴定,在2份样品中检测出了蘑菇毒素,在1份拟灰花纹鹅膏(Amanita fuligine⁃oides)干粉提取物中检出α⁃鹅膏毒肽、β⁃鹅膏毒肽和二羟鬼笔毒肽,含量分别为607、377、69.0 mg/kg;在1份口蘑科(Tricholomataceae)蘑菇中检出脱磷裸盖菇素,含量为12.6 mg/kg。The discovery and identification of mushroom toxins has long been an important area in the fields of toxicology and food safety.Mushrooms are widely favored for their culinary and medicinal value;however,the presence of potentially lethal toxins in some species poses a substantial challenge in ensuring their safe consumption.Therefore,the development of a robust and sensitive analytical method is necessary for accurately identifying the risks associat⁃ed with mushroom consumption.The study of mushroom toxins,which are characterized by their diversity and substantial variations in chemical structures,present a considerable chal⁃lenge for achieving precise and high⁃throughput analysis.To address this issue,the present study employed a robust approach combining a solid⁃phase extraction(SPE)purification technique with high performance liquid chromatography⁃tandem mass spectrometry(HPLC⁃MS/MS)to establish an analytical method for the detection and quantification of five amatoxins and two tryptamines(psilocybin and bufotenine)present in some mushrooms.Several optimization procedures were undertaken,including optimizing the chromatographic conditions,mass spectrometric parameters,and sample extraction and purifi⁃cation.The procedure involved the extraction of dry mushroom powder with methanol contai⁃ning 0.3%formic acid,followed by purification using a strong cation exchange cartridge(SCX).The analytes were separated on a T3 chromatographic column(100 mm×2.1 mm,1.8μm)using mobile phases of acetonitrile and 5 mmol/L ammonium acetate solution containing 0.1%formic acid.The multiple reaction monitoring(MRM)mode was employed for data acqui⁃sition.Amatoxins were quantified using matrix⁃matched standard calibration curves,whereas isotopic internal standards were used to quantify tryptamine.The results showed that all seven toxins exhibited good linearities(r^(2)>0.99)within the optimized concentration range.The limits of detection(LODs)for bufotenine,psilocybin,and amatoxins were determined as 2.0,5.0,an

关 键 词：高效液相色谱⁃串联质谱法 鹅膏毒肽 鬼笔毒肽 脱磷裸盖菇素 蟾蜍色胺 蘑菇 

分 类 号：O658[理学—分析化学]