尿液和血浆中蘑菇毒肽测定的方法学研究  

Methodology research on detection of phallotoxins in urine and plasma samples

作　　者：李帮锐[1] 冯家力[1] 曾栋[1] 丁力[1] 刘先军[1] 李谭瑶[1] 左家信[1] 范翔[1] 陈东洋[1] 

机构地区：[1]湖南省疾病预防控制中心理化检验科,湖南长沙410005

出　　处：《职业与健康》2016年第20期2786-2791,共6页Occupation and Health

基　　金：湖南省卫生计生委科技计划项目(C2015-065)

摘　　要：目的开展尿液和血浆中3种鹅膏毒肽和3种鬼笔毒肽测定的方法学研究。方法尿液和血浆样本经乙腈沉淀蛋白后,HLB固相萃取柱萃取净化,超高效液相色谱-串联质谱法测定α-鹅膏毒肽、β-鹅膏毒肽、γ-鹅膏毒肽、羧基二羟鬼笔毒肽、二羟鬼笔毒肽和羧基三羟鬼笔毒肽6种蘑菇毒肽,基质匹配制作工作曲线,外标法定量;对样本测定条件进行优化,减小基质干扰以提高方法的灵敏度;研究方法的适用性,考察方法的专一性、线性、灵敏度、精密度和加标回收率。结果尿液和血浆中6种毒肽的检出限均低于0.06 ng/ml;在0.2-20 ng/ml范围内,线性关系良好;相关系数R2均大于0.999 3;相对标准偏差均小于12.1%;对于上述两种基质,加标回收率在79.6%-104.8%之间。结论方法灵敏、准确、专一性好,适合于突发毒蕈中毒事件中α-鹅膏毒肽、β-鹅膏毒肽、γ-鹅膏毒肽、羧基二羟鬼笔毒肽、二羟鬼笔毒肽和羧基三羟鬼笔毒肽的确证分析,也适合对中毒患者临床治疗进行效果评价。[Objective]To carry out the methodology research on detection of 3 kind of amanita hemolysin and 3 kind of phalloidin in urine and plasma samples.[Methods]The urine and plasma sample were prepared by protein precipitation with ace tonitrile,and solid phase extraction（SPE） procedure on a HLB column,and were detected by ultra performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS） for the determination of α-amanitin,β-amanitin,γ-amanitin,phallisacin,phalloidin and phallacidin. The analyte quantifications were performed by external standard method with matrix-matched calibration curves. The sensitivity was increased by minimizing interferences due to optimized detection conditions. Linearity,sensitivity,specificity and precision and recovery were studied,in order to guarantee reliability in the analytical results.[Results]The developed method proved to be specific and sensitive,with LOD（Limit of Detection）values for 6 phallotoxins all under 0.06 ng/ml in urine and plasma samples. Linearity,in the range of 0.2-20 ng/ml,showed that coefficients of correlation（R2） were greater than 0.999 3. The relative standard deviations were lower than 12.1%. The recovery presented good results for the concentrations analyzed,with values ranging from 79.6 to 104.8% for both matrices.[Conclusion]The present method is applicable in emergency determination of α-amanitin,β-amanitin,γ-amanitin,phallisacin,phalloidin and phallacidin,and clinical assessment because of its accuracy and specificity with high sensitivity.

关 键 词：超高效液相色谱-串联质谱法 蘑菇毒肽 尿液 血浆 

分 类 号：R446.1[医药卫生—诊断学]