HPLC-MS-MS测定小鼠组织中氧化苦参碱及苦参碱含量.docVIP

HPLC-MS/MS测定小鼠组织中氧化苦参碱及苦参碱含量 　 作者：陈有军，刘奕明，赵瑞芝，刘丽娟 【摘要】 目的建立HPLC-MS/MS测定小鼠肝、心、脾、肺、肾组织中氧化苦参碱和苦参碱浓度的方法。方法利用肝脏组织进行了提取方法的研究，考察了提取溶剂、提取温度、匀浆配比等对氧化苦参碱提取回收率的影响;测定以非那雄胺为内标,用电喷雾离子化和正离子多离子反应监测方式检测氧化苦参碱和苦参碱。结果组织-水为1∶10，在冰水浴中匀浆，以2%正丁醇氯仿混合溶剂的液-液萃取法处理样品;氧化苦参碱和苦参碱线性范围均为42 000 ng·ml-1;定量下限均为4 ng·ml-1;各组织方法回收率均在87.1%110.3%;批内、批间精密度lt;14%;提取回收率gt;58.6%。结论该含量测定方法准确、灵敏、简便、省时，适用于小鼠各脏组织中氧化苦参碱和苦参碱的含量测定。 【关键词】 氧化苦参碱; 苦参碱; HPLC-MS/MS; 组织 　　Abstract：ObjectiveTo establish LC-MS/MS method for determination of oxymatrine and matrine in liver, heart, spleen, lung and kidney of rats.MethodsExtraction method was studied using rat liver, and the effect of solvent, temperature and the proportion of tissues-water on extraction efficacy of oxymatrine recoveries were investigated. Finasteride as an internal standard gas-auxiliary electrospray ionization source, oxymatrine and matrine were detected by the positive electrospray ionization (ESI)-MS method under multiple reaction monitor (MRM) mode. Results Tissues were homogenated in ice water bath with the tissue-water proportion of 1:10, extracted by 2% n-butanol and trichlormethane. The linearity of both oxymatrine and matrine ranged from 4 to 2000 ng·ml-1 ,the detection limits of them was 4ng·ml-1.The recovery of this method ranged from 87.1%110.3%.The values of RSD within day and between days were less than 14%. Mean recovery rates were more than 58.6%. ConclusionThis method is accurate，highly sensitive,simple and convenient, time saving for the simultaneous determination of oxymatrine and matrine concentrations in tissue of rats. 　　Key words：Oxymatrine; Matrine; HPLC-MS/MS; Tissue 　　氧化苦参碱(oxymatrine OMT)和苦参碱(matrine MT)是中药苦参中的主要活性成分，氧化苦参碱给药后，体内主要代谢为苦参碱[13]。两者均有抗肿瘤、抗菌、抗炎、抗风湿、抗病毒、抗寄生虫、抗心律失常等作用[4]。尽管关于氧化苦参碱与苦参碱分析方法的报道很多，但基本都是血液或体外样品含量测定，关于组织内两种成分同时测定还未见报道。组织成分复杂，有酶等多种生物活性物质存在，对氧化苦参碱有代谢作用，从而影响其含量测定，因此本文选择了药物主要代谢组织肝脏作为提取方法研究的对象，通过对提取溶剂、提取温度和匀浆配比等的考察，建立了稳定性好、专属性强、灵敏度高的HPLC-MS/MS同时测定小鼠脏器组织中氧化苦参碱和苦参碱含量的方法，对于氧化苦参碱体内分布及代谢研究等都有重要意义。 　　1 材料与仪器 　　1.1 动物广州中医药大学实验