建立同时检测大鼠组织中真武汤有效成分的LESA-MS/MS方法研究  

Research on establishing LESA-MS/MS method for simultaneous detection of active constituents in Zhenwu Decoction in rat tissues

作　　者：仇琪[1] 曹景琳 张芸楠 李静[1] 林阳[1] QIU Qi;CAO Jing-lin;ZHANG Yun-nan;LI Jing;LIN Yang(Beijing Anzhen Hospital,Capital Medical University,Beijing 100029,China)

机构地区：[1]首都医科大学附属北京安贞医院,北京100029

出　　处：《中华中医药杂志》2020年第3期1476-1479,共4页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：十三五“新药创制重大专项”(No.2017ZX09304017);国家自然科学基金项目(No.81403200);北京市医院管理局“青苗”计划(No.QML20150603)。

摘　　要：目的:建立同时检测大鼠组织中真武汤有效成分的表面液滴萃取-质谱联用(LESA-MS/MS)方法,为中药体内分布研究探索新的检测方法。方法:清洁级健康雄性SD大鼠40只,随机分为两组:正常对照组、真武汤组。真武汤组给予真武汤粉剂9.1g/kg,每日早晚各1次,均以0.5%羧甲基纤维素钠溶液灌胃给药,灌胃体积为10mL/kg,正常对照组大鼠根据体质量给予0.5%羧甲基纤维素钠溶液,连续灌胃给药60d。给药60d后取两组大鼠心脏、肾脏和肝脏组织制备冰冻切片。本研究以甲醇、水和氨水作为萃取溶液,采用LESA-MS/MS方法进行检测:通过工作站控制机器人在多重反应监测(MRM)模式下进行检测,负离子扫描模式,扫描范围120到1000Da,分辨率100000,采集时间2min。监测离子对:去甲乌药碱m/z 270.1→135.1,茯苓酸m/z 527.3→221.2,次乌头碱m/z 616.3→338.1。结果:稳定性考察显示,心脏、肾脏和肝脏组织中的待测物在反复复融3次后检测结果稳定,在-80℃冰箱中冷冻保存14d后检测结果稳定,在室温下放置24h后检测结果稳定。结论:本研究建立了同时测定大鼠组织中去甲乌药碱、茯苓酸和次乌头碱3种真武汤中有效成分的LESA-MS/MS分析方法。该方法样品前处理简单,省时省力,稳定性好,适于在临床医学领域推广和应用。Objective:To establish a new method for the detection of the active constituents of Zhenwu Decoction in rats by liquid extraction surface analysis mass spectrometry(LESA-MS/MS).Methods:Forty male SD rats were randomly divided into two groups:normal control group and Zhenwu Decoction group.Zhenwu Decoction group gave 9.1g/kg of Zhenwu Decoction powder,twice a day(in the morning and evening).Drug was administered by 0.5%sodium carboxymethylcellulose Dissolution and the volume of gastric perfusion was 10mL/kg.Rats from the normal control group were given 0.5%sodium carboxymethylcellulose according to their body weight.After 60 days of administration,sections from the heart,kidney and liver tissues were frozen and analyzed.In this study,methanol,water and ammonia were used as extraction solutions,and the LESA-MS/MS method was used for the detection:The detection was performed under the multiple reaction monitoring(MRM)mode by robot which was controlled by workstation,with negative ion scanning mode range 120 to 1000Da.And the resolution was 100000,acquisition time was 2 min.Monitoring ion pair:higenamine m/z 270.1→135.1,poria acid m/z 527.3→221.2,hypaconitine m/z 616.3→338.1.Results:The stability study showed that the test results in the heart,kidney and liver tissues were stable after repeated rewarming for 3 times,14 days of cryopreservation in the-80℃refrigerator,and placing for 24h at room temperature.Conclusion:This study established a LESA-MS/MS method for the simultaneous determination of the active constituents in three kinds of Zhenwu Decoction including higenamine,poria acid and hypaconitine in rat tissues.The method has simple sample preparation,saves time and labor with good stability which is appropriate for promotion and application in the field of clinical medicine.

关 键 词：表面液滴萃取-质谱联用 真武汤 有效成分 同时检测 

分 类 号：R285.5[医药卫生—中药学]