HPLC-MS/MS法同时测定龙胆泻肝丸中高毒成分马兜铃酸Ⅰ与鬼臼毒素

Simultaneous determination of aristolochic acid I and podophyllotoxin in Longdan Xiegan pills by HPLC-MS/MS

目的建立液相色谱-质谱联用法同时测定龙胆泻肝丸中高毒成分马兜铃酸Ⅰ与鬼臼毒素的研究方法,为龙胆泻肝丸的质量控制提供技术依据。方法采用Agilent InfinityLab Poroshell 120 EC-C_(18)色谱柱(100 mm×2.1 mm,2.7μm)分离,以0.1%甲酸溶液(含5 mmol·L^(-1)乙酸铵)-0.1%甲酸乙腈为流动相进行梯度洗脱,柱温35℃,流速0.3 mL·min-1,进样量为5μL。在电喷雾离子源正离子模式下,采用多反应监测方式测定,外标法定量。结果马兜铃酸Ⅰ与鬼臼毒素线性关系良好,线性范围分别为9.5~94.9 ng·mL^(-1)、10.0~99.7 ng·mL^(-1),r均大于0.9950。马兜铃酸Ⅰ与鬼臼毒素平均回收率大蜜丸分别为81.89%(RSD=8.55%)、84.34%(RSD=8.67%);小蜜丸分别为82.19%(RSD=7.63%)、96.05%(RSD=6.18%);浓缩丸分别为67.99%(RSD=6.70%)、94.98%(RSD=5.01%);水丸分别为75.98%(RSD=11.37%)、93.88%(RSD=6.01%)(n=18)。结论该方法操作简单,结果可行,可快速测定龙胆泻肝丸中高毒成分马兜铃酸Ⅰ与鬼臼毒素含量。

Objective An HPLC-MS/MS method was established for the simultaneous determination of aristolochic acid I and podophyllotoxin,the highly toxic components in Longdan Xiegan pills,so as to provide the technical basis for the quality control of Longdan Xiegan pills.Methods The samples were separated on an Agilent InfinityLab Poroshell 120 EC-C_(18)column(100 mm×2.1 mm,2.7μm)with a gradient elution of 0.1%formic acid solution(containing 5 mmol·L^(-1)ammonium acetate)-0.1%formic acid acetonitrile as the mobile phase at a column temperature of 35℃,a flow rate of 0.3 mL·min-1,and the injection volume of 5μL.Multi-reaction monitoring(MRM)was applied to the determination in positive ion mode of electrospray ion source,and the quantification was conducted by external standard method.Results Aristolochic acid I and podophyllotoxin showed a good linear relationship in the range of 9.5-94.9 ng・mL^(-1)and 10.0-99.7 ng・mL^(-1),respectively,and r was greater than 0.9950.The average recovery rates of aristolochic acid I and podophyllotoxin in Big Honeyed pills were 81.89%(RSD=8.55%)and 84.34%(RSD=8.67%);in the Small Honeyed pills were 82.19%(RSD=7.63%)and 96.05%(RSD=6.18%);in the Concentrated pills were 67.99%(RSD=6.70%)and 94.98%(RSD=5.01%);in the Water pills were 75.98%(RSD=11.37%)and 93.88%(RSD=6.01%)(n=18),respectively.Conclusion The method is simple and accurate,and could be used for the rapid determination of aristolochic acid I and podophyllotoxin,the highly toxic components in Longdan Xiegan pills.