一测多评法测定小活络丸中9种乌头类生物碱的含量

Determination of Nine Aconitum Alkaloids in Xiaohuoluo Pills with Quantitative Analysis of Multi-components by Single Marker

目的:建立一测多评法同时测定小活络丸中9种乌头类生物碱含量。方法:采用高效液相色谱四级杆飞行时间串联质谱(HPLC-QTOF-MS)法,采用Agilent ZORBAX Extend-C_(18)RRHT(2.1 mm×50 mm,1.8μm)色谱柱;流速为0.21 ml·min^(-1);柱温为30℃;以甲醇为流动相A,以水(含0.1%甲酸和2.5 mmol·L^(-1)醋酸铵溶液)为流动相B,梯度洗脱。以乌头碱为内参物,测定次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱、乌头原碱、次乌头原碱、新乌头原碱对于其的相对校正因子,并对相对校正因子进行重现性考察。利用相对校正因子计算其他8种生物碱成分的含量,同时利用外标法测定这9种成分,比较两种测定方法的差异,验证一测多评法的准确性和可行性。结果:在线性范围内,次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头碱、苯甲酰新乌头原碱、乌头原碱、次乌头原碱、新乌头原碱对乌头碱的相对校正因于分别为1.736、1.979、1.0471、0.9242、1.2901、1.3078、1.2859、1.0948,建立了QAMS法测定小活络丸中9种生物碱的含量,其测定值与计算值无显著差异。结论:本文方法可用于小活络丸中乌头碱、次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱、乌头原碱、次乌头原碱、新乌头原碱的含量测定。

Objective： To develop a method of quantitative analysis of multi-components by single marker（QAMS） for nine kinds of alkaloids in Xiaohuoluo pills. Methods： An HPLC-QTOF-MS method with an Agilent ZORBAX Extend-C18 RRHT （2.1 mm× 50 mm,1.8 μm） column was applied. The flow rate was 0.21 ml·min^-1. The column temperature was 30 ℃. The mobile phase was methanol （A）-water （B; containing 0.1% formic acid and 2.5 mmol·L^-1 ammonium acetate） with gradient elution. The aconitine was used as the internal standard, and the relative correction factor （RCFs） of hypaconitine, mesaconitine, benzoylaconine, berrtoyl- hypaconine, benzoylmesaconine, aconine, hypaconine and mesaconine was respectively established, and the reproducibility inspection on the RCF was performed. The contents of the other 8 kinds of aconitum alkaloids were calculated according to the RCF. At the same time, an external standard method （ESM） was performed for the content determination of the nine alkaloids. The results of the two methods were compared. The feasibility and accuracy of the QAMS method were verified. Results： Within a certain range,the RCF of hypacontine, mesacontine, benzoylaconine, benzoylhypaconine, benzoyl mesaconine, aconine, hypaconine and mesaconine to aconitine was 1. 736,1. 979,1. 0471,0. 9242,1. 2901,1. 3078, 1. 2859, and 1. 0948 ,respectively. The QAMS method was established for determining alkaloids. There were no significant differences between the results of the QAMS method and those of the＇external standard method （ESM）. Conclusion： With the validation of methodology, the method established in our study can be used for the content determina- tion of aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, aeonine, hypaconine and mesaconine in xiaohuoluo pills.