高效液相色谱串联质谱法测定当归芍药散提取物和含药血清中3种活性成分含量

Content Determination of Three Active Components in Danggui Shaoyao Powder Extracts and Its Drug-containing Serum by HPLC-MS/MS

目的建立测定当归芍药散提取物和含药血清中3种活性成分含量的高效液相色谱串联质谱方法。方法药物提取物样品经甲醇-水(1∶1,V/V)溶液溶解、离心、净化后过0.45μm滤膜,血清样品适当稀释、过膜后用高效液相色谱串联质谱法测定。色谱柱为Agilent Poroshell 120 EC-C18(100 mm×2.1 mm,2.7μm),茯苓酸流动相为乙腈-0.5mmol/L乙酸铵水溶液(9∶1,V/V),阿魏酸和芍药苷以乙腈-5mmol/L乙酸铵水溶液为流动相梯度洗脱,流速0.4 mL/min,柱温35℃,进样量5μL,采用电喷雾离子源,负离子模式,干燥气温度350℃,干燥气流速12L/min,检测方式为多反应监测。结果当归芍药散中阿魏酸、芍药苷和茯苓酸分别在0.1~20.0μg/mL、1~100μg/mL、0.1~20.0μg/mL范围内线性关系良好,加样回收率高,重复性好。当归芍药散提取物中阿魏酸、芍药苷和茯苓酸含量分别为(127.6±14.9)μg/g、(6081.8±468.0)μg/g和(14.7±0.75)μg/g,当归芍药散含药血清中阿魏酸和芍药苷含量分别为(0.026±0.004)μg/g和(0.61±0.07)μg/g。结论本研究建立的方法样品处理简单,方法特异性强,精确度高,重复性偏差小,回收率高,可用于当归芍药散提取物和含药血清中活性成分的质量控制。

Objective To establish a method for the determination of three active components in Danggui Shaoyao Powder extracts and its drug-containing serum by HPLC-MS/MS.Methods The extract samples were dissolved in methanol-water(1:1,V/V)solution,centrifuged and purified,and then passed through a 0.45μm filter membrane.Serum samples were appropriately diluted,passed through the membrane and determined by HPLC-MS/MS.The column was Agilent Poroshell 120 EC-C18(100 mm×2.1 mm,2.7μm).The mobile phase of porcine acid was acetonitrile-0.5 mmol/L ammonium acetate aqueous solution(9:1,V/V),and the mobile phase of ferulic acid and paeoniflorin was acetonitrile-5 mmol/L ammonium acetate aqueous solution(gradient elution).The flow rate was 0.4 mL/min;the column temperature was 35℃;the injection volume was 5μL.Electrospray ion source and negative ion mode were used;drying gas temperature was 350℃;drying gas flow rate was 12 L/min;detection method was multiple reaction monitoring.Results The linear relationship of ferulic acid,paeoniflorin and pachymic acid in Danggui Shaoyao Powder was good in the range of 0.1–20.0μg/mL,1–100μg/mL,and 0.1–20.0μg/mL,respectively.The standard recovery was high and the repeatability was good.The contents of ferulic acid,paeoniflorin and pachymic acid in Danggui Shaoyao Powder extracts were(127.6±14.9)μg/g,(6081.8±468.0)μg/g,and(14.7±0.75)μg/g,respectively.The contents of ferulic acid and paeoniflorin in Danggui Shaoyao Powdercontaining serum were(0.026±0.004)μg/g and(0.61±0.07)μg/g,respectively.Conclusion The method established in this study is simple in sample processing,strong in method specificity,high in accuracy,small in repeatability deviation,and high in recovery rate,and can be used for the quality control of the active components in the Danggui Shaoyao Powder extracts and drug-containing serum.