基于效液相色谱法测定参附注射液中人参皂苷的浓度

RdDetermination on the Concentration of Ginsenoside in Shenfu Injection by HPLC

目的:评价高效液相色谱法(HPLC)指纹图检测方法测试参附注射液中人参皂苷的浓度。方法:采用Agilengt-1100高效液相色谱仪进行试验研究,色谱柱选用Agilent的ZORBAXSB-C18柱,具体参数为5μm×250mm×4.6mm,柱温为30℃,进样量为10μL,选择流动相的条件是在A流动相水和B流动相乙腈中以1mL/min的流速进行HPLC研究。分别对HPLC方法学的严谨性进行检测,包括专属性试验、精密度试验、稳定性试验、重复性试验、线性回归试验、加样回收率试验等逐步建立方法的科学性;而后进一步采用HPLC对参附注射液中人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1、人参皂苷Rd的浓度进行具体的检测和统计分析。结果:专属性结果显示4种人参皂苷(Rg1、Re、Rb1、Rd)均具有良好的分离度,拖尾因子均在0.98~1.04范围。线性回归方程:人参皂苷Rg1为Y=11278X-54.71,人参皂苷Re为Y=3633X-36.34,人参皂苷Rb1为Y=8640X-4.18,人参皂苷Rd为Y=7088X+35.03,所有人参皂苷相关系数均>0.999,结果显示人参皂苷线性关系良好。人参皂苷Rg1、Re、Rb1及Rd峰面积的RSD分别为1.23%、1.28%、1.33%、1.20%、1.35%,结果表明HPLC具有良好的精密度。人参皂苷Rg1、Re、Rb1及Rd的峰面积RSD分别为0.44%、0.75%、0.25%、0.85%;结果表明参附注射液供试品溶液在24h内稳定。人参皂苷Rg1、Re、Rb1及Rd的RSD分别为1.18%、1.47%、1.86%、1.59%、1.30%,结果显示HPLC具有良好的重复性。加样回收率,人参皂苷Rg1、Re、Rb1及Rd的RSD分别为1.82%、1.04%、1.54%、2.07%,结果显示加样回收率均良好。不同批次参附注射液中人参皂苷Rg1、Re、Rb1及Rd的浓度存在较大差别。结论:高效液相色谱法检测不同批次参附注射液中人参皂苷的浓度存在较大差异。

Objective: To test the concentration of ginsenoside in Shenfu Injection by high performance liquid chromatography(HPLC). Methods: The Agilengt-1100 HLPC was used in this study.ZORBAX SB-C 18 column of the Agilent was selected as the chromatographic column,whose specific parameter was 5 μm × 250 mm × 4.6 mm,the column temperature was 30 ℃ and the sample volume was 10 μL.The condition of selecting the mobile phase was to study at the 1 mL/min velocity in the A flow phase water and the B flow phase acetonitrile.The rigor of the HPLC methodology was tested respectively.The scientificity of the method was gradually established by steps such as specificity test,precision test,stability test,repeatability test,linear regression test,sample recovery test and so on.Then the HPLC was used to perform specific detection and statistical analysis of the concentrations of ginsenoside Rg1,ginsenoside Re,ginsenoside Rb1 and ginsenoside Rd in the Shenfu Injection. Results: 1)The specificity outcome showed that the separation degrees of ginsenoside Rg1,ginsenoside Re,ginsenoside Rb1 and ginsenoside Rd were good.Their tailing factors were within the range of 0.98-1.04.The results showed that the HPLC method in the study of detecting the concentration of ginsenoside in the Shenfu Injection was of good specificity. 2)Linear regression equation:ginsenoside Rg1′s was Y=11 278X-54.71;ginsenoside Re′s was Y=3 633X-36.34;ginsenoside Rb1′s was Y=8 640X-4.18;and ginsenoside Rd′s was Y= 7 088X +35.03.All the correlation coefficients of the ginsenosides were higher than 0.999,and the results showed that the linear relationship of the ginsenoside was good. 3)The RSD of peak area of ginsenoside Rg1,Re,Rb1 and Rd were 1.23%,1.28%,1.33%,1.20% and 1.35% respectively.The results showed that the HPLC instrument had good precision.The RSD of peak area of ginsenoside Rg1,Re,Rb1 and Rd were 0.44%,0.75%,0.25% and 0.85% respectively.The test results showed that the sample solution of Shenfu Injection was stable within 24 hours.The RSD of ginsenoside Rg1,Re,Rb1 and Rd were 1.18%,1.47%,1.86%,1.59% and 1.30% respectively.The results showed that the HPLC method had good repeatability. 4)The HPLC was used for determining the sample recovery rate,and the results showed that the RSD of ginsenoside Rg1,Re,Rb1 and Rd were 1.82%,1.04%,1.54% and 2.07% respectively,which showed that the sample recovery rate was good. 5)The concentrations of ginsenoside Rg1,Re,Rb1 and Rd in different batches of Shenfu Injection were quite different. Conclusion: The concentration of ginsenoside determined by HPLC in Shenfu Injection of different batches is quite different.