基于效液相色谱法测定参附注射液中人参皂苷的浓度 罗宇文 杨亦炳 王文娟Summary 目的：評价高效液相色谱法（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ZORBAX SB-C18柱，具体参数为5 μm×250 mm×4.6 mm，柱温为30 ℃，进样量为10 μL，选择流动相的条件是在A流动相水和B流动相乙腈中以1 mL/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=11 278X-54.71，人参皂苷Re为Y=3 633X-36.34，人参皂苷Rb1为Y=8 640X-4.18，人参皂苷Rd为Y=7 088X+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24 h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Key 高效液相色谱法;参附注射;线性回归方程;加样回收率;人参皂苷Rg1;人参皂苷Re;人参皂苷Rb1;人参皂苷RdAbstract 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-C18 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.Key Words High performance liquid phase; Shenfu Injection; Linear regression equation; Sample recovery rate; Ginsenoside Rg1; Ginsenoside Re; Ginsenoside Rb1; Ginsenoside Rd：R284：Adoi：10.3969/j.issn.1673-7202.2019.08.012参附注射液源于古方“参附汤”，原方由红参、附子两味中药组成为一种常用的急救药物，在中医药理论指导下主治元气大亏，阳气暴脱等急症。
随着中药制剂的改革，参附注射液随之问世并广泛运用于临床，目前临床多用于心力衰竭类型的疾病[1-3]，用于增强心脏收缩功能和抗心力衰竭有研究显示，参附注射液中的有效成分之一是人参皂苷，人参皂苷按皂苷元的不同類型可分为两类，即原人参二醇型皂苷和原人参三醇型皂苷，其中根据含量的高低原人参二醇型皂苷的亚型Rb1、Rd等[4-5]，原人参三醇型皂苷的亚型根据含量高低排序依次为Re、Rg1等而近年来，人参皂苷显著的药理活性越来越被人们所熟知，研究报道显示，人参皂苷Rh2、Rg3和Rk1均具有抗肿瘤的药理活性[6-9]，人参皂苷Rg3具有神经保护作用，改善脑缺血损伤，人参皂苷Rg3、Rg5和Rk1具有羟自由基清除能力和抗炎等药理作用本研究为完善参附注射液的质量控制体系，基于高效液相色谱法（HPLC）测定参附注射液中人参皂苷浓度，具体选取原人参二醇型中皂苷含量较高的人参皂苷Rb1、Rd和原人参三醇型皂苷种含量较高的人参皂苷Rg1、Re等共4种人参皂苷成分进行分析研究现报道如下1 仪器与试剂1.1 仪器 Agilent高效液相色谱仪（产品型号：1260），色谱柱采用同色谱仪同厂家的匹配型号ZORBAX SB-C18柱，色谱仪产品配件：四元梯度泵，单元是DAD多波长检测器，Agilent系列产品均购于美国Agilent Techologies公司;精密BS210S型电子天平购于德国Sartorius公司。
1.2 试剂 对照品人参皂苷Rb1对照品（产品批号：110704-20092）、人参皂苷Rd对照品（产品批号：111818-201302）、人参皂苷Rg1对照品（产品批号：110703-201027）、人参皂苷Re对照品（产品批号：110754-200822）均购于中国食品药品检定研究院色谱纯乙腈，去离子水和分析纯均购买于湖北杜克化学科技有限公司1.3 分析样品 3个不同批次的参附注射液（国药准字Z51020664）购于华润三九（雅安）药业有限公司2 方法与结果2.1 色谱条件采用Agilengt-1100高效液相色谱仪进行试验研究，色谱柱选用Agilent的ZORBAX SB-C18柱，具体参数为5 μm×250 mm×4.6 mm，柱温为30 ℃，进样量为10 μL，以水和乙腈（以下分别称之为流动相A和流动相B）作为本次研究的流动相的条件，并以1 mL/min的流速开展参附注射液的HPLC人参皂苷浓度研究，根据高效液相色谱法（HPLC）的梯度洗脱程序进行试。