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Volume 40 Issue 1
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DAI Jianying, YOU Wei, TIAN Tian, HONG Zhanying. Simultaneous determination of five components in Anemarrhenae Rhizoma by HPLC-ELSD[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(1): 34-37. doi: 10.12206/j.issn.1006-0111.202109070
Citation: DAI Jianying, YOU Wei, TIAN Tian, HONG Zhanying. Simultaneous determination of five components in Anemarrhenae Rhizoma by HPLC-ELSD[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(1): 34-37. doi: 10.12206/j.issn.1006-0111.202109070

Simultaneous determination of five components in Anemarrhenae Rhizoma by HPLC-ELSD

doi: 10.12206/j.issn.1006-0111.202109070
  • Received Date: 2021-09-13
  • Rev Recd Date: 2022-01-05
  • Available Online: 2022-01-20
  • Publish Date: 2022-01-25
  •   Objective  To establish a method for the simultaneous determination of new mangiferin, mangiferin, artemisinin BⅡ, icariin and artemisinin A in Anemarrhenae Rhizoma by high performance liquid chromatography-evaporation light scattering detector (HPLC-ELSD).   Methods  The column was Agilent Poroshell 120 EC-C18. The mobile phase used acetonitrile-0.2% acetic acid water system with gradient elution. Column temperature was 30 ℃. Flow rate was 0.7 ml/min. Evaporative light scattering detector used nitrogen as atomizing gas. The atomizing gas temperature was 40 ℃ and the drift tube temperature was 90 ℃. The nitrogen volume flow rate was 2.00 L/min and the sample volume was 20 μl.   Results  The five components were able to achieve baseline separation. Neomangiferin, Mangiferin, Anemaponin BⅡ, Baohuoside I , Anemarrhena saponin AⅢwere determined as 24.1-386 μg/ml (r=0.999 3), 23.2-371 μg/ml (r=0.998 6), 54.2-867.2 μg/ml(r=0.995 6), 5.3-84.8 μg/ml (r=0.996 8), 10-160 μg/ml (r=0.998 9) respectively, which showed a good linear relationship within the concentration range. The average recovery rate of the five components was between 101.8% and 105.0%, and the repeatability RSD was less than 2.4%. The content of the above five components in Zhimu medicinal materials were 1.62%, 0.82%, 7.36%, 0.07%, 0.34%, respectively.   Conclusion  The method is simple, accurate, and highly sensitive, which could be used as the quantitative determination of multiple index components of Anemarrhenae Rhizoma.
  • [1] 朱建新, 李旺. 知母的研究进展[J]. 河北北方学院学报(自然科学版), 2018, 34(2):56-60.
    [2] 蔡飞, 王维泓, 高守红, 等. 知母皂苷及其苷元的药理作用研究进展[J]. 药学实践杂志, 2011, 29(5):331-335. doi:  10.3969/j.issn.1006-0111.2011.05.004
    [3] 国家药典委员会. 中华人民共和国药典(2020年版)一部[S]. 北京: 中国医药科技出版社, 2020: 222.
    [4] 朱岩. HPLC-ELSD同时测定知母药材中3种主要皂苷含量[J]. 化学工程师, 2018, 32(2):32-34,48.
    [5] 黄彬彬. 知母化学成分的研究[D]. 长春: 吉林大学, 2015.
    [6] 钟可, 王文全, 靳凤云, 等. HPLC-ELSD法同时测定河北产道地药材不同物候期知母中知母皂苷AⅢ和知母皂苷BⅡ的含量[J]. 中华中医药杂志, 2013, 28(6):1710-1713.
    [7] 苏晓楠, 季德, 周亚萍, 等. HPLC-DAD-ELSD同时测定知母中黄酮和皂苷类成分的含量[J]. 中国中药杂志, 2015, 40(1):108-111.
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Simultaneous determination of five components in Anemarrhenae Rhizoma by HPLC-ELSD

doi: 10.12206/j.issn.1006-0111.202109070

Abstract:   Objective  To establish a method for the simultaneous determination of new mangiferin, mangiferin, artemisinin BⅡ, icariin and artemisinin A in Anemarrhenae Rhizoma by high performance liquid chromatography-evaporation light scattering detector (HPLC-ELSD).   Methods  The column was Agilent Poroshell 120 EC-C18. The mobile phase used acetonitrile-0.2% acetic acid water system with gradient elution. Column temperature was 30 ℃. Flow rate was 0.7 ml/min. Evaporative light scattering detector used nitrogen as atomizing gas. The atomizing gas temperature was 40 ℃ and the drift tube temperature was 90 ℃. The nitrogen volume flow rate was 2.00 L/min and the sample volume was 20 μl.   Results  The five components were able to achieve baseline separation. Neomangiferin, Mangiferin, Anemaponin BⅡ, Baohuoside I , Anemarrhena saponin AⅢwere determined as 24.1-386 μg/ml (r=0.999 3), 23.2-371 μg/ml (r=0.998 6), 54.2-867.2 μg/ml(r=0.995 6), 5.3-84.8 μg/ml (r=0.996 8), 10-160 μg/ml (r=0.998 9) respectively, which showed a good linear relationship within the concentration range. The average recovery rate of the five components was between 101.8% and 105.0%, and the repeatability RSD was less than 2.4%. The content of the above five components in Zhimu medicinal materials were 1.62%, 0.82%, 7.36%, 0.07%, 0.34%, respectively.   Conclusion  The method is simple, accurate, and highly sensitive, which could be used as the quantitative determination of multiple index components of Anemarrhenae Rhizoma.

DAI Jianying, YOU Wei, TIAN Tian, HONG Zhanying. Simultaneous determination of five components in Anemarrhenae Rhizoma by HPLC-ELSD[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(1): 34-37. doi: 10.12206/j.issn.1006-0111.202109070
Citation: DAI Jianying, YOU Wei, TIAN Tian, HONG Zhanying. Simultaneous determination of five components in Anemarrhenae Rhizoma by HPLC-ELSD[J]. Journal of Pharmaceutical Practice and Service, 2022, 40(1): 34-37. doi: 10.12206/j.issn.1006-0111.202109070
  • 中药知母(Anemarrhenae rhizoma)为百合科植物知母(Anemarrhena asphodeloides Bge.)的干燥根茎,始载于《神农本草经》,具有清热泻火,肺热燥咳,滋阴润燥以及肠燥便秘等作用[1]。知母根茎中含有甾体皂苷类、黄酮类、双苯吡酮类、木质素类、多糖类和生物碱类等多种成分,甾体皂苷类是其首要药理活性成分。其中新芒果苷、芒果苷等黄酮苷类单体成分具有降低血糖的作用,知母皂苷BⅡ以及知母皂苷AⅢ等甾体皂苷类单体成分具有改善学习记忆、抗老年痴呆的作用[2]

    《中国药典》(2020年版)将芒果苷和知母皂苷BⅡ作为知母的质量评价指标[3],目前知母中芒果苷和新芒果苷的含量测定方法多采用高效液相-紫外检测法,而知母中另外一类重要的活性成分甾体皂苷类,由于不存在共轭结构、缺少紫外吸收,需通过衍生化技术将其转化成有紫外特征吸收的物质;但这种操作复杂、耗时,且准确度受衍生物稳定性的影响较大[4-5],因此紫外检测器并不适用于甾体皂苷类物质的检测。相比于紫外检测器,蒸发光散射检测器(ELSD)不要求化合物带有发色团或荧光基团,能够测定没有紫外吸收或为紫外末端弱吸收的样品;而且可以在多溶剂梯度的情况下获得稳定的基线和较高的分辨率。目前,已有应用HPLC-ELSD法对知母药材中单种成分或多成分含量测定的报道[6-7],但是所建立的方法往往分析时间较长,消耗试剂较多。

    本文建立了一种快速、简便、准确的HPLC-ELSD方法,可同时测定知母中3个黄酮类成分(新芒果苷、芒果苷、宝藿苷Ⅰ)和2个甾体皂苷类成分(知母皂苷BⅡ、知母皂苷AⅢ)的含量,可为知母药材质量控制提供参考依据。

    • 高效液相色谱仪(1260 InfinityⅡ Prime LC, 美国安捷伦公司);蒸发光散射检测器(G4260B,美国安捷伦公司);分析天平(XS205,梅特勒-托利多国际公司)、涡旋混合器(SCILOGEX MX-S,美国Scilogex公司)、超声波清洗器(2300TH型,上海安谱实验科技股份有限公司)。

    • 新芒果苷对照品(批号:3671)、芒果苷对照品(批号:5586)、知母皂苷BⅡ对照品(批号:1369)、宝藿苷Ⅰ对照品(批号:1869)、知母皂苷AⅢ对照品(批号:3892)购自上海诗丹德标准技术服务有限公司;知母药材由上海市食品药品检验所中药室赠予;色谱纯乙腈(北京迪科马科技有限公司);色谱纯异丙醇(上海安谱实验科技股份有限公司);分析纯乙醇和乙酸(国药集团化学试剂有限公司)。

    • 精密称取芒果苷、新芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ和知母皂苷AⅢ 对照品适量,置于5 ml容量瓶中,加50%乙醇溶液适量,超声溶解后,加入50%乙醇稀释并定容得到混合溶液,精密量取1 ml该混合溶液置于5 ml容量瓶中,加50%乙醇溶液定容,最终得含芒果苷464 μg/ml,新芒果苷482 μg/ml,知母皂苷BⅡ 1084 μg/ml,宝藿苷Ⅰ 106 μg/ml,知母皂苷AⅢ 200 μg/ml的混合对照品储备液,摇匀备用。

    • 取知母药材适量,干燥后粉碎,过3号筛得知母药材粉末。取药材粉末0.5 g,精密称定,置于50 ml具塞锥形瓶中,精密加入50%乙醇50 ml,称重,超声(功率300 W,60 Hz)处理30 min。超声结束后放置至室温后再次称定质量,使用50%乙醇补足所减失的质量,摇匀后滤过。取续滤液过0.22 μm微孔滤膜,作为供试品溶液。

    • 色谱柱为Agilent poroshell 120 EC-C18柱(2.1×100 mm,2.7 μm);流动相为乙腈(A)-0.2%醋酸水(B);梯度洗脱程序设定为0~3 min,4%~10%A;3~5 min,10%A;5~7 min,20%A;7~11 min,20%~25%A;11~14 min,25%~90%A;14~18 min,90%A;18~19 min,90%~4%A;19~20 min,90%~4%A。柱温为30 ℃;流速为0.7 ml/min。蒸发光散射器的参数设定雾化气温度为40 ℃;漂移管温度为90 ℃;氮气体积流量为2.0 ml/min。供试品进样量为20 μl。混合对照品和供试品色谱图见图1,新芒果苷、芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ、知母皂苷AⅢ 的保留时间分别为4.05、5.49、11.63、13.85、14.38 min,各成分之间分离度均>1.5。

    • 精密量取1.6 ml混合对照品储备液和0. 4 ml 50%乙醇溶液,涡旋30 s混匀,后使用50%乙醇依次对半稀释。按上述色谱条件进行测定,以峰面积(A)的对数值与进样浓度(C)的对数值进行线性回归,考察结果见表1,表明知母中各成分在相应的浓度范围内均呈现良好的线性关系。

      成分回归方程线性范围(μg/ml)r
      新芒果苷lgA=1.8601×lgC−1.687424.1~3860.9993
      芒果苷lgA=1.8445×lgC−1.395523.2~3710.9986
      知母皂苷BⅡlgA=1.43×lgC−0.18454.2~867.20.9956
      宝藿苷ⅠlgA=1.4974×lgC+0.33645.3~84.80.9968
      知母皂苷AⅢlgA=1.4808×lgC+0.238710~1600.9989
    • 精密吸取混合对照品20 μl,按上述色谱条件连续进样6次,计算5种成分峰面积的RSD值,结果显示,新芒果苷、芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ、知母皂苷AⅢ的RSD分别为1.3%、1.0%、0.9%、1.5%、1.0%、1.4%,表明仪器精密度良好。

    • 称取知母药材粉末0.5 g,精密称定,平行称取6份,按上述操作制备供试品溶液,进样测定,计算新芒果苷、芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ、知母皂苷AⅢ的含量分别为1.62%、0.82%、7.36%、0.07%、0.34%,RSD分别为1.8%、1.8%、1.4%、2.2%、2.4%,显示方法重复性良好。

    • 称取知母药材粉末0.5 g,按上述操作制备供试品溶液,分别于制备后0、2、4、6、8、12 h进样测定,结果显示新芒果苷、芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ、知母皂苷AⅢ的峰面积RSD分别为:2.3%、1.3%、0.9%、3.1%、1.8%,说明样品在12 h内稳定。

    • 称取知母药材粉末0.25 g,精密称定,平行称取6份,分别加入与知母药材粉末各成分含量相近的混合对照品溶液,按上述操作制备供试品溶液,进样测定,计算加样回收率。结果见表2,知母中5种成分的平均回收率为101.8%~105.0%,表明该方法准确度良好。

      成分原有量
      m/mg)
      加入量
      m/mg)
      测得量
      m/mg)
      回收率
      (%)
      平均回收率
      (%)
      RSD
      (%)
      新芒果苷4.064.809.01103.2101.81.5
      4.114.808.99101.9
      4.194.809.07101.8
      3.994.808.87101.8
      4.074.809.04103.5
      4.094.808.8398.69
      芒果苷2.082.134.26102.8102.2

      2.5
      2.102.134.2098.73
      2.142.134.39105.3
      2.042.134.2710.0
      2.082.134.1999.07
      2.092.134.28102.7
      知母皂苷BⅡ18.4510.8029.88105.77104.911.28
      18.6510.829.93104.5
      19.0410.8030.59107.0
      18.1410.8029.55105.6
      18.5010.8029.65103.2
      18.5910.8029.75103.4
      宝藿苷Ⅰ0.170.210.39103.4102.01.8
      0.170.210.39102.2
      0.180.210.39102.6
      0.170.210.39100.9
      0.170.210.3998.57
      0.170.210.39104.1
      知母皂苷AⅢ0.871.172.07103.4103.21.0
      0.881.172.07102.7
      0.901.172.08101.7
      0.861.172.05102.5
      0.871.172.08104.3
      0.881.172.09104.7
    • 称取知母药材粉末0.5 g,精密称定,按上述操作进样测定,计算各成分含量,最终计算得新芒果苷、芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ、知母皂苷AⅢ的平均含量分别为1.62%、0.82%、7.36%、0.07%、0.34%。

    • 本研究首先采用乙腈-水作为流动相,结果发现新芒果苷、芒果苷峰形不佳,色谱峰出现分叉现象。考虑到该两种物质呈弱酸性,故采用乙腈-0.2%醋酸水溶液作为流动相以抑制其解离,结果显示其分叉现象消失,各峰之间的分离度良好,且基线平稳,因此最终采用乙腈-0.2%醋酸水溶液作为最终流动相系统。结果如图2所示。

    • 流动相流出色谱柱后在载气压力的作用下与雾化气体(氮气)在雾化室内混合形成细小的液滴(气溶胶),气体流量越低,形成的液滴越大,散射光越多,分析灵敏度增加,反之越小。但是较低的气体流量会造成溶剂难以蒸发,因此本研究考察了氮气流速分别为1.5、2.0、2.5 L/min时各待测物质的响应值,结果显示,当氮气流速为2.0 L/min 时待测物质响应值最大,信号噪音比率最大,适合分析。

    • 漂移管温度的设置主要取决于流动相的构成、样品的挥发性以及流动相的流速。一般来讲,对于有机溶剂含量比例高、流速低的流动相,其要求的漂移管温度也低。本研究使用含新芒果苷386 μg/ml、芒果苷371 μg/ml、知母皂苷BⅡ 867.2 μg/ml、宝藿苷Ⅰ 84.8 μg/ml、知母皂苷AⅢ 160 μg/ml的混合对照品溶液,考察了漂移管温度分别为80、90、100 ℃时各待测物质的响应值,结果表明当漂移管温度为90 ℃时各物质峰面积响应值均较大,信号噪音比率最大,适合分析,因此最终漂移管温度采用90 ℃。结果如图3所示。

      本研究建立了一种同时测定知母药材中包括芒果苷、知母皂苷BⅡ在内的5种活性成分含量的HPLC-ELSD方法。该法快速、简便、准确度高,可为知母的质量评价和质量标准的提升提供科学依据,为知母有效成分的体内动态变化研究提供测定方法。

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