CN104458995A - 一种采用hplc快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法 - Google Patents
一种采用hplc快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法 Download PDFInfo
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Abstract
本发明属于中药成分分析领域。即一种采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法。其色谱分析条件如下：（1）色谱柱采用反相C18色谱柱；（2）流动相组成为：甲醇-水-四氢呋喃-酸，流动相中各成分的体积比为：四氢呋喃与甲醇比例为0.03～0.1，四氢呋喃+甲醇比例为30～40%，酸的比例为水相的0.1-2%；（3）紫外检测波长为348nm；（4）色谱柱温度为：35～45℃。在等度洗脱的条件下，20min内就可以同时分离分析15个以上的色谱峰，与从前的方法相比，不但解决了木犀草素-7-O-β-D-葡萄糖苷与木犀草素-7-O-β-D-葡萄糖醛酸苷较难分离的问题，并且苦碟子注射液中的酚酸类色谱峰也很好的分离出来，而且分析时间大大缩短。
Description
技术领域
本发明属于中药成分分析领域。即一种采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法。
背景技术
苦碟子注射液是以菊科草本植物抱茎苦荬菜为原料提取精制而成的静脉注射剂，其主要成分有酚酸类、黄酮类等。苦碟子注射液列入国家药品标准产品（国药准字Z20025450），由通化华夏药业有限责任公司生产。因其具有活血止痛、清热祛瘀的功效，而被广泛用于心脑血管疾病的治疗，长期的临床实践证明其使用安全，疗效确切。木犀草素-7-O-β-D葡萄糖醛酸苷属于黄酮苷类，在苦碟子药材中含量较高且比较稳定，其在苦碟子注射液中含量也较高，因此可作为苦碟子注射液质量控制的指标成分。但是因为木犀草素-7-O-β-D葡萄糖醛酸苷与木犀草素-7-O-β-D葡萄糖苷的结构相近，目前采用HPLC方法分析苦碟子注射液时，上述两种成分的分离度不够，分离时间大大延长，并且因为采用梯度洗脱，分析色谱图容易出现波动（对比文献：HPLC-DAD法同时测定苦碟子注射液中六种黄酮类成分的方法，申请号：201310071067.X）。
发明内容
本发明针对现有苦碟子注射液分析方法的缺陷，提供了一种采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法。
一种采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法，其色谱分析条件如下：（1）色谱柱采用反相C18色谱柱；（2）流动相组成为：甲醇-水-四氢呋喃-酸，流动相中各成分的体积比为：四氢呋喃与甲醇比例为0.03～0.1，四氢呋喃+甲醇比例为30～40%，酸的比例为水相的0.1-2%；（3）紫外检测波长为348nm；（4）色谱柱温度为：35～45℃。优选色谱柱温度为40～45℃。
流动相中各成分的体积比优选为为：四氢呋喃与甲醇比例为0.05～0.1，四氢呋喃+甲醇比例为35-38%；酸的比例为水相的0.5-1%。
酸包括但不限于甲酸、冰乙酸、三氟乙酸、三氯乙酸、磷酸、磷酸二氢钠、磷酸二氢钾、硫酸、柠檬酸、苹果酸、酒石酸。酸优选为甲酸或冰乙酸。
一种采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法，其特征在于色谱分析条件如下：（1）色谱柱采用反相C18色谱柱；（2）流动相组成为：四氢呋喃与甲醇比例为0.05～0.1，四氢呋喃+甲醇比例为35-38%；甲酸的比例为水相的0.5-1%；（3）紫外检测波长为348nm；（4）色谱柱温度为：40～45℃。
通过中心复合设计的方法，优化色谱分析条件，可以明显提高各色谱峰分离效果，尤其是木犀草素-7-O-β-D葡萄糖醛酸苷与木犀草素-7-O-β-D葡萄糖苷的分离效果，缩短分析时间，色谱分析图谱重现性好。
中心复合设计(central composite design， CCD)方法是近年来发展起来的实验设计方法。该方法的优点是实验点距中心点的距离相等，自变量的极端水平为实验所允许的极大极小值，一次设计即可获得较佳条件，用非线性数学模型拟合相关系数较高，预测性好。为了明确各因素对HPLC分析结果的影响，本发明采用中心复合设计方法对苦碟子注射液中木犀草素-7-O-β-D葡萄糖醛酸苷的高效液相分析方法进行了优化，得出最佳色谱条件。
下面结合实施例对本发明的效果作进一步的说明，下述各实施例仅用于说明本发明，对本发明没有限定。
附图说明
图1－图3是表2中各分析条件下的HPLC图谱。
图4是各指标（Y）与影响因子（X）关系图。
图5是优化的色谱分析条件下苦碟子注射液的HPLC分析图谱。
具体实施方式
实施例1
采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法:
1．仪器与材料
岛津LC-2010A高效液相色谱仪(SHIMADZU公司)，UV检测器，色谱柱UltimateTMAQ-C18 (4.6×250 mm ，5 μm) (Welch Materials公司)。苦碟子注射液(通化华夏药业有限责任公司。批号 91112)，色谱甲醇，色谱乙腈，色谱四氢呋喃，色谱甲酸(山东禹王公司)，双蒸水(实验室自制)。
2．色谱条件
色谱柱：UltimateTMAQ-C18 (4.6×250 mm，5μm)(Welch Materials 公司)；流动相：A相有机相，B相为1%(v/v)的甲酸水溶液；流速为1 mL·min-1；检测波长为348 nm；进样体积为10 μL。
流动相：A相为四氢呋喃与甲醇溶液的混合溶液，四氢呋喃的体积比变化范围为 0.03-0.1 (v/v) ，B相为1% (v/v) 的甲酸水溶液，A相变化范围为30%-40%；柱温变化范围35-45℃。
3．样品测定
取苦碟子注射液，经过0.45 μm微孔滤膜，按各色谱条件测定。
4．色谱分析条件优化试验
4.1．试验设计与结果
影响色谱行为的因素有很多，根据范第姆特速率理论，HPLC的实验条件应该是：①小粒度、均匀的球形化学键合相；② 低粘度流动相，流速不宜过快；③ 柱温适当。当色谱柱固定后，影响色谱行为的因素包括：流动相的种类、流动相的配比、柱温以及流速。为减少试验次数，本实验采用常用流速1 mL·min-1。综合以上因素，最终确定了温度 (X1) 、四氢呋喃与甲醇比例 (X2) 、有机相（甲醇+四氢呋喃）比例 (X3) 为影响色谱行为的关键因素。以其作为考察对象，每个因素设3个水平。考察指标采用4、5号峰分离度 (Y1) ，5、6号峰分离度 (Y2) ，7号峰保留时间 (Y3) 。因素及水平表详见 表1；试验安排及结果详见表2。各实验结果色谱详见图1－图3, 纵坐标为mAU，横坐标为min。图1中的（1）是表2中run 1的实验结果图。依此类推，各图的分析条件与表2相对应。
每个试验重复2次， 为了4、5、6号峰达到彼此分离，我们认为分离度Y1、 Y2大于2（一般要求大于1.5即可）是理想的；为了提高工作效率，7号峰的出峰时间应控制在20分钟之内，即Y3 < 20 min。
表1 影响因素与水平及真实值与代码值
表2 实验设计与结果
4.2．模型拟合
将表2中的数据用软件采用最小二乘法拟合预测模型Y1、Y2及Y3，模型的二项式通式为Y =b0 + b1·x1 + b2·x2 + b3·x3 + b4·x1·x2+ b5·x1·x3+ b6·x2·x3 +b7·x2 1+ b8·x2 2+ b9·x2 3
根据以上模型Y1Y2Y3的拟合结果，去除估算系数P值大于0.05的项，拟合方程为：
Y1 = 4.085 + 0.411x1 - 3.096x2-0.372x3 + 0.836x1x2 + 1.5326x2x3 (R2=0.9986，P<0.01)
Y2 = 2.365 - 0.275x1 + 0.356x2 - 0.575x3 + 0.070x1x3 - 0.122x2x3 - 0.104x2 2 (R2=0.9995，P<0.01)
Y3 =25.268 -5.187x1 - 1.505x2 - 16.000x3 + 3.473x1x3 + 1.060x2x3 + 5.858x2 3 (R2=0.9991，P<0.01)
根据上述方程绘制X1、X2和X3对各评价指标Y1,Y2和Y3的预测效应图(图4) 。
由拟合结果可以看出，随着温度（X1）的升高，4、5号峰分离度（Y1）逐渐上升，而5、6号峰分离度（Y2）与7号峰的保留时间（Y3）均逐渐下降；随着四氢呋喃与甲醇比例（X2）的增加，4、5号峰分离度（Y1）急剧下降，而5、6号峰分离度（Y2）明显提高，7号峰的保留时间（Y3）变化不显著；随着有机相比例（X3）的提高，4、5号峰分离度（Y1）逐渐上升，而5、6号峰分离度（Y2）与7号峰的保留时间（Y3）均急剧下降。为使4、5、6号峰达到良好分离（分离度大于2），并使7号峰的保留时间小于20 分钟，通过预测效应图得出的优化范围是：温度为35～45℃，较优40～45℃；四氢呋喃与甲醇比例为0.03～0.1，较优0.05～0.1；有机相比例为30～40%，较优为35-38%。
实施例2
采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法:
选取色谱分析优化范围内条件进行苦碟子注射液的分析实验。（1）仪器与材料。岛津LC-2010A高效液相色谱仪(SHIMADZU公司)，UV检测器， UltimateTMAQ-C18色谱柱 (4.6×250 mm，5 μm，Welch Materials公司)。苦碟子注射液(通化华夏药业有限责任公司，批号 91112)，色谱甲醇，色谱乙腈，色谱四氢呋喃，色谱甲酸(山东禹王公司)，双蒸水(实验室自制)。（2）色谱条件。Ultimate AQ-C18色谱柱 (4.6×250 mm，5μm，Welch Materials 公司)；柱温度40℃；流动相：A相有机相，B相为1%(v/v)的甲酸水溶液；流速为1 mL·min-1；检测波长为348 nm；四氢呋喃与甲醇比例为0.0825、有机相比例为37.5%。（3）样品测定。取苦碟子注射液10μl，按上述条件注入液相色谱仪进行分析测定。色谱见附图5，模型验证结果见表3。
表3 各平价指标的预测值与实验值
注:偏离差 (%) = (预测值- 试验值) /预测值×100 %
从 表3中可以看出，所得各拟合方程具有良好的预测性，优化范围内的分析条件满足实验要求。
本发明的效果。以本发明优化得到的色谱分析条件，在等度洗脱的条件下，20min内就可以同时分离分析15个以上的色谱峰，通过化合物对照品确认了其中的1-7号峰，分别是(-)3, 4-二羟基咖啡酰基酒石酸（1）、咖啡酸（2）、木犀草素-7-O-β-D-葡萄糖(1→2)葡萄糖苷（3）、菊苣酸（4）、木犀草素-7-O-β-D-葡萄糖苷（5）、木犀草素-7-O-β-D-葡萄糖醛酸苷（6）和芹菜素-7-O-β-D-葡萄糖醛酸苷（7）；与从前的方法相比，不但解决了木犀草素-7-O-β-D-葡萄糖苷与木犀草素-7-O-β-D-葡萄糖醛酸苷较难分离的问题，并且苦碟子注射液中的酚酸类色谱峰也很好的分离出来，而且分析时间大大缩短，因为采用了等梯度洗脱的方法，重现性良好。
Claims (6)
1.一种采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法，其特征在于色谱分析条件如下：（1）色谱柱采用反相C18色谱柱；（2）流动相组成为：甲醇-水-四氢呋喃-酸，流动相中各成分的体积比为：四氢呋喃与甲醇比例为0.03～0.1，四氢呋喃+甲醇比例为30～40%，酸的比例为水相的0.1-2%；（3）紫外检测波长为348nm；（4）色谱柱温度为：35～45℃。
2.如权利要求1中所述的采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法，其特征在于流动相中各成分的体积比为：四氢呋喃与甲醇比例为0.05～0.1，四氢呋喃+甲醇比例为35-38%；酸的比例为水相的0.5-1%。
3.如权利要求1或2中所述采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法，其特征在于所述的酸包括但不限于甲酸、冰乙酸、三氟乙酸、三氯乙酸、磷酸、磷酸二氢钠、磷酸二氢钾、硫酸、柠檬酸、苹果酸、酒石酸。
4.如权利要求3所述采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法，其特征在于所述的酸为甲酸或冰乙酸。
5.如权利要求1中所述的采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法，其特征在于色谱柱温度为40～45℃。
6.一种采用HPLC快速分析苦碟子注射液中黄酮苷及酚酸类有效成分的方法，其特征在于色谱分析条件如下：（1）色谱柱采用反相C18色谱柱；（2）流动相组成为：四氢呋喃与甲醇比例为0.05～0.1，四氢呋喃+甲醇比例为35-38%；甲酸的比例为水相的0.5-1%；（3）紫外检测波长为348nm；（4）色谱柱温度为：40～45℃。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106706809A (zh) * | 2016-12-26 | 2017-05-24 | 河北神威药业有限公司 | 一种同时测定舒血宁注射液中多组分含量的方法 |
| CN106770828A (zh) * | 2016-12-26 | 2017-05-31 | 河北神威药业有限公司 | 一种同时测定银杏叶提取物及其制剂中多组分含量的方法 |
| CN114689729A (zh) * | 2020-12-31 | 2022-07-01 | 鲁南制药集团股份有限公司 | 一种荆防颗粒中黄酮苷类成分的检测方法 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101032541A (zh) * | 2006-07-28 | 2007-09-12 | 成都地奥九泓制药厂 | 一种紫锥菊提取物及其制备方法和含量测定方法 |
| CN103115985A (zh) * | 2013-03-07 | 2013-05-22 | 通化华夏药业有限责任公司 | Hplc-dad法同时测定苦碟子注射液中六种黄酮类成分的方法 |
| CN103163243A (zh) * | 2013-03-07 | 2013-06-19 | 通化华夏药业有限责任公司 | Hplc-dad法同时测定苦碟子注射液中五种有机酸类成分含量的方法 |
-
2014
- 2014-12-17 CN CN201410777085.4A patent/CN104458995B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101032541A (zh) * | 2006-07-28 | 2007-09-12 | 成都地奥九泓制药厂 | 一种紫锥菊提取物及其制备方法和含量测定方法 |
| CN103115985A (zh) * | 2013-03-07 | 2013-05-22 | 通化华夏药业有限责任公司 | Hplc-dad法同时测定苦碟子注射液中六种黄酮类成分的方法 |
| CN103163243A (zh) * | 2013-03-07 | 2013-06-19 | 通化华夏药业有限责任公司 | Hplc-dad法同时测定苦碟子注射液中五种有机酸类成分含量的方法 |
Non-Patent Citations (7)
| Title |
|---|
| FOAUD SALEIH R AL-SUEDE 等: "Marked antitumor activity of cat"s whiskers tea (Orthosiphon stamineus) extract in orthotopic model of human colon tumor in nude mice", 《J BIOCHEM TECH》, vol. 3, no. 5, 28 December 2012 (2012-12-28), pages 170 - 176 * |
| G. G. ZAPESOCHNAYA 等: "Dicaffeolyquinic acids from Helichrysum italicum and Achillea cartilaginea", 《CHEMISTRY OF NATURAL COMPOUNDS》, vol. 28, no. 1, 31 January 1992 (1992-01-31), pages 40 - 44 * |
| GHASEM HAGHI 等: "Simultaneous Quantification of Flavonoids and Phenolic Acids in Plant Materials by a Newly Developed Isocratic High-Performance Liquid Chromatography Approach", 《J. AGRIC. FOOD CHEM》, vol. 58, no. 20, 4 October 2010 (2010-10-04), pages 10812 - 10816 * |
| MJA SIDDIQUI 等: "Simultaneous Analysis of Bioactive Markers from Orthosiphon Stamineus Benth Leaves Extracts by Reverse Phase High Performance Liquid Chromatography", 《TROP J PHARM RES》, vol. 10, no. 1, 28 February 2011 (2011-02-28), pages 97 - 103 * |
| 何雅君: "HPLC 同时测定山楂提取物中绿原酸", 《中国中药杂志》, vol. 37, no. 6, 31 March 2012 (2012-03-31), pages 829 - 831 * |
| 潘海峰 等: "HPLC 测定山楂叶中 7 种成分的含量", 《中药材》, vol. 35, no. 6, 30 June 2012 (2012-06-30), pages 925 - 927 * |
| 闵春艳: "正交试验比较金银花药材中绿原酸与木犀草苷的乙醇提取工艺", 《中成药》, vol. 33, no. 10, 20 October 2011 (2011-10-20), pages 1815 - 1818 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106706809A (zh) * | 2016-12-26 | 2017-05-24 | 河北神威药业有限公司 | 一种同时测定舒血宁注射液中多组分含量的方法 |
| CN106770828A (zh) * | 2016-12-26 | 2017-05-31 | 河北神威药业有限公司 | 一种同时测定银杏叶提取物及其制剂中多组分含量的方法 |
| CN106706809B (zh) * | 2016-12-26 | 2019-05-24 | 河北神威药业有限公司 | 一种同时测定舒血宁注射液中多组分含量的方法 |
| CN106770828B (zh) * | 2016-12-26 | 2019-07-02 | 河北神威药业有限公司 | 一种同时测定银杏叶提取物及其制剂中多组分含量的方法 |
| CN114689729A (zh) * | 2020-12-31 | 2022-07-01 | 鲁南制药集团股份有限公司 | 一种荆防颗粒中黄酮苷类成分的检测方法 |
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