高效液相色谱/蒸发光散射检测法（HPLC/ELSD）测定三七药材及其制剂中三七皂苷R_1和人参皂苷R_(g1)、R_(b1)的含量
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摘要
三七Panax notoginseng(Burk．) F.H.Chen是我国名贵的中药材，具有化瘀止血、活血定痛的功效，被誉为血症良药、伤科要药。三七含三七皂苷、黄酮苷、槲皮素、槲皮苷、β-谷甾醇和氨基酸等。三七的药理活性成分的研究主要集中在三七皂苷成分，具有广泛的药理作用。三七中所含的皂苷与人参相似，为达玛烷系四环三萜类皂苷，水解后主要得到人参三醇和人参二醇。总皂苷含量可达8～12％，主要为人参皂苷R_(b1)、R_(g1)、R_(g2)，并含少量R_a、R_(b2)、R_d、R_e等，另含三七皂苷R_1、R_2等。三七常用于复方中，对三七皂苷成分分析，成为了评价其药品质量的重要方法。复方丹参滴丸是由丹参、三七和冰片等三味中药，经科学方法提取有效成分精制而成，具有活血化瘀、理气止痛的功效，是治疗心血管疾病的常用药。
高效液相色谱-蒸发光散射检测法(HPLCfELSD)为近年来分析测定皂苷类成分的新方法。ELSD为质量型检测器，它对缺乏紫外吸收结构成分的检测有独到之处。本研究工作采用高效液相色谱-蒸发光散射检测法，对三七药材及复方丹参滴丸中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1进行了含量测定。在样品预处理中，采用固相萃取技术(SPE)直接对样品水溶液进行净化和富集。研究结果可为含三七制剂的成分测定及质量控制提供有益的借鉴。
一、三七药材中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1的含量测定
建立三七药材中人参皂苷R_(g1)和R_(b1)，三七皂苷R_1的含量测定方法。应用高效液相色谱法-蒸发光散射检测器(HPLC—ELSD)，结合固相萃取技术对样品水溶液进行预处理，Hypersil NH_2柱，流动相为乙腈-异丙醇-10mmol·L~(-1)醋酸铵水溶液(冰醋酸调pH5.0)(75：20：5)；流速：0.6ml·min~(-1)，测定了三七药材中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1的含量。该方法测定此三种成分的线性范围为1.0～10.01μg，加样回收率为95.5～102.5％，日内精密度≤2％、日间精密度≤4％。实验方法简便准确，可为三七的含量测定方法提供借鉴。
二、复方丹参滴丸中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1的含量测定
建立中药制剂中人参皂苷R_(g1)和R_(b1)，三七皂苷R_1的含量测定方法。色谱条件同上，对复方丹参滴丸中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1进行了测定。该方法测定此三种皂苷的线性范围为1.0～10.0μg，加样回收率为95.3～100.4％，日内精密度≤2％、日间精密度≤4％。复方丹参滴丸以水溶性成分为主，待测成分极性亦较大，如何除去其他成分对测定的干扰，是预处理中首要解决的问题。采用固相萃取技术对样品进行预处理，除去干扰成分的效果较好，而且具有样品用量少、操作步骤简单、快捷等特点。此方法简便准确，可为三七制剂含量测定的预处理方法之一。
摘 要
三、使用次数对固相革取柱柱效影响的考察
取同一样品的续滤液，加于5根OSD固相革取柱，进行预处理。然后，用
甲醇 10ml对柱进行冲洗，再进行下一轮使用，连续重复使用固相革取柱 8次。
以人参皂昔 Rb；为指标，进行测定，考察其峰面积与使用次数之间的关系。使用
单因素方差分析对结果进行统计，方差齐性检验：P二0．348＞0刀5，认为总体方差
相等；不同使用次数测得的峰面积之间卜0刀刃＞0刀5，可认为在实验所采用的次
数内，不影响测定结果。
Radix notoginseng is a rare traditional Chinese herb widely used in the oriental world with the indication of promoting blood circulation to remove blood stasis and stopping bleeding, alleviating pain. It is an important traditional drug for various bleeding or blood stasis and tranma. The pharmacological research was concentrated on the saponins and many activity of them have been tested. Radix notoginseng contains a mixture of structurally similar saponins which represents the main active contituents of it. The most saponins in it are classed into dammarane type 4 rings triterpenes, hydrolyzates of which are panaxadiol and panaxatriol. Notoginseng contains about 8~12% by weight of saponins which comprise of ginsenosides such as Rg1> R^ Rb1> Rb2, Ras Rd, Re and notoginsenosides such as R,> R2. Notoginseng is usually used as an ingredient in a traditional Chinese complex prescription. The analysis of saponins is an important method to evaluate the quality of notoginseng and its Chinese medicine preparations. Denshen Dropping Pill in complex prescription is composed of Radix Salviae Miltiorrhizae, Radix Notoginseng and Borneolum Syntheticum, manufactured by modern pharmaceutics. It possesses the pharmacological action of promoting blood circulation to dissipate blood stasis, regulating Qi and relieving pain. In clinical therapy it is an useful drug for cardiovascular diseases.
High performance liquid chromatography-evaparative light scattering detector(HPLC/ELSD) is a new method to analyse saponins. ELSD is a universal detector with superiority to determining chemical constituents without ultraviolet absorption structure. In this paper a HPLC/ELSD method has been established to assay ginsenoside Rg1- Rb1 and notoginsenoside R, in Radix Notoginseng and its preparation. In sample pretreament Solid Phase Extract(SPE) is used to purify the aqueous extract. This work can be an useful example for analysis and quality controll of preparations containing notoginseng. 1. Assay of ginsenoside Rg1> Rb1 and notoginsenoside R, in Radix Notoginseng A method has been established to assay Ginsenoside Rg1 and Rb1,Notoginsenoside R, in Radix Notoginseng by HPLC/ELSD with Solid Phase Extraction. The three saponins can be separated from each other and determinated on a Hypersil NH2 column(200mmX4.0mm15p m) with the mobile phase acetonitrile-isopropyl alcohol-10mM ammonium acetate buffer
solution(pH 5.0)(75:20:5);flow rate 0.6mL/min. The linear range was from 1.0 to 10.0|jg for ginsenoside Rg1 and Rb1,notoginsenoside R^The average recoveris for them were 95.5~102.5%.The RSD for inter-day and intra-day were less than 2% and 4%. The method is concise and accurate.
2. Assay of Ginsenoside Rg1and Rb1,Notoginsenoside R, in Danshen Dropping Pill by HPLC/ELSD
A method has been established for purifying and assay. Ginsenoside Rg1 and Rb1, notoginsenoside R, in Danshen Dropping Pill were derivatizid with SPE, then assayed by HPLC/ELSD.The chromatogrphic and ELSD conditions all are the same as above, he linear range was from 1.0 to 10.Dug for ginsenoside Rg1 and Rb1, notoginsenoside R^ The average recoveris for them were 95.3~100.4%.The RSD for them for inter-day and intra-day were less than 2% and 4%.
The main constituents in Danshen Dropping Pill are water soluble and the object analytes are polar compounds. How to remove the interference is the first question to solute in sample pretreament. To purify the sample with SPE we can obtain the good performance with superiority of using fewer sample dosage and solvent, concise operation.
3. Study on the re-usability of the SPE cartridges
We took the same sample and processed them in 8 times in the same SPE cartridge. The peak area of ginsenoside Rb1 was determined. The data of peak area and the re-using times of cartridge was processed by one-way ANOVA. The result showes that in 8-time experiments the efficacy SPE cartridge has not significant different. A considerable cost-saving may be achieved by re-using the SPE cartridges.
高效液相色谱-蒸发光散射检测法(HPLCfELSD)为近年来分析测定皂苷类成分的新方法。ELSD为质量型检测器，它对缺乏紫外吸收结构成分的检测有独到之处。本研究工作采用高效液相色谱-蒸发光散射检测法，对三七药材及复方丹参滴丸中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1进行了含量测定。在样品预处理中，采用固相萃取技术(SPE)直接对样品水溶液进行净化和富集。研究结果可为含三七制剂的成分测定及质量控制提供有益的借鉴。
一、三七药材中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1的含量测定
建立三七药材中人参皂苷R_(g1)和R_(b1)，三七皂苷R_1的含量测定方法。应用高效液相色谱法-蒸发光散射检测器(HPLC—ELSD)，结合固相萃取技术对样品水溶液进行预处理，Hypersil NH_2柱，流动相为乙腈-异丙醇-10mmol·L~(-1)醋酸铵水溶液(冰醋酸调pH5.0)(75：20：5)；流速：0.6ml·min~(-1)，测定了三七药材中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1的含量。该方法测定此三种成分的线性范围为1.0～10.01μg，加样回收率为95.5～102.5％，日内精密度≤2％、日间精密度≤4％。实验方法简便准确，可为三七的含量测定方法提供借鉴。
二、复方丹参滴丸中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1的含量测定
建立中药制剂中人参皂苷R_(g1)和R_(b1)，三七皂苷R_1的含量测定方法。色谱条件同上，对复方丹参滴丸中人参皂苷R_(g1)、R_(b1)和三七皂苷R_1进行了测定。该方法测定此三种皂苷的线性范围为1.0～10.0μg，加样回收率为95.3～100.4％，日内精密度≤2％、日间精密度≤4％。复方丹参滴丸以水溶性成分为主，待测成分极性亦较大，如何除去其他成分对测定的干扰，是预处理中首要解决的问题。采用固相萃取技术对样品进行预处理，除去干扰成分的效果较好，而且具有样品用量少、操作步骤简单、快捷等特点。此方法简便准确，可为三七制剂含量测定的预处理方法之一。
摘 要
三、使用次数对固相革取柱柱效影响的考察
取同一样品的续滤液，加于5根OSD固相革取柱，进行预处理。然后，用
甲醇 10ml对柱进行冲洗，再进行下一轮使用，连续重复使用固相革取柱 8次。
以人参皂昔 Rb；为指标，进行测定，考察其峰面积与使用次数之间的关系。使用
单因素方差分析对结果进行统计，方差齐性检验：P二0．348＞0刀5，认为总体方差
相等；不同使用次数测得的峰面积之间卜0刀刃＞0刀5，可认为在实验所采用的次
数内，不影响测定结果。
Radix notoginseng is a rare traditional Chinese herb widely used in the oriental world with the indication of promoting blood circulation to remove blood stasis and stopping bleeding, alleviating pain. It is an important traditional drug for various bleeding or blood stasis and tranma. The pharmacological research was concentrated on the saponins and many activity of them have been tested. Radix notoginseng contains a mixture of structurally similar saponins which represents the main active contituents of it. The most saponins in it are classed into dammarane type 4 rings triterpenes, hydrolyzates of which are panaxadiol and panaxatriol. Notoginseng contains about 8~12% by weight of saponins which comprise of ginsenosides such as Rg1> R^ Rb1> Rb2, Ras Rd, Re and notoginsenosides such as R,> R2. Notoginseng is usually used as an ingredient in a traditional Chinese complex prescription. The analysis of saponins is an important method to evaluate the quality of notoginseng and its Chinese medicine preparations. Denshen Dropping Pill in complex prescription is composed of Radix Salviae Miltiorrhizae, Radix Notoginseng and Borneolum Syntheticum, manufactured by modern pharmaceutics. It possesses the pharmacological action of promoting blood circulation to dissipate blood stasis, regulating Qi and relieving pain. In clinical therapy it is an useful drug for cardiovascular diseases.
High performance liquid chromatography-evaparative light scattering detector(HPLC/ELSD) is a new method to analyse saponins. ELSD is a universal detector with superiority to determining chemical constituents without ultraviolet absorption structure. In this paper a HPLC/ELSD method has been established to assay ginsenoside Rg1- Rb1 and notoginsenoside R, in Radix Notoginseng and its preparation. In sample pretreament Solid Phase Extract(SPE) is used to purify the aqueous extract. This work can be an useful example for analysis and quality controll of preparations containing notoginseng. 1. Assay of ginsenoside Rg1> Rb1 and notoginsenoside R, in Radix Notoginseng A method has been established to assay Ginsenoside Rg1 and Rb1,Notoginsenoside R, in Radix Notoginseng by HPLC/ELSD with Solid Phase Extraction. The three saponins can be separated from each other and determinated on a Hypersil NH2 column(200mmX4.0mm15p m) with the mobile phase acetonitrile-isopropyl alcohol-10mM ammonium acetate buffer
solution(pH 5.0)(75:20:5);flow rate 0.6mL/min. The linear range was from 1.0 to 10.0|jg for ginsenoside Rg1 and Rb1,notoginsenoside R^The average recoveris for them were 95.5~102.5%.The RSD for inter-day and intra-day were less than 2% and 4%. The method is concise and accurate.
2. Assay of Ginsenoside Rg1and Rb1,Notoginsenoside R, in Danshen Dropping Pill by HPLC/ELSD
A method has been established for purifying and assay. Ginsenoside Rg1 and Rb1, notoginsenoside R, in Danshen Dropping Pill were derivatizid with SPE, then assayed by HPLC/ELSD.The chromatogrphic and ELSD conditions all are the same as above, he linear range was from 1.0 to 10.Dug for ginsenoside Rg1 and Rb1, notoginsenoside R^ The average recoveris for them were 95.3~100.4%.The RSD for them for inter-day and intra-day were less than 2% and 4%.
The main constituents in Danshen Dropping Pill are water soluble and the object analytes are polar compounds. How to remove the interference is the first question to solute in sample pretreament. To purify the sample with SPE we can obtain the good performance with superiority of using fewer sample dosage and solvent, concise operation.
3. Study on the re-usability of the SPE cartridges
We took the same sample and processed them in 8 times in the same SPE cartridge. The peak area of ginsenoside Rb1 was determined. The data of peak area and the re-using times of cartridge was processed by one-way ANOVA. The result showes that in 8-time experiments the efficacy SPE cartridge has not significant different. A considerable cost-saving may be achieved by re-using the SPE cartridges.
引文
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