CN101078712A - 扶正化瘀植物药血浆丹参素和丹酚酸b的测定方法 - Google Patents

扶正化瘀植物药血浆丹参素和丹酚酸b的测定方法 Download PDF

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CN101078712A
CN101078712A CNA200710040137XA CN200710040137A CN101078712A CN 101078712 A CN101078712 A CN 101078712A CN A200710040137X A CNA200710040137X A CN A200710040137XA CN 200710040137 A CN200710040137 A CN 200710040137A CN 101078712 A CN101078712 A CN 101078712A
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马越鸣
王天明
张永煜
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SHANGHAI MODERN CHINESE TRADITIONAL MEDICINE TECHNOLOGY DEVELOPMENT Co Ltd
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Priority to CNA200710040137XA priority Critical patent/CN101078712A/zh
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Priority to US12/451,147 priority patent/US7993928B2/en
Priority to CA2685358A priority patent/CA2685358C/en
Priority to PCT/CN2008/000866 priority patent/WO2008131648A1/zh
Priority to EP08748425.9A priority patent/EP2157427B1/en
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/9453—Cardioregulators, e.g. antihypotensives, antiarrhythmics
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    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray

Abstract

本发明公开了扶正化瘀植物药血浆丹参素和丹酚酸B的测定方法，包括：(1)哺乳类血浆样品预处理方法，将含药血浆加入已用甲醇和水活化后的Waters Oasis HLB小柱，经淋洗和洗脱后，洗脱液挥干富集，用流动相复溶后用UPLC/MS法进行检测，(2)UPLC/MS测定方法：色谱条件：色谱柱：Acquity UPLC BEH C18，2.1×100mm，流动相A：Water－Acetonitrile－Formic acid 95∶5∶0.1v/v/v，流动相B：Acetonitrile－Formic acid 100∶0.1v/v；质谱条件：电喷雾离子源(ESI)，采用负离子模式检测，质量扫描范围m/z 150～800。本发明适合用于扶正化瘀植物药的丹参素和丹酚酸B药代动力学研究。

Description

扶正化瘀植物药血浆丹参素和丹酚酸B的测定方法
技术领域
本发明属药物代谢动力学领域，特别是涉及扶正化瘀植物药血浆丹参素和丹酚酸B的测定方法。
背景技术
扶正化瘀植物药由丹参、桃仁、五味子等复方组成，具有治疗肝、肺、肾纤维化的作用，但对扶正化瘀制剂缺乏药物代谢动力学的研究，因而对体内的药效成分不清楚，难以为质量控制及指导临床合理用药提供依据，阻碍了该药进入国际市场。
到目前为止，未见有关扶正化瘀植物药药物代谢动力学的研究报道，无该复方在生物样品(包括血浆样品)中丹参素和丹酚酸B测定方法。
发明内容
本发明所要解决的技术问题是提供扶正化瘀植物药血浆丹参素和丹酚酸B的测定方法，该方法用于药代动力学研究，阐明扶正化瘀植物药丹参素和丹酚酸B的药代动力学规律。
本发明解决的技术问题通过以下技术方案来实现：
扶正化瘀植物药血浆丹参素和丹酚酸B的测定方法，包括下列步骤：
(1)哺乳类血浆样品预处理
a.将哺乳类给予扶正化瘀植物药后的含药血浆，加入加2-5mol/L磷酸混匀后，血浆与磷酸之间的体积比为1∶2-3，加入已用甲醇和水活化后的Waters Oasis HLB小柱，经水及80-100％甲醇淋洗和0.2-1％氨水甲醇洗脱后，洗脱液在25-30℃条件下挥干富集，用流动相复溶。
b.流动相复溶后用UPLC/MS法检测
色谱条件：色谱柱：Acquity UPLC BEH C18，2.1×100mm，流动相A：Water-Acetonitrile-Formic acid 95∶5∶0.1v/v/v，流动相B：Acetonitrile-Formic acid 100∶0.1v/v；质谱条件：电喷雾离子源(ESI)，采用负离子模式检测，质量扫描范围m/z 150～800。
所述步骤(1)淋洗条件为第一步用水，第二步用80-100％甲醇，洗脱条件为：0.2-1％氨水甲醇。
所述步骤(2)负离子模式检测，脱溶剂气流量为440L/h，脱溶剂气温度为300℃，锥孔气流量为50L/h，离子源温度为100℃，喷雾毛细管电压为3800V，取样锥孔电压为30V，萃取锥孔电压为2.00V，透镜电压0.1V。
本发明在固相萃取过程中，固相对分析物的吸附力大于样品母液，当样品通过固相萃取柱时，分析物及一些相近似的成分被吸附在固体表面，其他组分则随样品母液通过柱子，先用极性较大的溶剂淋洗柱子，将一些无关的成分清洗去除，最后用适当的溶剂将分析物洗脱下来，洗脱液抽干富集；利用UPLC系统，将分析物与洗脱液中的其他成分进行分离，最后通过质谱检测仪进行检测。
本发明的丹参素和丹酚酸B均为水溶性成分，采用固相萃取方法，可充分将因浆中的丹参素和丹酚酸B萃取出来，再加上使用UPLC/MS系统进行检测，使丹参素和丹酚酸B与样品中其他成分的分离度明显提高，且分析方法更加灵敏和快捷，以利于进行药代动力学研究中丹参素和丹酚酸B的血药浓度测定。
附图说明
图1.丹参素UPLC-MS色谱图。
图2.丹酚酸B UPLC-MS色谱图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
                                 实施例1
扶正化瘀植物药血浆丹参素和丹酚酸B的测定方法包括：
1、血浆样品预处理方法：将哺乳类给予扶正化瘀植物药的含药血浆先加2.17mol/L磷酸混匀后，生物样品与磷酸之间的体积比为：1∶2-3；再加入已用甲醇和水活化后的WatersOasis HLB小柱，经水及80-100％甲醇淋洗和0.2-1％氨水甲醇洗脱后，洗脱液在25℃条件下挥干富集，用流动相复溶。
2、UPLC/MS测定方法：本发明采用UPLC/MS法的分析条件色谱条件：色谱柱：AcquityUPLC BEH C18，2.1×100mm，流动相A：Water-Acetonitrile-Formic acid 95∶5∶0.1v/v/v，流动相B：Acetonitrile-Formic acid 100∶0.1v/v，按下列梯度洗脱：
Time(min)   Flow Rate(mL/min) ％A ％B
  05.00   0.3000.300   10085.0   015.0
  10.0020.0030.0035.0035.0138.00   0.3000.3000.3000.3000.3000.300   70.040.020.020.0100.0100.0   30.060.080.080.00.00.0
质谱条件：电喷雾离子源(ESI)，采用负离子模式检测；脱溶剂气流量为440L/h，脱溶剂气温度为300℃，锥孔气流量为50L/h，离子源温度为100℃，喷雾毛细管电压为3800V，取样锥孔电压为30V，萃取锥孔电压为2.00V，透镜电压0.1V。质量扫描范围m/z 150～800。
测定结果：哺乳类灌服扶正化瘀植物药后血浆中可测出丹参素和丹酚酸B(见图1-2)

Claims (3)

1.扶正化瘀植物药血浆丹参素和丹酚酸B的测定方法，包括下列步骤：
(1)哺乳类血浆样品预处理
a.将哺乳类给予扶正化瘀制剂后的含药血浆，加入加2-5mol/L磷酸混匀后，血浆与磷酸之间的体积比为1∶2-3，加入已用甲醇和水活化后的Waters Oasis HLB小柱，经水及80-100％甲醇淋洗和0.2-1％氨水甲醇洗脱后，洗脱液在25-30℃条件下挥干富集，用流动相复溶。
b.流动相复溶后用UPLC/MS法检测
色谱条件：色谱柱：Acquity UPLC BEH C18，2.1×100mm，流动相A：Water-Acetonitrile-Formic acid 95∶5∶0.1 v/v/v，流动相B：Acetonitrile-Formic acid 100∶0.1v/v；
质谱条件：电喷雾离子源(ESI)，采用负离子模式检测，质量扫描范围m/z 150～800。
2.根据权利要求1所述的扶正化瘀植物药血浆丹参素和丹酚酸B的测定方法，其特征在于：所述步骤(2)负离子模式检测，脱溶剂气流量为440L/h，脱溶剂气温度为300℃，锥孔气流量为50L/h，离子源温度为100℃，喷雾毛细管电压为3800V，取样锥孔电压为30V，萃取锥孔电压为2.00V，透镜电压0.1V。
3.根据权利要求1所述的扶正化瘀植物药血浆丹参素和丹酚酸B的测定方法，其特征在于：所述步骤(1)淋洗条件为第一步用水，第二步用80-100％甲醇，洗脱条件为：0.2-1％氨水甲醇。
CNA200710040137XA 2007-04-27 2007-04-27 扶正化瘀植物药血浆丹参素和丹酚酸b的测定方法 Pending CN101078712A (zh)

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CNA200710040137XA CN101078712A (zh) 2007-04-27 2007-04-27 扶正化瘀植物药血浆丹参素和丹酚酸b的测定方法
US12/451,147 US7993928B2 (en) 2007-04-27 2008-04-28 Detection of blood plasma Danshensu and Salvianolic acid B dissipating blood stasis botanical
CA2685358A CA2685358C (en) 2007-04-27 2008-04-28 Method of detecting blood plasma danshensu and salvianolic acid b after administration of fuzheng huayu (fzhy)
PCT/CN2008/000866 WO2008131648A1 (fr) 2007-04-27 2008-04-28 Détection de danshensu de plasma sanguin et d'acide salvianolique b d'un extrait de plante éliminant l'hémostase
EP08748425.9A EP2157427B1 (en) 2007-04-27 2008-04-28 Detection of 3-(3,4-dihydroxyphenyl)-lactic acid (danshensu) and salvianolic acid b in blood plasma after administering a botanical extract

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CN101042380A (zh) 2007-04-27 2007-09-26 上海现代中医药技术发展有限公司 扶正化淤植物药血浆五味子乙素的测定方法
CN101078712A (zh) 2007-04-27 2007-11-28 上海现代中医药技术发展有限公司 扶正化瘀植物药血浆丹参素和丹酚酸b的测定方法
CN101040907B (zh) 2007-04-27 2011-01-05 上海现代中医药技术发展有限公司 扶正化瘀植物药中丹参原料指纹图谱质量检测方法
CN101045092A (zh) * 2007-04-29 2007-10-03 上海现代中医药技术发展有限公司 一种扶正化瘀植物药组合物的应用
CN100573140C (zh) 2007-04-29 2009-12-23 上海现代中医药技术发展有限公司 扶正化瘀植物药中五味子原料指纹图谱质量检测方法

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US7989212B2 (en) 2007-04-27 2011-08-02 Shanghai Sundise Detection of blood plasma schizadrin B of dissipating blood stasis botanical
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US8334110B2 (en) 2007-04-27 2012-12-18 Shanghai Sundise Chinese Medicine Technology Development Co., Ltd. Detection of blood plasma amygdalin of dissipating blood stasis botanical
US9161959B2 (en) 2007-04-29 2015-10-20 Shanghai Sundise Chinese Medicine Technology Development Co., Ltd Use of a vegetable drug composition in the manufacturing of pharmaceutical preparation for the treatment of portal hypertension caused by hepatocirrhosis

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