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CN101334386B - 扶正化淤植物药血浆苦杏仁苷的测定方法 - Google Patents

扶正化淤植物药血浆苦杏仁苷的测定方法 Download PDF

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CN101334386B
CN101334386B CN2007100401399A CN200710040139A CN101334386B CN 101334386 B CN101334386 B CN 101334386B CN 2007100401399 A CN2007100401399 A CN 2007100401399A CN 200710040139 A CN200710040139 A CN 200710040139A CN 101334386 B CN101334386 B CN 101334386B
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马越鸣
王天明
张永煜
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Abstract

本发明涉及扶正化淤植物药血浆苦杏仁苷的测定方法，包括步骤：(1)将哺乳类给予扶正化淤植物药后的含药血浆，先加2-5mol/L磷酸混匀后，血浆与磷酸体积比为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；(2)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 acid100∶0.1v/v；质谱条件：电喷雾离子源(ESI)，采用正离子模式检测，质量扫描范围m/z 150～800；本发明适合用于扶正化淤植物药苦杏仁苷的药代动力学研究。

Description

扶正化淤植物药血浆苦杏仁苷的测定方法
技术领域
本发明属药物代谢动力学领域，特别是涉及扶正化淤植物药血浆苦杏仁苷的测定方法。
背景技术
扶正化淤植物药由丹参、桃仁、五味子等复方组成，具有治疗肝、肺、肾纤维化的作用，但对扶正化淤植物药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，未见有关扶正化淤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)中苦杏仁苷的测定方法。
发明内容
本发明所要解决的技术问题是提供扶正化淤植物药血浆苦杏仁苷的测定方法，该方法用于药代动力学研究，阐明扶正化淤植物药苦杏仁苷的药代动力学规律。
本发明解决的技术问题通过以下技术方案来实现：
扶正化淤植物药血浆苦杏仁苷的测定方法，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淤植物药后的含药血浆，先加2-5mol/L磷酸混匀后，血浆与磷酸体积比为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.UPLC/MS法检测；
(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 acid100∶0.1v/v；质谱条件：电喷雾离子源(ESI)，采用正离子模式检测，质量扫描范围m/z 150～800。
所述步骤(2)电喷雾离子源，采用正离子模式检测；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。
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。
本发明的苦杏仁苷为水溶性成分，采用固相萃取方法，可充分将样品中的苦杏仁苷萃取出来，再加上使用UPLC/MS系统进行检测，使苦杏仁苷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苦杏仁苷的血药浓度测定。
附图说明
图1苦杏仁苷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
一种血浆中扶正化淤植物药的苦杏仁苷测定方法包括：
1、血浆样品预处理方法：将哺乳类给予扶正化淤植物药后的含药血浆先加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：Acquity UPLCBEH 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：
Figure G200710040139901D00031
质谱条件：电喷雾离子源(ESI)，采用正离子模式检测；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：哺乳类灌服扶正化淤植物药后血浆中可测出苦杏仁苷(见图1)

Claims (2)

1.扶正化瘀植物药血浆苦杏仁苷的测定方法，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，先加2.17mol/L磷酸混匀后，血浆与磷酸体积比为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℃条件下挥干富集，用流动相复溶；
b.UPLC/MS法检测；
(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，按下列梯度洗脱：
Figure FSB00000726055400011
质谱条件：电喷雾离子源(ESI)，采用正离子模式检测，质量扫描范围m/z150～800。
2.根据权利要求1所述的扶正化瘀植物药血浆苦杏仁苷的测定方法，其特征在于：所述步骤(2)电喷雾离子源，采用正离子模式检测；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/z150～800。
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