CN112362782B - 一种分离维格列汀与其手性异构体的hplc方法 - Google Patents

一种分离维格列汀与其手性异构体的hplc方法 Download PDF

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CN112362782B
CN112362782B CN202011243417.2A CN202011243417A CN112362782B CN 112362782 B CN112362782 B CN 112362782B CN 202011243417 A CN202011243417 A CN 202011243417A CN 112362782 B CN112362782 B CN 112362782B
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Abstract

本发明公开了一种分离维格列汀与其手性异构体的HPLC方法。本发明提供的色谱条件以常规的C18色谱柱即可将维格列汀与其R型异构体有效分离，无需依赖较为昂贵的手性色谱柱，显著优于现有技术。

Description

一种分离维格列汀与其手性异构体的HPLC方法
技术领域
本发明属于药物分析领域，具体涉及一种分离维格列汀与其手性异构体的HPLC方法。
背景技术
糖尿病是由于胰岛素分泌及作用缺陷引起的以血糖升高为特征的慢性代谢疾病，口服的降糖药包括胰岛素增敏剂(噻唑烷二酮类和双胍类)、胰岛素分泌促进剂(非磺酰脲类、磺酰脲类胰岛素分泌促进剂)、α-葡萄糖苷酶抑制剂等。近年来，噻唑烷二酮类药物的几个主要品种包括曲格列酮、罗格列酮、吡格列酮等在临床应用过程中发现了肝损伤、心血管风险及诱发癌症风险等毒副作用，限制了这类药物的应用。新型降糖药二肽基肽酶-4抑制剂类药物具有较好的安全性和耐用性，总不良反应与安慰剂相似。维格列汀是一种口服有效的二肽基肽酶-4抑制剂，可有效改善2型糖尿病患者的血糖控制，其药理活性、降糖机理以及临床疗效已通过相关试验得到了证实，为糖尿病患者提供了一种新的用药选择。
维格列汀是一种手性化合物，其手性异构体难以避免。为了控制维格列汀的质量，有必要分析监控其手性异构体杂质的含量。维格列汀及其R型异构体的化学结构如下。
Figure BDA0002769103620000011
维格列汀及其R型异构体的分离难度非常大，现有技术都是借助于手性色谱柱(高效液相色谱法测定维格列汀R型异构体的含量，天津药学，2019年)。但是，手性色谱柱比较昂贵，一套分析方法学开发下来通常要损失若干根手性色谱柱，分析成本高。
在流动相中添加手性添加剂是一种有可能将手性异构体分离开的方法，但是该方法开发难度极高，也并非所有手性化合物都能顺利寻找到合适的手性添加剂。
发明内容
本发明旨在克服现有技术的不足，提供一种分离维格列汀与其手性异构体的HPLC方法。
本发明目的通过下面的技术方案实现：
一种分离维格列汀与其R型异构体的HPLC方法，色谱条件如下：
色谱柱：C18色谱柱(4.6mm×250mm，5μm)；
流动相A相：先配制含20μM S-(4-硝基苯基)-L-半胱氨酸的5％乙腈水溶液，再用磷酸调节pH值至3.5，临用现配；
流动相B相：95％乙腈水溶液；
洗脱模式和比例：A相和B相按照体积比70:30等度洗脱；
流速：1.0mL/min；
检测波长：210nm；
柱温：30℃；
维格列汀与其R型异构体的化学结构式如下：
Figure BDA0002769103620000021
优选地，所述的HPLC方法中进样量为10μL。
优选地，所述C18色谱柱为Agilent Eclipse Plus C18(4.6mm×250mm，5μm)。
有益效果：
本发明提供的色谱条件以常规的C18色谱柱即可将维格列汀与其R型异构体有效分离，无需依赖较为昂贵的手性色谱柱，显著优于现有技术。
附图说明
图1为本发明色谱条件对维格列汀与其R型异构体混合对照品的分离色谱图；
图2为对比色谱条件对维格列汀与其R型异构体混合对照品的分离色谱图。
具体实施方式
以下实施例是为了具体本发明的实质性技术内容，不能以下实施例记载的具体常规细节限定本发明的保护范围。
一、仪器与试药
1、仪器
美国Agilent公司1260液相色谱仪(真空脱气机、二元泵、自动进样器)。
Mettler toledo电子天平(XS105)。
2、试剂
维格列汀对照品、维格列汀R型异构体对照品的纯度均≥99％。
手性添加剂S-(4-硝基苯基)-L-半胱氨酸购自麦克林试剂，纯度≥98％。
乙腈为色谱纯，水为超纯水，乙腈水溶液的百分浓度为乙腈的体积百分浓度。
二、方法和结果
1、色谱条件
色谱柱：Agilent Eclipse Plus C18(4.6mm×250mm，5μm)；
流动相A相：先配制含20μM S-(4-硝基苯基)-L-半胱氨酸的5％乙腈水溶液，再用磷酸调节pH值至3.5，临用现配；
流动相B相：95％乙腈水溶液；
洗脱模式和比例：A相和B相按照体积比70:30等度洗脱；
流速：1.0mL/min；
检测波长：210nm；
柱温：30℃；
进样量：10μL。
2、溶液配制
混合对照品溶液：取适量流动相A相和B相，按照体积比70:30混合作为溶剂，配制成含有0.5mg/mL维格列汀对照品和0.5mg/mL维格列汀R型异构体对照品的混合对照品溶液。
维格列汀对照品溶液：取适量流动相A相和B相，按照体积比70:30混合作为溶剂，配制成含有0.1mg/mL维格列汀对照品的维格列汀对照品溶液。
维格列汀R型异构体对照品溶液：取适量流动相A相和B相，按照体积比70:30混合作为溶剂，配制成含有0.1mg/mLR型异构体对照品的维格列汀R型异构体对照品溶液。
3、进样分析
分别精密量取10μL混合对照品溶液、维格列汀对照品溶液、维格列汀R型异构体对照品溶液注入液相色谱仪，按照上述色谱条件分析，记录色谱图。结果如图1所示，在该色谱条件下，维格列汀与维格列汀R型异构体可以达到基线分离，分离度大于2.0。
为了体现手性添加剂S-(4-硝基苯基)-L-半胱氨酸对维格列汀和维格列汀R型异构体的分离效果，同时设置了对比试验，该对比试验的唯一区别是色谱条件中的流动相A相中不含有S-(4-硝基苯基)-L-半胱氨酸，其他完全一致。结果如图2所示，在该色谱条件下，维格列汀与维格列汀R型异构体共洗脱，无法分离。
上述试验结果表明，本发明提供的色谱条件以常规的C18色谱柱即可将维格列汀与其R型异构体有效分离，无需依赖较为昂贵的手性色谱柱，显著优于现有技术。
上述实施例是为了具体本发明的实质性技术内容，不能以上述实施例记载的具体常规细节限定本发明的保护范围。

Claims (2)

1.一种分离维格列汀与其R型异构体的HPLC方法，其特征在于，色谱条件如下：
色谱柱：Agilent Eclipse Plus C18，规格为4.6mm×250mm、5μm；
流动相A相：先配制含20μM S-(4-硝基苯基)-L-半胱氨酸的5％乙腈水溶液，再用磷酸调节pH值至3.5，临用现配；
流动相B相：95％乙腈水溶液；
洗脱模式和比例：A相和B相按照体积比70:30等度洗脱；
流速：1.0mL/min；
检测波长：210nm；
柱温：30℃；
维格列汀与其R型异构体的化学结构式如下：
Figure FDA0003455628480000011
2.根据权利要求1所述的HPLC方法，其特征在于：进样量为10μL。
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