HPLC柱后衍生化法在伏格列波糖质量控制中应用的研究
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摘要
糖尿病是以慢性高血糖为特征的一组异质性代谢性疾病,由某种原因造成胰岛素绝对或相对缺乏所引起,以慢性高血糖伴碳水化合物、脂肪和蛋白质的代谢障碍为特征,糖尿病的患者人数也随着人们生活水平的提高而逐年上升。目前处于研究阶段的口服糖尿病药物有口服胰岛素制剂、促胰岛素分泌剂、胰岛素增敏剂、α-葡萄糖苷酶抑制剂等四类。
伏格列波糖是一种新型的α-葡萄糖苷酶抑制剂,由日本武田药品工业株式会社开发研制,并于1994年上市,其商品名为倍欣。与其它α-葡萄糖苷酶抑制剂相比,伏格列波糖具有活性高、剂量小、胃肠道内副作用小等优点。由于分子中并无共轭结构和生色团,伏格列波糖无法采用HPLC/UV、HPLC/FLD等分析方法进行检测。国内外研究者曾采用过HPLC/UV、HPLC/ELSD、HPLC/RID、柱前衍生-GC、柱前衍生-HPLC、HPLC柱后衍生荧光分析法等分析法。与其它分析方法相比,HPLC柱后衍生荧光分析法具有选择性好、灵敏度高等优点,适合用于伏格列波糖及其制剂的质量控制中。
本文建立了一种用于伏格列波糖及其制剂质量控制的HPLC柱后衍生荧光分析方法,并对可能影响伏格列波糖峰面积的柱后衍生因素进行了考查。
本研究主要分为两个部分：第一部分主要是HPLC柱后衍生荧光分析方法的建立及优化；第二部分是HPLC柱后衍生因素的考查。这两部分的主要内容如下：1.HPLC柱后衍生荧光分析方法的建立及优化
采用Zorbax SB-aq4.6*250mm5μm;含不同浓度辛烷磺酸钠的磷酸盐缓冲液为流动相；荧光检测器检测(激发波长为350nm,发射波长430nm,PMT设为16)；牛磺酸和高碘酸钠为衍生试剂；反应浴温度100℃,衍生反应管长20m(内径,0.5mm)；冷却浴温度15℃,冷却管长2m(内径,0.3mm)
2. HPLC柱后衍生因素的考查
本部分采用单因素实验设计考查了T形三通接法、反应浴温度、冷却浴温度、衍生试剂浓度、伏格列波糖浓度、流动相与衍生试剂流速及二者的流速比等柱后衍生因素对伏格列波糖峰面积的影响,结论如下：
T形三通的接法对伏格列波糖峰面积影响不大,但对其理论塔板数有一定的影响。
在一定范围内,伏格列波糖峰面积随着反应浴温度的升高而变大。
在一定范围内,冷却浴温度对伏格列波糖峰面积基本无影响。
伏格列波糖峰面积随着衍生试剂浓度的增加先增加再减小,随着伏格列波糖浓度的增加而变大。流动相流速、衍生试剂流速对伏格列波糖峰面积的影响较为明显。
Diabetes is a series of heterogeneous metabolic diseases with the feature of chronic high blood sugar, which is caused by absolute or relative lack of insulin for some reason and characterized by chronic high blood sugar with carbohydrates, and fat and protein metabolism disorder. With people's living standards improving year by year, the number of patients is also increasing. Currently, there are four categories of oral diabetes drugs in the research phase, and they are oral insulin preparations, insulin secretion, insulin sensitizers and a-glucosidase inhibitors.
Voglibose is a new type of a-glucosidase inhibitors, which is developed and listed in1994by Japan Takeda Pharmaceutical Industries, Ltd., and named Basen. Compared with other a-glucosidase inhibitors, voglibose has high activities, small doses and few side effects in the gastrointestinal tract. Due to no conjugate structure and chromophore group in the molecular, the voglibose could not be detected by analytical methods of HPLC/UV and HPLC/FLD. Domestic and foreign researchers have used the analysis methods of HPLC/UV, HPLC/ELSD, HPLC/RID, pre-column derivatization-GC, pre-column derivatization-HPLC and HPLC post-column derived fluorescence. Compared with other analytical methods, HPLC post-column derived fluorescence analysis is of better selectivity and higher sensitivity, which makes it suitable for the quality control of voglibose and other preparations.
In this study, a HPLC post-column derivatization fluorescence method is established for the quality control of voglibose, which may have impact on the testing of post-column derivatization factors for the peak area of voglibose.
This study is divided into two parts:the first part is mainly focused on the establishment and optimization of the HPLC post-column derived fluorescence analysis method; the second part is on the study of factors derived in the HPLC column. The main contents of these two parts are as follows:
1Establishment and optimization of the HPLC post-column derivatization fluorescence method
Zorbax SB-aq4.6*250mm5μm column is applied, with phosphate buffer containing different concentrations of sodium octanesulfonate as the mobile phase, fluorescence detector(excitation wavelength of350nm and emission wavelength of430nm, the PMT is set to16), taurine and sodium periodate as derivatization reagent, reaction bath temperature of100℃, the derivative reaction tube length of20m (inner diameter,0.5mm), cooling temperature of15℃and the cooling pipe length of2m (internal diameter,0.3mm).
2Study of factors derived in the HPLC column
This section uses the single-factor experimental design to examine the T-shaped three-way connection, the reaction temperature, the cooling temperature, derivatization reagent concentration, flow rate of voglibose and the mobile phase, and other post-column derived factors on the voglibose peak area. The conclusions are as follows:
The T-shaped three connection has few effect on the voglibose peak area, but certain impact on the theoretical plates.
Within a certain range, the voglibose peak area increases with the temperature of the reaction bath.
Within a certain range, the cooling temperature has no effect on the voglibose peak area.
The voglibose peak area is getting larger first and then smaller in accordance with the increasing derivatization reagent concentration, and larger with the concentration of voglibose increasing. The flow rate of mobile phase and the derivatization reagent have significant influences on the voglibose peak area.
伏格列波糖是一种新型的α-葡萄糖苷酶抑制剂,由日本武田药品工业株式会社开发研制,并于1994年上市,其商品名为倍欣。与其它α-葡萄糖苷酶抑制剂相比,伏格列波糖具有活性高、剂量小、胃肠道内副作用小等优点。由于分子中并无共轭结构和生色团,伏格列波糖无法采用HPLC/UV、HPLC/FLD等分析方法进行检测。国内外研究者曾采用过HPLC/UV、HPLC/ELSD、HPLC/RID、柱前衍生-GC、柱前衍生-HPLC、HPLC柱后衍生荧光分析法等分析法。与其它分析方法相比,HPLC柱后衍生荧光分析法具有选择性好、灵敏度高等优点,适合用于伏格列波糖及其制剂的质量控制中。
本文建立了一种用于伏格列波糖及其制剂质量控制的HPLC柱后衍生荧光分析方法,并对可能影响伏格列波糖峰面积的柱后衍生因素进行了考查。
本研究主要分为两个部分：第一部分主要是HPLC柱后衍生荧光分析方法的建立及优化；第二部分是HPLC柱后衍生因素的考查。这两部分的主要内容如下：1.HPLC柱后衍生荧光分析方法的建立及优化
采用Zorbax SB-aq4.6*250mm5μm;含不同浓度辛烷磺酸钠的磷酸盐缓冲液为流动相；荧光检测器检测(激发波长为350nm,发射波长430nm,PMT设为16)；牛磺酸和高碘酸钠为衍生试剂；反应浴温度100℃,衍生反应管长20m(内径,0.5mm)；冷却浴温度15℃,冷却管长2m(内径,0.3mm)
2. HPLC柱后衍生因素的考查
本部分采用单因素实验设计考查了T形三通接法、反应浴温度、冷却浴温度、衍生试剂浓度、伏格列波糖浓度、流动相与衍生试剂流速及二者的流速比等柱后衍生因素对伏格列波糖峰面积的影响,结论如下：
T形三通的接法对伏格列波糖峰面积影响不大,但对其理论塔板数有一定的影响。
在一定范围内,伏格列波糖峰面积随着反应浴温度的升高而变大。
在一定范围内,冷却浴温度对伏格列波糖峰面积基本无影响。
伏格列波糖峰面积随着衍生试剂浓度的增加先增加再减小,随着伏格列波糖浓度的增加而变大。流动相流速、衍生试剂流速对伏格列波糖峰面积的影响较为明显。
Diabetes is a series of heterogeneous metabolic diseases with the feature of chronic high blood sugar, which is caused by absolute or relative lack of insulin for some reason and characterized by chronic high blood sugar with carbohydrates, and fat and protein metabolism disorder. With people's living standards improving year by year, the number of patients is also increasing. Currently, there are four categories of oral diabetes drugs in the research phase, and they are oral insulin preparations, insulin secretion, insulin sensitizers and a-glucosidase inhibitors.
Voglibose is a new type of a-glucosidase inhibitors, which is developed and listed in1994by Japan Takeda Pharmaceutical Industries, Ltd., and named Basen. Compared with other a-glucosidase inhibitors, voglibose has high activities, small doses and few side effects in the gastrointestinal tract. Due to no conjugate structure and chromophore group in the molecular, the voglibose could not be detected by analytical methods of HPLC/UV and HPLC/FLD. Domestic and foreign researchers have used the analysis methods of HPLC/UV, HPLC/ELSD, HPLC/RID, pre-column derivatization-GC, pre-column derivatization-HPLC and HPLC post-column derived fluorescence. Compared with other analytical methods, HPLC post-column derived fluorescence analysis is of better selectivity and higher sensitivity, which makes it suitable for the quality control of voglibose and other preparations.
In this study, a HPLC post-column derivatization fluorescence method is established for the quality control of voglibose, which may have impact on the testing of post-column derivatization factors for the peak area of voglibose.
This study is divided into two parts:the first part is mainly focused on the establishment and optimization of the HPLC post-column derived fluorescence analysis method; the second part is on the study of factors derived in the HPLC column. The main contents of these two parts are as follows:
1Establishment and optimization of the HPLC post-column derivatization fluorescence method
Zorbax SB-aq4.6*250mm5μm column is applied, with phosphate buffer containing different concentrations of sodium octanesulfonate as the mobile phase, fluorescence detector(excitation wavelength of350nm and emission wavelength of430nm, the PMT is set to16), taurine and sodium periodate as derivatization reagent, reaction bath temperature of100℃, the derivative reaction tube length of20m (inner diameter,0.5mm), cooling temperature of15℃and the cooling pipe length of2m (internal diameter,0.3mm).
2Study of factors derived in the HPLC column
This section uses the single-factor experimental design to examine the T-shaped three-way connection, the reaction temperature, the cooling temperature, derivatization reagent concentration, flow rate of voglibose and the mobile phase, and other post-column derived factors on the voglibose peak area. The conclusions are as follows:
The T-shaped three connection has few effect on the voglibose peak area, but certain impact on the theoretical plates.
Within a certain range, the voglibose peak area increases with the temperature of the reaction bath.
Within a certain range, the cooling temperature has no effect on the voglibose peak area.
The voglibose peak area is getting larger first and then smaller in accordance with the increasing derivatization reagent concentration, and larger with the concentration of voglibose increasing. The flow rate of mobile phase and the derivatization reagent have significant influences on the voglibose peak area.
引文
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