摘要
第一部分基于中空纤维液相微萃取的4种莨菪碱活性成分含量测定及优势构象确定
目的：以氢溴酸山莨菪碱、硫酸阿托品、氢溴酸东莨菪碱、丁溴酸东莨菪碱为模型化合物优化液相微萃取前处理条件,建立莨菪碱的液相微萃取一高效液相色谱含量测定方法；为复杂样品的分离、纯化和浓缩提供一种有效、简便、环境友好的样品前处理方法,讨论模型化合物的萃取规律及萃取机理；利用协同液相微萃取方法初步探讨氢溴酸山莨菪碱、硫酸阿托品、氢溴酸东莨菪碱和丁溴酸东莨菪碱的优势构象；探讨中空纤维与莨菪碱形成电荷转移超分子对萃取效果的影响以及4种莨菪碱结构中N原子的亲电能力。
方法：利用简易液相微萃取装置,优化被分析物的液相微萃取条件,包括溶剂载体中空纤维的种类、萃取溶剂、供相与接受相、搅拌速度和萃取时间。模型化合物在优化的液相微萃取条件下被萃取后,收集接受相进入高效液相色谱仪进行分析。模型化合物氢溴酸山莨菪碱、硫酸阿托品、氢溴酸东莨菪碱、丁溴酸东莨菪碱的优化液相微萃取条件：聚丙烯纤维作溶剂载体,二甲苯：正庚醇(7：3)混合溶剂作萃取溶剂,10-3 mol／L氨水样品溶液为供相,5×10-3 mol／L盐酸溶液为接受相,搅拌速度为1800r／min,萃取时间为100min；色谱条件为：C18柱,流动相为甲醇：水(含0.04mol／L醋酸钠,0.015%冰醋酸,0.02%三乙胺)为35：65,流速0.8 ml／min,柱温30℃,检测波长215nm。
结果：氢溴酸山莨菪碱、硫酸阿托品、氢溴酸东莨菪碱、丁溴酸东莨菪碱分别在0.05μg／ml～5μg／ml,5ug/ml～25μg/ml范围内呈线性,相关系数大于0.99,精密度小于7%,消旋山莨菪片和颠茄片平均回收率分别为95%～119%和93%～95%,氢溴酸山莨菪碱和丁溴酸东莨菪碱检测限为0.03μg／ml,硫酸阿托品和氢溴酸东莨菪碱检测限为0.01μg／ml,消旋山莨菪片中山莨菪碱含量为4.9mg/片,颠茄片中山莨菪碱含量为20.3μg/片,阿托品含量为72.8μg/片,东莨菪碱含量为2.1μg/片。
结论：该方法能有效去除制剂中杂质的干扰,提高了选择性,进而测定制剂中的化合物,纯化和浓缩效果好,有机溶剂消耗少,是一种快速、有效、环境友好的成分分析前处理方法。
第二部分液相微萃取在硫酸阿托品和氢溴酸东茛菪碱蛋白结合参数测定中的应用
目的：建立中空纤维液相微萃取(HF-LPME)-高效液相色谱法(HPLC)测定硫酸阿托品和氢溴酸东莨菪碱蛋白结合率及蛋白结合参数的方法。
方法：以聚丙烯为中空纤维,正庚醇：二甲苯(30：70)混合溶剂为萃取溶剂,pH7.4的磷酸盐缓冲样品溶液为供相,5×10-2mol／L盐酸溶液为接受相,在数控保温箱37℃静置5个小时进行微萃取,然后通过HPLC测定药物与蛋白结合后游离的浓度,进而计算出药物蛋白结合率,并利用Klotz方程,得到药物与BSA的结合常数和结合位点数。
结果：硫酸阿托品和氢溴酸东莨菪碱均在1～25μg／mL范围内线性关系良好,r>0.99。硫酸阿托品和氢溴酸东莨菪碱蛋白结合率范围分别为17%～30%,11%～34%,与BSA的结合常数分别为：2899.1、626.1,结合位点数分别为：0.3、0.6。
结论：该方法简便易行,能够有效测定硫酸阿托品和氢溴酸东莨菪碱蛋白结合率及蛋白结合参数。
Determination of Four Hyoscyamine Active Components and Study on Preferred Conformation on the Basis of Hollow Fiber Liquid Phase Microextraction
Objectives:The method of determination of hyoscyamine in praeparatum on the basis of the compounds such as anisodamine hydrobromide, atropine sulphate, scopolamine hydrobromide, scopolamine butylbromide by liquid phase microextraction with back extraction (LPME/BE) combined with high performance liquid chromatography (HPLC) was established in order to supply a effective, convenient and environment-friendly method of pre-processing for complicated sample separating, clearing, enriching. Besides, the regularity and mechanism of microextraction were researched. The preferred conformations of anisodamine hydrobromide, atropine sulphate, scopolamine hydrobromide, scopolamine butylbromide were researched by cooperative liquid phase microextraction (CLPME) initially. The effect of charge transfer supermolecule formed by hollow fiber and hyoscyamines on the extraction efficiency was researched in the extraction procedure and the electrophilic capability of N atom in formation of the four compounds was studied.
Methods:A simple LPME system was adopted to select and optimize the LPME conditions, such as the solvent carrier-hollow fiber, organic solvent, donor and acceptor phase, stirring rate and extraction time. The analytes were extracted by this self-made LPME system and analyzed by HPLC. The optimal extraction conditions of anisodamine hydrobromide, atropine sulphate, scopolamine hydrobromide, scopolamine butylbromide were as follow, polypropylene fiber as solvent carrier, mixture of dimethyl benzene:n-enanthol (7:3) as organic solvent,10-3 mol/L ammonia water as the donor phase,5×10-3 mol/L acid hydroc as the acceptor solution, a stirring rate of 1800 r/min and extraction time of 100 min. The HPLC column was a C18 column. The optimized mobile phase of HPLC was a mixture of methanol and water (35:65, v/v, including 0.04mol/L natrium aceticum,0.015% acetic acid,0.02% triethylamine) flowing at a rate of 0.8 ml/min and the temperature of the column was 30℃. The analytes were analyzed at 215 nm.
Results:The linear calibration curves of anisodamine hydrobromide, atropine sulphate, scopolamine hydrobromide, scopolamine butylbromide were obtained in the concentration ranges of 0.05μg/ml～5μg/ml,5μg/ml～25μg/ml, r>0.99, RSD was lower than 7%. The relative recoveries of raceanisodamine tablets and belladonna tablets were in the range of 95%～119% and 93%～95%, respectively. The detection limits of anisodamine hydrobromide and scopolamine butylbromide were 0.03μg/ml and the detection limits of atropine sulphate, scopolamine hydrobromide were 0.01μg/ml. The content of anisodamine hydrobromide in raceanisodamine tablets was 4.9 mg/tablet and the content of anisodamine hydrobromide, atropine sulphate, scopolamine hydrobromide in belladonna tablets were 20.3μg/tablet,72.8μg/tablet,2.1μg/tablet, respectively.
Conclusion:LPME was applied to eliminate the interference of matrices, decrease waste of organic solvent and increase the selectivity, and the results showed the possibility of this method for the determination of compounds in praeparatum. A good effect of purification and concentration was obtained by LPME. Being friendly to environment, it is a quick and effective technique for sample pre-processing.
Application of Liquid Phase Microextraction on Determination of Protein Binding Parameters of Atropine Sulfate and Scopolamine Hydrobromide
Objective:To establish the method of hollow fiber-liquid phase microextraction-high performance liquid chromatography (HF-LPME-HPLC) to determine the protein binding rates and protein binding parameters of atropine sulfate and scopolamine hydrobromide.
Method:Polypropylene (PP) was used as hollow fiber; o-enanthol-dimethyl benzene (30:70) as extractive solvent; pH7.4 phosphate buffered solution (PBS) of sample as donor phase; 5×10-2 mol/L hydrochloric acid as acceptor phase. The drugs were extracted without stirring in 37℃for 5 hours, then determined the concentration of dissociation by high performance liquid chromatography to calculate the protein binding rates. The binding constant and the number of binding sites of drug and BAS were obtained by the Klotz equation.
Result:The linear correlation of atropine sulfate and scopolamine hydrobromide were good in range of 1～25μg/mL, r>0.99. The protein binding rates of atropine sulfate and scopolamine hydrobromide were 17%～30%,11%～34%, respectively. The binding constants with BAS were 2899.1,626.1, respectively. The number of binding sites were 0.3,0.6, respectively.
Conclusion:The method was simple and convenient. The protein binding rates and protein binding parameters of atropine sulfate and scopolamine hydrobromide were determined with the method effectively.
引文
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