【作者】 安普丽；

【导师】 蒋晔；

【作者基本信息】 河北医科大学 ， 药物分析学， 2009， 硕士

【摘要】 BPs是近20年来发展起来的抗代谢性骨病的一类新药,临床上主要用于治疗骨质疏松症、变形性骨炎、恶性肿瘤骨转移引起的高钙血症和骨痛症等。由于BPs具有极性强、易电离、大部分无生色团、不挥发、生物利用度低等性质,给该类药物的分析带来一定的困难。目前,在该类药物的质量控制中仍然采用钼蓝比色法,该方法是将BPs氧化成正磷酸盐后,进行磷钼蓝比色,其有关物质（磷酸盐、亚磷酸盐）及还原性辅料对测定结果均有影响,专属性差。虽然已有文献报道专属的HPLC-ELSD方法,但其灵敏度仍然较低,仅适用于该类药物的原料药或制剂的分析,不能满足生物样品中痕量BPs检测的要求。少数含伯胺基的BPs,如Alen、Pami等,通过对其氨基衍生,进行色谱分析,提高了灵敏度,能够满足痕量生物样品分析的要求。但这些方法不适合绝大多数不含衍生基团的BPs,且操作繁琐、费时。本文课题的目的是建立专属的、灵敏的BPs分析方法,为该类药物的质量控制提供可靠的、更完善的分析手段。本课题主要从以下两个部分进行了研究:第一部分新型OPM的形成及其在药物分析中的应用一、新型OPM的形成机理及性质的研究目的:研究新型OPM的形成机理及其性质,如配比、摩尔吸收系数等。方法:酸性条件下,BPs与MoO反应生成具有较强紫外吸收的OPM;采用紫外分光光度计扫描,确定最大吸收处波长;采用Job法和平行移动法确定OPM分子结构中BPs和Mo的配比。结果:新型OPM较稳定;OPM分子结构中BPs和Mo的配比均为1:5,Alen, Pami, Zole, Eti, Inca和Iban与MoO形成的OPM的摩尔吸收系数分别为8.61×10³、9.58×10³、6.89×10³、7.53×10³、8.08×10³、8.80×10³ (L·mol·1·cm^-1)。不论BPs分子结构中是否含有氨基,BPs与MoO在酸性条件下反应均能形成OPM。结论:在酸性条件下,BPs与MoO反应生成稳定的、强紫外吸收的络合物-OPM。OPM分子的中心原子是有机膦原子。二、基于新型OPM的分光光度法在BPs制剂分析中应用目的:建立灵敏的、专属的分光光度法,分析制剂中BPs含量和小剂量伊班膦酸钠片剂的溶出度。方法:依据BPs和MoO在酸性条件下反应生成的OPM具有较强紫外吸收的性质,采用紫外分光光度法,检测波长为254nm。结果:OPM较稳定,室温放置24h最大吸收强度没有变化;以试剂空白为参比,OPM在254nm处的吸收强度与BPs浓度成良好的线性关系,Alen、Pami、Zole、Eti、Inca和Iban线性范围分别为5.00～50.0、4.92～49.2、5.26～52.6、5.00～50.0、5.16～51.6、4.92～49.2μg·ml-1 （r≥0.9989）,检测限分别为0.677、0.916、0.710、0.607、0.810、0.781μg·mL^-1,定量限分别为0.203、0.275、0.213、0.182、0.243、0.234μg·mL^-1,平均回收率为96.8～102.6% （n=3）,RSD小于1.5%;Iban片剂在8分钟时溶出度达到86%,10分钟时基本完全溶出。结论:该方法专属性好、灵敏度高、简便快速,适用于BPs制剂的分析和小剂量Iban片剂溶出度的分析。该方法与传统的磷钼蓝比色法相比较,不但不受磷酸盐、亚磷酸盐和还原性辅料的干扰,而且简便快速,不需要复杂的样品预处理。第二部分BPs的色谱分析方法的应用研究一、CE-间接紫外检测法测定Eti和Iban片剂含量目的:建立简便、快速、经济的CE-间接紫外检测方法,用于Eti和Iban的制剂分析。方法:以未涂层熔融石英毛细管柱为分离通道;背景电解质为7mmol·L^-1苯甲酸钠溶液;运行缓冲溶液为7mmol·L^-1磷酸二氢钾（pH 8.0）;分离电压为20kV;进样电压为10kV,进样时间为5s;检测波长为224nm。结果:Eti和Iban在62.60～1002μg·mL^-1和61.00～975μg·mL^-1的浓度范围内线性关系良好（r均大于0.999）,检测限分别为7.825μg·mL^-1和7.744μg·mL^-1,平均回收率在99.5%～100.3%之间,RSD小于1.5%（n=3）。结论:采用CE-间接紫外分离检测Eti和Iban的方法简便、快速、经济,适用于Eti和Iban制剂的常规分析,为该药物的质量控制提供了一个新的、专属的、灵敏的分析方法。二、离子对RP-HPLC-ELSD法测定Iban制剂的含量目的:建立专属的、灵敏的分析Iban制剂含量的离子对RP-HPLC-ELSD方法。方法:以UltimateTM XB-C18（4.6×250mm, 5μm）色谱柱为固定相,流动相为20mM三乙胺（用冰醋酸调pH至8.5）-甲醇-乙腈（90:2:8）,流速为1.0ml·L^-1,柱温为室温,检测器为ELSD。结果:Iban在30.70～980.0μg·L^-1的浓度范围内线性关系良好,回归方程为lgA=1.960 lgC+2.047 （r=0.999）,最低检测限为7.675μg·mL^-1,平均回收率为98.9～100.5%,RSD小于1.1%。结论:本文建立的RP-HPLC-ELSD方法专属性好、灵敏度高。与文献报道的RP-HPLC-ELSD方法相比较,由于采用了分子量小、挥发性更强的三乙胺作为离子对试剂,提高了检测灵敏度,为Iban的生产及制剂过程中的质量控制提供了可靠的分析手段。三、在线柱后光化学反应离子对RP-HPLC-UV法测定Zole制剂的含量目的:建立灵敏的、专属的在线柱后光化学衍生离子对RP-HPLC-UV法测定Zole制剂含量的方法。方法:以Phenomenon C18色谱柱为固定相,流动相为三乙胺（20mM用冰醋酸调pH为7.0）-甲醇（99:1）,流速为1mL·min^-1,柱温为室温。色谱流出液与过硫酸钾混合后流经一聚四氟乙烯盘管制成的光化学反应器时,Zole被氧化成正磷酸盐,正磷酸盐再与MoO、维生素C反应生成磷钼杂多络合物,用紫外-可见检测器检测,检测波长为650nm。结果:实现了Zole及其有关物质（磷酸盐和亚磷酸盐）分离和检测。Zole在20.00～250.0μg·mL^-1浓度范围内线性关系良好（r=0.999）,检测限为1μg·mL^-1。对于注射用Zole的平均回收率为98.7～101.0%,RSD小于2.1%,对于Zole注射液回收率为99.6～100.4%,RSD小于1.6%。结论:本文建立的离子对RP-HPLC-UV专属性好、灵敏度高、分析速度快,不需要繁琐的样品预处理过程,适用于Zole制剂的分析,为该药物制剂的常规的分析及质量控制提供了有效地可靠的分析手段。更多还原

【Abstract】 BPs are new category of bone resorption inhibitor drugs developing in the recent 20 years, which had been widely used in the management of skeletal disorder, including malignant hypercalcemia, postmenopausal osteoporosis, Paget’s disease and so on.The assays for BPs presented some difficulties, since the flowing characters: strong polar, ionic, low bioavailability and so on. Molybdenum blue spectrophotometry method is still used in routine quality control analysis of BPs, in which BPs were oxidized to orthophosphate ions firstly, so it was inevitable that trace phosphate, phosphite and excipients interfere with analysis of BPs, thus the precision and accuracy were low relatively. Although ion-pair HPLC-ELSD method for identification and routine determination of BPs was established, the sensitivity was so low that was not suitable for trace quantity biological specimen. Many indirect methods based on derivatization have been reported for the chromatographic separation and determination of BPs, which contain primary or secondary amine groups, for example alendronate and pamidronate, but the indirect methods were tedious, time-consuming and can be used for only analysis a few of BPs, as majority of BPs without derivative group, therefore there is still a need for specific methods for assay of BPs.The aim of our research is to introduce specific, simple and sensitive methods for routine analysis and quality control of BPs, which is helpful for developing BPs quality. Here, we present specific, simple and sensitive methods for assay of BPs according to the flowing the two parts:PART 1 Formation of Novel OPM and Analytical Application in PharmaceuticalⅠ. Studies on the Mechanism of Novel OPM Formation and Characters of OPMObjective: To study the formation mechanism of novel OPM and some characters of OPM, such as stoichiometric ratio and molar absorptivity.Methods: OPM were formed directly when mixing BPs with MoO in acidic media; their characters were studied by UV spectrophotometry method. Job’s method of consecutively variation and continuous variation method were applied for definition of stoichiometric ratio of BPs to Mo in OPM, according to the strong absorbance of OPM in wave range of 220～300nm.Results: The novel OPM are obtained, which are stable and have strong absorbance at 254nm. The stoichiometric ratio of BPs to Mo in OPM was determined to be 1:5. The molar absorptivity of OPM are 8.61×10³、9.58×10³、6.89×10³、 7.53×10³、8.08×10³、8.80×10³ (L·mol^-1·cm^-1) of Alen, Pami, Zole, Eti, Inca and Iban respectively. The forming of the OPM does not depend on the presence of N-substituendum in BPs.Conclusion: Novel OPM are obtained by reacting of BPs with MoO in acidic media, which are stable and have strong absorbance. The organo-phosphorus is coordination center in the OPM molecular structure.Ⅱ. Assay of BPs in Pharmaceutical by Spectrophotometry Based on the Novel OPMObjective: To establish a novel spectrophotometric method for quantitative analysis of BPs and for dissolution test of low dose Iban tablet with high accuracy and precision.Methods: Basing on OPM formed directly mixing BPs with MoO in acidic media, UV spectrophotometry was developed with a detection wavelength of 254nm.Results: Good linear relationship between absorbance and the concentrations of Alen, Pami, Zole, Eti, Inca and Iban were established in the range of 5.00～50.0, 4.92～49.2, 5.26～52.6, 5.00～50.0, 5.16～51.6 and 4.92～49.2μg·ml-1 （r≥0.9989）, respectively. The LOD of Alen, Pami, Zole, Eti, Inca and Iban were 0.677, 0.916, 0.710, 0.607, 0.810 and 0.781μg·mL^-1 and LOQ were 0.203, 0.275, 0.213, 0.182, 0.243 and 0.234μg·mL^-1 respectively. The average recovery were 96.8～102.6% （n=3）, RSD were less than 1.5%. The dissolution was quite fast: 86% of Iban was dissolved within 8min and the drug dissolution was complete dissolved about 10min.Conclusions: A specific and sensitive method was developed to assay of BPs in Pharmaceutical, and it can be conveniently adopted for dissolution test of low dose Iban tablets. Compared with molybdenum blue method, it could not be interferenced byphosphate, phosphite or common excipients, what’s more, it is so simple and rapid without any pretreatment.PART 2 Study of Chromatographic Method for Assay of BPsⅠ. Assay of Eti and Iban by HPCE with Indirect UV Detection Objective: To establish HPCE with indirect UV detection for assay of Eti and Iban.Method: An uncoated fused-silica capillary column was used, and its electrolyte system contained sodium benzoate (7mmol·L^-1)-KH2PO4 (7mmol·L^-1) （pH 8.0）, applied voltage 20kV and detection wavelength 224nm.Results: Good linear relations of Eti and Iban were obtained in the range of 62.60～1002μg·mL^-1 and 61.00～975μg·mL^-1 （r>0.999）, with the detection limit of 7.825μg·mL^-1 and 7.744μg·mL^-1 respectively. The average recoveries of Eti and Iban during 99.5%～100.3%, RSD were less than 1.5% （n=3）. Conclusion: The method is simple, rapid and economic, which provides a new reliable means for quality control of Eti and Iban.Ⅱ. Analysis of Iban by Ion Pair RP-HPLC-ELSDObjective: To establish a sensitive and specific RP-HPLC–ELSD method for analysis of Iban and its preparation.Method: Separations were performed on an UltimateTM XB-C18 （4.6×250mm, 5μm） column an isocratic mobile phase: acetonitrile-methanol-20mM triethylamine （pH 8.5, adjusted by acetic acid）-（ 90:2:8）. The mobile phase flow rate was 1.0ml·L^-1, at room temperature. Analyses were detected by ELSD.Results: In quantitative analysis, the method showed that the calibration curve was linear in the range of 30.70～980μg·mL^-1, regression equation is lgA=1.960 lgC+2.047 （r=0.999）, with the detection limit of 7.675μg·mL^-1. The average recoveries of Iban tablets were 98.8%, 99.2% and 100.5% and RSD were less than 1.1%.Conclusion: A sensitive and specific RP-HPLC-ELSD method was developed for analysis of Iban and its preparation. It is more sensitive than reported RP-HPLC-ELSD method since micro Mol. Wt and stronger volatile ion pair diethylamide was selected. It provides a new reliable means for quality control of Iban. Ⅲ. Determination of Zole Dosage Formulation by Ion Pair RP-HPLC-UV with Post-column Photochemical DerivatizationObjective: To develop a sensitive and specific ion pair RP-HPLC-UV method with post-column photochemical derivatization for the determination of Zola dosage formulation and its related substance.Methods: They were separated by Phenomenon C18 column and eluant of triethylamine （20mM adjusted to pH 7.0 with acetic acid）-methanol （99:1） at flow rate of 1.0ml·min^-1 at room temperature. The sensitive detection of Zole was based on its oxidation to orthophosphate by the on-line peroxydisulfate- assisted photolysis flow by post-column reaction with molybdate and Vitamin C to yield phosphomolybdate, then measured by UV detection.Results: Zole was successfully separated from its related substance （phosphate and phosphite）. A good linear relation was obtained in the range of 20.00～500.0μg·mL^-1 （r=0.999）, with the detection limit of 1μg·mL^-1. The average recoveries of Zole for injection and Zole injection were 98.7～101.0% and 99.6～100.4% respectively with RSD less than 2.1%.Conclusion: The ion pair RP-HPLC-UV method is simple, accurate and specific,and tedious sample pretreatment is unnecessary. The ion pair RP-HPLC-UV method provides an approach for the routine analysis and quality control of Zole in pharmaceutical.更多还原