【作者】 刘磊；

【导师】 白钢；

【作者基本信息】 南开大学 ， 微生物学， 2013， 博士

【摘要】 在现代体内药物分析工作中,80%左右的工作量及研究经费用于生物样品的前处理及分析样品制备等步骤。本文基于在线固相萃取前处理技术,实现了样品前处理的自动化,建立了系列“准确、灵敏、高通量”的分析方法,为体内药物监测及药代动力学研究提供有力的技术支持。首先,本研究探讨了全自动在线固相萃取-高效液相法用于测定小鼠血浆中补骨脂二氢黄酮甲醚的浓度。本方法简化了血浆样品繁琐的前处理步骤,提高了分析的速度及效率,实现了对目标化合物的较高回收率。本研究采用的CapcellMF Ph-1柱可有效去除血浆样品中的基质,且对补骨脂二氢黄酮甲醚的富集效果较好；在小鼠血浆中补骨脂二氢黄酮甲醚浓度测定过程中,最低定量限可低至20ng/ml,其选择性、线性、精密度、准确度及回收率均较好。补骨脂二氢黄酮甲醚在小鼠体内表现为吸收较快、清除较快、生物利用度较低的药代动力学特征。在线固相萃取-高效液相生物分析方法的多参数优化是方法学建立中的至关重要的步骤。本论文应用响应面分析法(RSM)对影响在线固相萃取过程中的若干因素进行了系统优化；按照影响程度由大到小依次为：洗脱剂pH值(pH)>转移时间(t(T))>基质洗脱时间(t(M))>洗脱剂流速(F)；该方法得到的最优条件组合大大提高了分析结果的准确性与可靠性。在响应面优化的基础上建立的在线固相萃取-高效液相色谱分析法能够快速、准确的测定小鼠血浆中高活性生物碱WM-5的浓度,整个分析时间8min;WM-5的最低定量限为20ng/ml。本方法简化了血浆样品繁琐的前处理步骤,实现了全自动化生物分析过程,提高了分析的速度及效率。在上述“准确、高效、快速”的单个目标分析物的分析方法基础上,本文针对临床应用最多的、具有不同理化参数的系列心血管类药物,系统地探讨了在线固相萃取-高效液相色谱串联质谱系统方法学的适应性。基于心血管类药物物理化学参数的差异(如分子量,1ogP,pKa等),本文系统考察了不同在线固相萃取柱的提取效率及相应的色谱分析与质谱参数,并进行了条件优化。实验结果显示：采用反目LiChrosphe(?)RP-18ADS柱作为在线固相萃取柱,可有效实现目标化合物群的保留与分离,且所用的分析条件温和(pH9.5醋酸铵缓冲液上样,pH3.5醋酸铵缓冲液洗脱),避免了血浆中蛋白的沉淀现象；经反冲后,对分析柱系统进行梯度洗脱,可有效实现10个不同极性的心血管类药物的同步色谱分离；整个分析时间11min,未见内源性基质与其他物质的干扰：其最低定量限、精确度、准确度、回收率与基质效应均优于文献报道的普通液相色谱分析结果。最后,本文利用在线固相萃取-高效液相色谱串联质谱系统对当前药物分析领域中一类较难分析的化合物-核苷类抗病毒药物进行了方法学的系统研究。由于此类化合物的极性极大、水溶性好,其药代动力学研究存在多种困难,特别是样品前处理过程中的基质去除、目标分析物的有效提取等。针对临床治疗艾滋病的“鸡尾酒”处方中常用的核苷类逆转录酶抑制剂(齐多夫定、拉米夫定、去羟肌苷、恩曲他滨),建立了高效的在线固相萃取-高效液相色谱串联质谱分析系统：以混合型阳离子交换柱Oasis(?) MCX为在线固相萃取柱,优化了流动相系统、固相萃取系统与分析系统,实现四个化合物有效保留与色谱分离；整个分析时间10min,未见内源性基质与其他物质的干扰；其最低定量限、精确度、准确度、回收率与基质效应均满足FDA对生物样本分析的要求。同时,针对抗病毒治疗药物-利巴韦林及其前药Taribavirin,本文建立了一种新型的在线固相萃取-高效液相色谱串联质谱分析法：自主设计了基于半化学吸附的在线固相萃取柱-苯硼酸固相萃取柱;经对色谱及质谱条件的优化,实现生物样品中的利巴韦林与Taribavirin的有效保留,并利用酸性洗脱剂有效实现了两者的色谱分离。该系统所用有机相比例小,有效避免了血浆中的蛋白沉淀,其最低定量限、精确度、准确度、回收率与基质效应均优于文献报道的普通色谱分析结果。更多还原

【Abstract】 Sample pretreatment and sample preparations are critical steps in bioanalysis of active pharmaceutical principals in in vivo study. It is estimated that about80%of the efforts and research funding have been spent on the sample pretreatment and sample preparation steps in pharmacokinetic (PK) study. The objectivs of this research is focused on automation of sample pretreatment and sample preparation to enable accurate, sensitive and high-throughput bioanalytical methods for PK study by application of on-line solid phase extraction (SPE) technology.First of all, a fully automated online SPE coupled with high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for determination of bavachinin in mouse plasma. Analytical process was performed on two reversed-phase columns (Capcell MF Ph-1cartridge&analytical column) connected via a Valco6-port switching valve. The lowest limit of quantification (LLOQ) of the assay was20ng/ml. The established automated online SPE-HPLC-DAD method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision and accuracy. It was successfully utilized to quantify bavachinin in mouse plasma to enable PK study. The PK properties of bavachinin were characterized as rapid oral absorption, high clearance, and poor absolute bioavailability.Optimization of multiparameters in SPE-HPLC bioanalytical system is a challenging task. Response surface methodology (RSM) was utilized for rapid and systematic optimization of on-line SPE parameters to maximize the response and separation of WM-5. The optimization was performed with Box-Behnken designs. Four major parameters were investigated for their contributions to the response and separation of WM-5, with a total of29experiments being performed for each instrument, respectively. Quantitative determination of WM-5in mouse plasma was performed to evaluate the statistical significance of the parameters on chromatographic response. A fully automated online SPE-HPLC-DAD method was developed for the determination of WM-5in mouse plasma. The single run was within8min, and LLOQ of WM-5was20ng/ml. The optimized method demonstrated good performance in terms of specificity, LLOQ, linearity, recovery, precision and accuracy.A fully automated online solid phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometry (online SPE-HPLC-MS/MS) method was described for the simultaneous determination of a variety of cardiovascular drugs in rat plasma. Following a simple centrifugation step, a10μl aliquot of the plasma sample was injected directly onto the HPLC system. The LiChrospher(?) RP-18ADS cartridge was chosen as the online SPE cartridge, the analytes were quantified by measurement of an API4000+triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode. The procedure was easier to execute and required significantly less sample handling than methods currently described in the literature. The correlation study of the physicochemical parameters and the analytical parameters of analytes may provide a solid basis for SPE cartridge selection and method optimization for the therapeutic drug monitoring and PK study of cardiovascular drugs.Nucleosides are a class of chemical structrues having widely clinical applications. However, the bioanalysis of nucleosides is very challenging due to their high polarity and high solubility. The matrix clearance and the extraction of target analytes from the matrix samples in in vivo PK study have attracted great attention in bioanalytical research. In this study, online SPE technology has been applied to address the difficulities in sample pretreatment and sample preparation of nucleosides in human plasma. A fully automated online SPE-HPLC-MS/MS method was established for the simultaneous determination of a variety of nucleoside reverse transcriptase inhibitors (Lamivudine, Zidovudine, Didanosine and Emtricitabine) in human plasma. After injection of10μl aliquot of the plasma sample onto the online SPE-HPLC-MS/MS system, the Oasis (?)MCX cartridge was performed as the online SPE cartridge, the analytes were quantified by measurement of an API4000+triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode, and the analysis was finished within10min with no significant matrix effect. The procedure was easier to execute and required less sample handling than methods currently described in the literature. The established automated online SPE-HPLC-MS/MS method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision, accuracy, recovery and matrix effect, which were within the criteria of FDA requirement of biological sample analysis. Moreover, a fully automated online SPE-HPLC-MS/MS method was established for the simultaneous determination of Ribavirin and Tribavirin in human plasma based on their physical and chemical properties. Because of the high polarity of Ribavirin and Tribavirin, a Bond Elut Phenylboronic acid SPE cartridge was developed and used in this study. Through optimization of chromatography and mass spectrometry, Ribavirin and Taribavirin were trapped in the SPE cartridge successfully, and thereafter they were eluted by acidic mobile phase. The precipitation of plasma proteins was effectively avoided by using low proportion of organic mobile phase. In conclusion, the procedure was easier to execute and required less sample handling than methods currently described in the literature. The established automated online SPE HPLC-MS/MS method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision, accuracy, recovery, and matrix effect.更多还原