請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78886
標題: | 以液相層析質譜儀開發藥物與代謝體於採血卡
之分析方法 Development of liquid chromatography-mass spectrometry methods for pharmaceutical and metabolomics analysis using dried blood spot |
作者: | Divyabharathi Chepyala 錢笛雅 |
指導教授: | 郭錦樺(Ching-Hua Kuo) |
關鍵字: | 採血卡,濫用藥物,離子加壓器,抗愛滋病藥物,柱後輸注內標,血液體積,血比溶,採血卡上之代謝體研究, Dried blood spot,drug abuse,ion-booster,anti-HIV drugs,post column infused internal standard,blood volume,Hematocrit effect,DBS-based metabolomics, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | The dried blood spot (DBS) sampling technique contains numerous advantages compared to the traditional plasma sampling technique. However, due to critical challenges, its applications in real world analysis except in relation to newborn screening are still minimal.
In this dissertation, we propose several approaches to mitigate the DBS associated challenges including low sensitivity and spotted blood volume to increase its applicability in pharmaceutical and metabolomics analysis. We first reported an ultra-high-performance liquid chromatography-ion booster-quadrupole time-of-flight mass spectrometry (UPLC-IB-QTOF) method for sensitive screening of abused drugs in DBS samples. An 80% acetonitrile solvent with a 5-min extraction by Geno grinder was used for sample extraction. A Poroshell column was used to provide efficient separation, and under optimal conditions, the analytical times were 15 and 5 min in positive and negative ionization modes, respectively. Ionization parameters of both electrospray ionization source and ion booster (IB) source containing an extra heated zone were optimized to achieve the best ionization efficiency of the investigated abused drugs. In spite of their structural diversity, most of the abused drugs showed an enhanced signal response with the high temperature ionization from an extra heated zone of IB source. Compared to electrospray ionization, the ion booster (IB) greatly improved the detection sensitivity for 86% of the analytes by 1.5 to 14-fold, and allowed the developed method to detect trace amounts of compounds on the DBS cards. The validation results showed that the coefficients of variation of intra-day and inter-day precision in terms of the signal intensity were lower than 19.65%. The extraction recovery of all analytes was between 67.21 and 119.38%. The limits of detection of all analytes were between 0.2 and 35.7 ng/mL. The stability study indicated that 7% of compounds showed poor stability (below 50%) on the DBS cards after 6 months of storage at room temperature and -80 °C. The reported method provides a new direction for abused drug screening using DBS. In order to improve the DBS applicability in drug monitoring, we developed a post column infused-internal standard (PCI-IS) combined with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) method for the simultaneous quantification of 6 HIV medicines including tenofovir, emtricitabine, cobicistat, darunavir, ritonavir and elvitegravir for blood concentration estimation in DBS samples. Darunavir-d9 was selected as the PCI-IS for both blood estimation and drug quantification. Whole blood spots were extracted with 70% methanol for 10 min and compounds were separated by a T3 C18 column within 9.5 min with optimized gradient profile. The method was validated within the concentration range of 2.5–2500 ng/mL for all the drugs. The developed method fulfilled the FDA and EMA validation criteria. All the analytes were stable at the tested storage conditions except for cobicistat which degraded up to 25% in DBSs at room temperature within 7 days. Linear correlation coefficients between paired DBS and plasma sample concentrations were used to predict plasma concentrations from DBSs. Bland−Altman plots showed above 95% agreement between predicted plasma and measured plasma concentrations, confirming the suitability of DBSs for cART monitoring. DBS-based metabolomics analysis is a powerful tool for investigating new biomarkers for clinical use. To improve data quality for DBS-based metabolomics studies, we developed a PCI-IS assisted LC-ESI-MS/MS analysis method. An efficient sample preparation protocol with 80% acetonitrile as the extraction solvent was first established to improve the metabolite recovery. The PCI-IS assisted LC-ESI-MS method was used to simultaneously estimate the blood volume and correct the signal change caused by ion source contamination and the matrix effect to evaluate the spot volume effect and hematocrit (Hct) variation effect on target metabolites in DBSs. D8-phenylalanine was selected as the single PCI-IS to correct the matrix effect. For calibration of errors caused by the blood volume difference, 95% of the test metabolites showed good correlation between spot volume and signal intensity after PCI-IS correction. The spot volume was further calibrated by the same PCI-IS. Investigation of the Hct variation effect on target metabolites revealed that it affected the concentrations of metabolites in the DBS samples depending on their abundance in the red blood cell (RBC) or plasma; it is essential to pre-investigate the distribution of metabolites in blood to minimize the comparison bias in metabolomics studies. The PCI-IS-assisted strategy is anticipated to improve data quality for metabolomics studies using DBSs and benefit various clinical research applications. In conclusion, we demonstrated that UPLC-IB-QTOF and PCI-IS assisted LC-ESI-MS/MS analytical methods could solve the low sensitivity and spotted blood volume challenges of DBS sampling technique and could increase the it’s applicability in real world analysis. We anticipate that these developed methods could improve the simplicity and accuracy for forensic, pharmaceutical and meabolomics analysis and benefit precision medicine. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78886 |
DOI: | 10.6342/NTU201804215 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-107-F01423027-1.pdf 目前未授權公開取用 | 8.54 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。