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Title: | 藥物分析與代謝體研究 第一部分: 開發QuEChERS萃取方法與毛細管膠束電泳法分析人體血漿中 Co-trimoxazole藥物 第二部分: 利用標的代謝體學方法建立糖尿病之預測模型 第三部分: 人體血漿中核苷與核苷酸的LC-MS/MS分析方法開發 Pharmaceutical Analysis and Metabolomics Study Part I: Development of QuEChERS Extraction and Micellar Electrokinetic Chromatography Method for the Analysis of Co-trimoxazole in Human Plasma Part II: Using Targeted Metabolomics Method to Establish a Prediction Model for Diabetes Part III: Development of an LC-MS/MS Method for Nucleoside and Nucleotide Analysis in Human Plasma |
Authors: | Yi-Hao Wu 吳翊豪 |
Advisor: | 郭錦樺(Ching-Hua Kuo) |
Keyword: | QuEChERS,新生糖尿病,代謝體,LC-MS/MS,血液,核?,核甘酸, QuEChERS,new onset diabetes,metabolomics,LC-MS / MS,plasma,nucleoside,nucleotide, |
Publication Year : | 2017 |
Degree: | 碩士 |
Abstract: | 近年來,隨著儀器的靈敏度的提升與萃取技術的優化,準確且精密的分析方法在臨床研究十分具有發展潛力。本論文運用儀器分析開發藥物與代謝物在血液的分析方法,論文分成三個部分。
QuEChERS是近年來發展的新萃取技術,被應用於蔬果的農藥殘存檢測。在本論文的第一部分修改並且最佳化原本QuEChERS的萃取流程並與Capillary Electrophoresis with UV Detection (CE-UV)平台結合,開發出一種經濟、方便、靈敏的分析方法測量人體血漿中的Sulfamethoxazole與Trimethoprime藥物。本論文針對CE-UV平台與QuEChERS萃取法分別作方法開發。最後利用最佳化的掃集法-場強放大樣品堆積毛細管電泳(Sweeping-Field Amplified Sample Stacking,FASS)分析平台搭配Modified QuEChERS法,能夠在人體血液樣品中檢測到0.1 μg mL-1的Sulfamethoxazole與Trimethoprime血中藥物濃度,達到臨床上檢測的需求。 本論文第二部分為使用targeted metabolomics的研究方法針對12個有助於識別與Type 2 Diabetes(T2D)罹患風險的代謝物開發LC-ESI-MS/MS分析方法平台,並用於預測新生糖尿病的研究。本研究以最適化LC-MS/MS分析平台測量236個世代研究的人體血漿樣品的分析結果分別利用isotope internal standards 以及z-score對基質效應及批次效應校正後,此平台成功的尋找到5個在控制組與病例組有統計上顯著差異的標的代謝物。分別為leucine/isoleucine, valine, 5-methylcytidine, 以及glutamylleucine。5-methylcytidine的濃度與T2D的發生呈現負相關,而leucine/ isoleucine, valine, 5-Methylcytidine, 以及glutamylleucine則是與T2D的發生呈現正相關。我們分別對這些標的代謝物對於臨床表徵做Pearson correlation,討論其生理角色與可能相關的代謝途徑,最後以ROC curve建立預測新生糖尿病的預測模型,Clinical risk factors模型的曲線下面積(AUC)為0.66,而metabolites 加入clinical risk factors的模型預測可使模型的曲線下面積上升到0.72,顯示合併代謝物濃度評估,可提高模型預測未來新生糖尿病的能力。本研究所提出之標的代謝物有助於提升新生糖尿病的預測能力,並增進對於新生糖尿病發生的病理機轉的了解。 核苷與核苷酸通常被認為主要存在於細胞核中,而近年來的研究發現血液循環系統中的核苷與核苷酸的濃度與一些代謝性以及免疫性疾病有關。本論文的第三部分使用LC-MS/MS開發人體血漿中核苷與核苷酸的分析方法。我們開發了萃取人類血漿樣品中核鹼基、核苷以及核苷酸的方法並使用LC-MS/MS建立分析平台,比較了不同萃取液的萃取效率,以及Reversed phase、HILIC、ANP、Ion-pair chromatography對此類化合物峰型以及滯留的影響。實驗結果顯示,在所有測試的條件中Cogent Diamond Hydride與SeQuant ZIC-pHILIC分別在核鹼基與核苷的方法開發以及核苷酸的方法開發中有最好的選擇性與峰型。在核鹼基與核苷的分析中使用Cogent Diamond Hydride管柱成功地建立了分析11個化合物標準品的平台,在核苷酸的分析中使用了zic-pHILIC管柱成功地建立了分析16個化合物標準品的平台。我們並把此實驗平台應用在人體血漿樣品的分析,在人類血漿中成功地分析了11個核鹼基與核苷化合物與10個核苷酸相關化合物。本實驗所開發的核鹼基、核苷與核苷酸分析平台使用簡單的移動相與萃取方法,整體分析時間短,涵蓋的分析物範圍多,未來可應用於與疾病的相關性研究。 In recent years, with the improvement of the sensitivity of the instrument and the advancement of extraction technology, accurate and sophisticated analytical methods have great values in clinical researches. In this thesis, we used instrumental analysis technology to develop analytical methods for drugs and metabolites in human blood samples, and our achievements are divided into three sections for discussion. QuEChERS is a novel extraction technology developed in recent years and has been applied to the detection of pesticide residues in fruits and vegetables. In the first section of this thesis, we modified and optimized the extraction process of QuEChERS and combined with Capillary Electrophoresis with UV Detection (CE-UV) platform to develop an economical, convenient and sensitive method for measuring sulfamethoxazole and trimethoprim concentration in human plasma. Under the optimized Sweeping-Field Amplified Sample Stacking (FASS) combined with CE method and the modified QuEChERS sample pretreatment, the detection sensitivity could reach 0.1 μg mL-1 of trimethoprime and sulfamethoxazole in human blood samples, which could satisfied with clinical requirement. In the second section of this thesis, we applied targeted metabolomics strategy to develop LC-ESI-MS/MS analytical platform for the detection of 12 selected metabolites that could be helpful to identify the risks associated with Type 2 Diabetes (T2D). We used 236 human plasma samples from a cohort study and measured by the optimized LC-MS / MS analysis platform. After using the isotope internal standards and z-score to adjust the matrix effect and batch effect, we successfully found 5 targeted metabolites including: leucine / isoleucine, valine, 5-methylcytidine, and glutamylleucine, with statistically significance between the case and control group. The concentration of 5-methylcytidine in blood was negatively correlated with the occurrence of T2D, whereas leucine / isoleucine, valine, 5-Methylcytidine, and glutamylleucine were positively correlated with T2D. Pearson correlation of targeted metabolites and clinical characteristics were further used to investigate the possible physiological and metabolic pathways of these markers in T2D. Finally, the model for predicting new onset diabetes was established with ROC curve. The area under the curve (AUC) using clinical risk factors was 0.66, while incorporating with clinical risk factors and aforementioned five metabolites, the AUC of the model could rise up to 0.72. These results indicate that combining metabolite concentration assessment could improve the prediction ability for the new onset diabetes. These five metabolites presented by this study could help to improve the predictive and enhance the understanding of the pathogenesis of new onset diabetes. Nucleosides and nucleotides are considered to be predominantly present in the cell nucleus, and in recent studies, researchers have found that the concentration of nucleosides and nucleotides in the circulating system is associated with some metabolic and immunological disorders. In the third section of this thesis, we used LC-MS / MS to develop analytical method of nucleosides and nucleotides in human plasma. We developed the method for extracting nucleobases, nucleosides and nucleotides from human plasma samples and established the analytical platform by using LC-MS / MS. The extraction efficiency of different extraction solvents was compared, and the peak shape and retention ability of these compounds were also examined by Reversed phase, HILIC, ANP, and Ion-pair chromatography. The experimental results show that the best selectivity and peak shape could be obtained by using Cogent Diamond Hydride for nucleobase and nucleoside, and SeQuant ZIC-pHILIC for nucleotide. Platform for analyzing 11 nucleobases and nucleosides standards was successfully established by using a Cogent Diamond Hydride column; and 16 nucleotides standards was successfully established by using a zic-pHILIC column. We also applied this experimental platform to human plasma samples, and successfully analyzed 11 nucleobases and nucleoside compounds and 10 nucleotide-related compounds in human plasma. Notably, simple mobile phase and extraction method were used in this nucleobases, nucleoside and nucleotide analysis platform. The overall analysis time is short, but still covering a wide range of analytes. Future works could be done by applying this platform to the relevance of nucleosides and nucleotides with varies disease. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67249 |
DOI: | 10.6342/NTU201702692 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 藥學系 |
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