第一部分、開發Eicosanoids之LC-MS/MS分析方法並應用於Atorvastatin治療急性缺血性中風的研究 第二部分、建立測量人體血漿中5-Fluorouracil的LC-MS/MS分析方法

Abstract

在近代基於質譜儀 (Mass spectrometer, MS) 技術的進步，徹底改善我們分析生物樣品中複雜混合物的能力，讓我們得以靈敏且專一的測量生物樣品中的分析物。這種高靈敏度和高通量分析工具已被廣泛應用於各種生醫研究的領域，包括代謝體學以及藥物分析。 本研究第一部分以超高壓液相層析串聯式質譜儀 (Ultra High Performance Liquid Chromatography, UHPLC-MS/MS) 建立靈敏之分析方法測定生物檢體中的類花生酸 (Eicosanoids)，以助於研究其在健康和疾病路徑中的作用，進而能更深入地了解這些類花生酸在生物體所扮演的角色。由於類花生酸具有結構相似度高和內生性濃度極低的特性，分析生物檢體中的類花生酸是極具挑戰性的。為了提升檢測靈敏度，本研究的第一部分開發了兩種分析類花生酸的方法。 第一種分析方法優化液相層析條件、質譜參數，並利用固相萃取做為樣品前處理步驟來減少基質的干擾，再搭配大體積進樣的方法提升質譜儀偵測靈敏度。本研究進一步將最適化分析條件進行準確度、精密度、線性、定量極限和樣品萃取回收率和安定性的確效。 第二種分析方法同樣利用固相萃取做為樣品前處理步驟來減少基質的干擾，並以3-(Aminomethyl)pyridine (3-PA) 進行衍生以增加類花生酸的靈敏度。衍生後的類花生酸的訊號感度顯著地提高。在標準品溶液有6至396倍的信號增強；而在標準品添加血漿溶液中觀察到1.4至425倍的信號增強。最後，因發展衍生化方法時有觀察到儀器零件壽命縮短的現象，且未添加標準品的檢品可測量到的類花生酸數量並未顯著提高，故最終選擇未衍生法應用分析生物檢品中的類花生酸。 本研究以最適化分析方法測定小鼠腦組織中的類花生酸之含量，研究MCAO手術造成的傷害以及Atorvastatin的影響。研究發現MCAO後三小時主要影響氧化壓力、抗血小板凝集、血纖維蛋白溶酶原活化和血壓調節的路徑，它們大多屬於Prostaglandin (PG) 類型的類花生酸。Atorvastatin在這段期間主要影響抗血小板凝集、血壓調節、氧化壓力和細胞毒性的路徑，且Atorvastatin可逆轉 TXB2、PGF2α 因MCAO所造成的變化。MCAO後二十四小時主要為影響氧化壓力、血小板凝集、血壓調節和具細胞毒性物質的類花生酸，它們大多屬於Hydroxyeicosatetraenoic acid (HETE) 類型的類花生酸。Atorvastatin在這段期間對類花生酸影響較不顯著。但這些推測的機轉在未來需要更多的研究與驗證。 本研究的第二部分開發定量人類血漿中5-Fluorouracil (5-FU) 的超高壓液相層析串聯式質譜儀分析方法。本分析法採用簡單的去蛋白法進行樣品前處理，分析時間僅需六分鐘。方法經專一性、回收率、安定性、線性、精密度和準確度的確效，驗證此方法可用於準確測量人體血漿中的5-FU。

The technological advances in modern mass spectrometer (MS) have revolutionized our ability to analyze complex mixtures in biological samples with good sensitivity and specificity. With the development of this high sensitivity and high throughput analysis tool, it has been applied to variety of biomedical analysis fields, including metabolomics and drug analysis. In the first part, in order to gain more understanding of the biological roles of eicosanoids and to facilitate the studying of pathway change of eicosanoids in disease state, two sensitive analytical methods were established for quantifying eicosanoids in biological samples by ultra-high pressure liquid chromatography mass spectrometry (LC-MS/MS). Eicosanoids are characterized by structurally similarity and very low endogenous abundance, and the development of the analysis method is very challenging. In order to improve the detection sensitivity of the mass spectrometer, two methods for analyzing eicosanoids have been developed in the first part of this study. The first analytical method has been optimized for chromatographic conditions, mass parameters, and the solid phase extraction procedure used as the sample pretreatment step to reduce the interference from the matrix. Large volume injection was applied for improving detection sensitivity of mass spectrometer. The method was validated in terms of accuracy, precision, linearity, limit of quantification, extraction recovery and stability. The second analytical method also used the solid phase extraction as the sample pretreatment step to reduce matrix interference. Eicosanoids were derivatized by using 3- (Aminomethyl) pyridine (3-PA) to improve detection sensitivity. The results displayed that there are 6 to 396-folds signal enhancement for eicosanoid standard solutions, and 1.4 to 425-folds signal enhancement in plasma spiked samples after derivatization. However, it was noticed that the life of the instrument parts were shorten and the number of detectable eicosanoids was not significantly increased, we finally choose the first analytical method for application. In this study, the contents of eicosanoids in mouse brain tissue were determined by the optimized method to study the damage caused by MCAO and the effect of Atorvastatin. The results shown that three hours after MCAO mainly affected eicosanoids involved in the pathways of oxidative stress, anti-platelet aggregation, plasminogen activation and blood pressure regulation. Most of them belong to prostaglandin (PG). Atorvastatin mainly affected eicosanoids involved in the anti-platelet aggregation, blood pressure regulation, oxidative stress and cytotoxic pathways during this period. In addition, Atorvastatin reversed the changes of TXB2 and PGF2α due to MCAO.Twenty-four hours after MCAO mainly affected eicosanoids involved in the pathways of oxidative stress, platelet aggregation, blood pressure regulation and cytotoxic substances. Most of them belong to the class hydroxyeicosatetraenoic acid (HETE). Atorvastatin showed less effect on eicosanoids during this period. However, these observations need more investigation and verification in the future. In the second part of this thesis, a method for the analysis of 5-Fluorouracil (5-FU) in human plasma by LC-MS/MS was developed. This assay uses a simple deproteinization method for sample preparation, which takes only six minutes in LC-MS analysis. The method was validated by specificity, recovery, stability, linearity, accuracy and precision and shown can be used to accurately measure 5-FU in human plasma.