- 學位論文
利用液相層析離子阱質譜儀裂解技術測定化學衍生化長鏈不飽和脂肪酸中碳-碳雙鍵的位置
Chemical Derivatization Coupled with Multistage Fragmentation of Ion Trap Mass Spectrometry for The Positional Determination of Carbon-Carbon Double Bond in Long-Chain Unsaturated Fatty Acid
摘要
高效能且大規模地分析不飽和脂肪酸（Unsaturated fatty acids, UFA）在脂質代謝體學（Lipidomics）中扮演著至關重要的角色。不飽和脂肪酸由碳氫鍵和羧酸基組成，會依碳鏈長度、雙鍵數和雙鍵位置的不同其作用有所改變。其中，碳-碳雙鍵位置對細胞膜功能、訊號傳遞和生物轉換有關。液相層析-串聯式質譜儀（LC-MS/MS）是確定結構的有力工具，但在典型的碰撞誘導解離（CID）下，電荷驅動的碎裂僅發生在不飽和脂肪酸的帶電羧酸鹽上，並且丟失了CO2或H2O，無法提供足夠的資訊來確定碳-碳雙鍵的位置。 本研究使用了衍生化試劑2-(2-aminoethyl amino) pyridine (AEAP)，應用於長鏈脂肪酸的分析，包括：（A）監測發炎前後之巨嗜細胞中花生四烯酸（Arachidonic acid, AA）和前列腺素E2（Prostaglandin E2, PGE2）消長情形及（B）分析橄欖油中不飽和脂肪酸的種類，並進行方法確效。 本研究也利用AEAP修飾搭配多階段碎裂模式(Multistage fragmentation，MSn)，以電荷遠程裂解，用於測定碳-碳雙鍵在不飽和脂肪酸的位置。本策略的重點在於利用AEAP修飾在不飽和脂肪酸上並使之帶一正電荷，在MS3模式下以促進電荷遠程裂解，並生成碎裂離子，用於決定不飽和脂肪酸上的碳-碳雙鍵。修飾後的幾種單元不飽和脂肪酸［如：油酸（Oleic acid, C18:1∆9）、反式油酸（Elaidic acid, C18:1∆9）、棕櫚油酸（Palmitoleic acid, C16:1∆9）、二十碳烯酸（Eicosenoic acid, C20:1∆11）、芥酸（Erucic acid, C22:1∆13）及神經酸（Nervonic acid, C24:1∆9）］利用MS3實驗試圖推導出在其雙鍵位置。 結果也顯示，修飾後的不飽和脂肪酸在CID碎裂下，可斷裂部分修飾基團(質荷比-94)，生成關鍵碎片。此關鍵碎片再經碎裂，可在MS3圖譜中獲得解析雙鍵位置之資訊。據推測，這些MS3碎片可以透過兩種碎裂模式－Endo-1,3-H shift及Exo-1,3-H shift而生成。 關鍵字：不飽和脂肪酸、碳-碳雙鍵位置、液相層析-串聯式質譜儀、多階段碎裂模式、化學衍生化
並列摘要
An efficient and large-scale analysis of unsaturated fatty acids (UFA) plays a crucial role in lipidomics. The UFAs consist of a hydrocarbon chain and a carboxylic acid group, which are diverse in terms of chain length, the number of double bonds, and double-bond positions. The carbon-carbon double bond position has been demonstrated to have potent effects on cellular membrane and tissues function, signal transduction, and bioenergy conversion. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been regarded as a powerful tool for structure determination. However, under a typical collision-induced dissociation (CID), the charge-driven fragmentation only occurred at the charged carboxylate of UFAs and lost CO2 or H2O to provide insufficient information for the determination of carbon-carbon double bond position. In this study, we used the derivatization reagent 2-(2-aminoethyl amino) pyridine (AEAP) to qualitative and quantitative analysis of long-chain fatty acids including the applications on the monitoring of the relative abundance of arachidonic acid (AA) and prostaglandin E2 (PGE2) during inflammation and the determination of fatty acids in olive oil. The method validation was also achieved in terms of accuracy, precision, and limit of detection/limit of quantitation. This method also applied to the determination of carbon-carbon double position on UFAs, which focused on the design of a positive charge on AEAP-modified UFAs to promote charge-remote fragmentation and the production of diagnostic ions for an efficient analysis of carbon-carbon double bonds on UFAs under multiple-stage fragmentation (MSn). Several monounsaturated fatty acids, such as oleic acid (C18:1∆9), elaidic acid (C18:1∆9), palmitoleic acid (C16:1∆9), eicosenoic acid (C20:1∆11), erucic acid, C22:1∆13), and nervonic acid (C24:1∆9), were used to demonstrate this method’s performance. The result indicated that the modified UFAs will lose 94 Da to form the key fragments under CID fragmentation. The further fragmentation of the key fragment can provide valuable information for the determination of the double bond position in MS3 spectrum. We proposed the fragmentation was mainly based on two fragmentation modes：Endo-1,3-H shift and Exo-1,3-H shift. Key words：Unsaturated fatty acids, carbon-carbon double bond position, liquid chromatography-tandem mass spectrometry (LC-MS/MS), multi-stage fragmentation (MSn), chemical derivatization