臺灣博碩士論文加值系統

膽酸為膽固醇主要的代謝產物，因此當膽固醇的反應異常導致膽酸濃度過高或過低時，人體會引發不同種類病狀，例如原發性膽汁性肝硬化、膽結石、乳房癌及大腸癌等。除此之外，膽酸也應用於治療疾病，例如chenodexycholic acid和ursodeoxycholic acid取代開刀治療膽結石的病人。近年來亦有利用膽酸結合樹脂(Bile acid sequestrant resin)，主要功能為阻止腸胃道內膽酸被吸收，增加肝臟利用膽固醇製造膽酸，以平衡人體內膽酸濃度，以減少肝細胞中膽固醇的含量。
利用液相層析串聯質譜儀(LC-MS)偵測市售牛肝標準品與生物樣品中(豬腎以及豬、牛、雞、鴨、鵝肝臟)中微量膽酸包括cholic acids (CA)、deoxycholic acid (DCA)、lithocholic acid (LCA)、ursodeoxycholic acid (UDCA)、chenodexycholic acid (CDCA)以及hyodeoxycholic acid (HDCA)等六種膽酸與其甘胺酸共軛膽酸，實驗過程中因為有生物樣品中酯質、蛋白質等干擾問題，因此結合固相萃取(solid phase extraction)方式樣品基質干擾並可同時減少有機溶劑使用，並以質譜儀MRM(Multiple Reaction Monitoring)模式進行偵測膽酸先驅離子(Precursor ion)斷裂之產物離子(Product ion)訊號，提高偵測膽酸的靈敏度，可成功定性及定量生物樣品中膽酸含量，膽酸與膽酸衍生物偵測極限為0.53-3.53 ng/mL (S/N=3)，RSD，生物肝臟樣品回收率均有88%以上。本研究萃取方法簡單且使用少量的有機溶劑且可快速偵測膽酸，可應用於測定市售生物樣品中膽酸與膽酸衍生物含量。

Cholesterol is the major bile acid metabolite. Therefor bile acid concentration abnormality will lead to different types of disease in the human body. For example primary biliary cirrhosis, gallstones, breast cancer, colorectal cancer, etc. Bile acid is also used to treat diseases. Chenodexycholic acid and ursodeoxycholic acid is used to replace the patient surgical treatment of gallstones.
In our study, we used liquid chromatography tandem mass spectrometer (LC-MS) to detect trace amounts of bile acids including cholic acid (CA), deoxycholic acid (DCA), lithcholic acid (LCA), ursodeoxycholic acid (UDCA), chenodexycholic acid (CDCA), hyodeoxycholic acid (HDCA) and their glycine-conjugated form in livers. We combined liquid-liquid extraction, solid phase extraction to save solvent and matrix interferences and multiple reaction monitoring mode (MRM) in mass spectrometer to detect product ion signal cleaved from precursor ions can successful qualitative and quantitative bile acids content in biological samples. Limit of detection 0.53-5.53 ng/mL (S/N=3). Recoveries of bile acids were more than 88% for real samples. In this work, the extraction method is simple and small amount of organic solvents can quickly detect bile acids in biological samples.

目錄 I
摘要 III
Abstract V
圖目錄 VI
表目錄 VIII
第一章 緒論 1
1-1. 膽酸介紹 1
1-2. 膽酸異常疾病 14
1-3. 文獻中膽酸的分析方法 16
1-4. 研究目的 26
第二章 實驗部分 28
2-1. 儀器設備和條件： 28
2-2. 其他裝置 28
2-3. 化學試劑和生物樣品 29
2-4. 溶液配製 31
2-5. 標準品校正取線製備 32
2-6. 組織樣品乾燥與粉末製備步驟 33
2-7. 實驗步驟 33
第三章 膽酸與膽酸衍生物之液相層析質譜儀測定 35
3-1. 膽酸分析物質譜探討 35
3-2. HPLC移動相與質譜儀分析 35
3-3. 多重反應偵測模式(Multiple Reaction Monitoring, MRM)偵測 38
3-4. 液相層析質譜儀對膽酸穩定度測試 57
第四章 生物樣品分析 59
4-1. 樣品前處理 59
4-2. 固相萃取(SPE)裝置萃取膽酸最佳化條件探討 60
4-3. 生物樣品之膽酸測定 67
4-4. 結論 81
參考文獻 82
附錄 A1-A90

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