臺灣博碩士論文加值系統

此篇論文主在建立以二維液相層析儀同時分離乳酸 (Lactate)與3-羥基丁酸 (3-Hydroxybutyrate)鏡像異構物。將螢光衍生化試劑
4-(N-Chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-COCl)加入含有乳酸以及 3-羥基丁酸的溶液，將混合液置於60˚C 下加熱 15 分鐘，進行螢光衍生化反應後取 20 μL 注入液相層析
儀。在第一維的液相層析儀中，利用 capillary monolithic ODS 管柱可以成功分離乳酸與 3-羥基丁酸。在第二維的液相層析儀中利用QD-AX、KSAACSP-001S 管柱及不同比例之氰甲烷、甲醇與三氟乙酸所組成的移動相，來進行乳酸與 3-羥基丁酸鏡像異構物的分離。所得之最佳分離係數分別是 1.14 與 1.08，偵測極限(LOD)大約為 2 微毫莫爾 (2 fmol/inj)。利用此方法成功定量了生物檢體，如在健康人的血漿、尿液以及唾液中發現，無論是乳酸或是 3-羥基丁酸的鏡像異構物均存在人體中。未來此方法可望應用在釐清乳酸與 3-羥基丁酸的鏡像異構物與疾病之間的關聯性與其個別的生理意義。

A two-dimensional HPLC system has been established for the
simultaneous determination of lactate and 3-hydroxybutyrate enantiomers. Lactate and 3-hydroxybutyrate were derivatized with NBD-COCl, and heated at 60˚C for 15 min. In the first dimension, the lactate and 3-hydroxybutyrate were separated by a capillary monolithic ODS column. In the second dimension, various chiral stationary phases had been tested.
The lactate enantiomers can be separated by the QD-AX and the 3-hydroxybutyrate enantiomers can be separated by the KSAASCP-001S. The mobile phases were composed of acetonitrile, methanol and TFA. The separation factors are 1.14 and 1.08 respectively. The detection limit of lactate and 3-hydroxybutyrate are 2 fmol/injection. Now by using the 2D-HPLC system, we can separate lactate and 3-hydroxybutyrate enantiomers at the same time. We also examined this method in the clinical healthy human samples, and we can find that both the lactate and 3-hydroxybutyrate enantiomers exist on healthy human serum, urine and saliva.
In the end, we hope this method can be applied to various clinical disease samples and that will help us to clarify the role of these enantiomers.

目錄
目錄 ...................................... I
表目錄 .................................... V
圖目錄 ....................................VI
縮寫表 ................................... VII
中文摘要... ... .. .. .. .. .. .. .. .. ..VIII
Abstract ... .. .. .. .. .. .. .. .. .. .. ..IX
第一章 緒論 ............................... 1
第二章 文獻回顧 ........................... 3
第一節 乳酸.......................................................................... 3
2.1.1 乳酸的基本性質 ........................... 3
2.1.2 乳酸的來源 ............................... 4
2.1.3 乳酸的代謝與排除 ......................... 4
2.1.4 與 D-Lactate 相關的疾病.................... 5
第二節 3-羥基丁酸............................................................... 8
2.2.1 3-羥基丁酸的基本性質 ...................... 8
2.2.2 酮酸的來源 ............................... 9II
2.2.3 3-羥基丁酸的代謝與排除 ................... 10
2.2.4 與 3-羥基丁酸相關的疾病 .................. 10
第三節 乳酸與 3-羥基丁酸鏡像異構物之關聯性............. 12
第四節 乳酸與 3-羥基丁酸鏡像異構物之分析方法......... 14
2.4.1 乳酸鏡像異構物的分析方法 ................ 15
2.4.2 3-羥基丁酸鏡像異構物的分析方法 ........... 15
第三章 研究目的 ...........................17
第四章 實驗材料與方法 ......................18
第一節 實驗試料與儀器..................................................... 18
4.1.1 實驗試藥................................. 18
4.1.2 實驗儀器................................. 18
第二節 實驗方法 ................................................................ 21
4.2.1 實驗條件參數分析 ........................ 21
4.2.2 乳酸與 3-羥基丁酸鏡像異構物的分析方法 .... 22
4.2.3 分析方法之確效 .......................... 28
4.2.4 生物檢品之製備分析方法 .................. 30
第三節 統計分析方法 ........................................................ 31III
第五章 實驗結果 ...........................32
第一節 實驗條件參數分析結果......................................... 32
5.1.1 N-methylmorpholine 濃度測試結果 .......... 32
5.1.2 衍生化反應溫度條件結果 .................. 33
5.1.3 衍生化反應溫度條件結果 .................. 34
第二節 分析管柱分離結果................................................. 35
5.2.1 1D-HPLC ................................ 35
5.2.2 Enantiomers separation .................... 37
第三節 實驗分析方法確效結果......................................... 41
5.3.1 檢量線線性（Linearity）................. 41
5.3.2 精密度 (Precision) ...................... 44
5.3.3 準確度 (Accuracy) ....................... 44
第四節 生物檢品分析結果 ........................ 47
5.4.1 健康人血漿中乳酸與 3-羥基丁酸的表現 ...... 47
5.4.2 健康人尿液中乳酸與 3-羥基丁酸的表現 ...... 48
5.4.3 健康人唾液中乳酸與 3-羥基丁酸的表現 ...... 49
第六章 討論 ...............................50IV
第一節 利用 2D-HPLC分離乳酸與 3-羥基丁酸鏡像異構物
方法之建立.......................................................................... 50
第二節 利用 2D-HPLC測量乳酸與 3-羥基丁酸鏡像異構物
在健康人體內的表現 .......................................................... 53
6.2.1 健康人血漿中乳酸與 3-羥基丁酸的表現 .............. 53
6.2.2 健康人尿液中乳酸與 3-羥基丁酸的表現 ............... 54
6.2.3 健康人唾液中乳酸與 3-羥基丁酸的表現 ....... 55
第三節 乳酸與 3-羥基丁酸鏡像異構物之間的關聯性 . 56
第七章 結論 ...............................58
參考文獻 ..................................59

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