臺灣博碩士論文加值系統

本研究利用螢光偵測導向之衍生反應，配合高效能液相層析法，對代謝疾病或污臭有關指標物isovaleric acid與valeric acid，建立簡單高靈敏之分析方法。本法利用2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES)為衍生試劑，以甲苯為反應溶媒，於碳酸鉀及18-crown-6 ether之催化下，將isovaleric acid與valeric acid加以衍生成螢光性之naphthoxyethyl衍生物，利用C-18固定相及甲醇：水：四氫呋喃為72：22：6（v/v）混合液為移動相進行分離，對所得待測物以螢光偵測器（lex: 235nm; lem: 350nm）檢測。所得結果顯示本法靈敏性高，Isovaleric acid與valeric acid之定量範圍爲0.5 ~10.0 mM，偵測極限0.07 mM (S/N = 3；20 mL)。
本研究對影響isovaleric acid與valeric acid衍生化反應之重要因素，包括試劑需要量、鹼性催化劑、反應時間及溫度等，皆詳加探討。本法對病人尿液中微量isovaleric acid與valeric acid之分析應用正探討中。

Analytical derivatization coupled with high-performance liquid chromatography was used for the analysis of isovaleric and valeric acids as biomarkers in metabolic acidosis or as malodorant in organic pollution. The method is based on the derivatization of isovaleric acid and valeric acid with 2-(2-naphthoxy)ethyl 2-(piperidino) ethanesulfonate (NOEPES) in toluene, using potassium carbonate and 18-crown-6 ether as reaction activators. The resulting naphthoxyethyl derivative of isovaleric acid and valeric acid were separated on a C-18 column, using a mixed solvent of methanol : water : tetrahydrofuran (72：22：6, v/v) as the mobile phase. The separated isovaleric acid and valeric acid derivatives were monitored with a fluorimetric detector (lex: 235nm; lem: 350nm), and the peak-areas of isovaleric and valeric acids derivatives were used for quantitative analysis.
The linear range of the method for the determination of isovaleric acid and valeric acid derivative were over 0.5 ~ 10.0 mM. The detection limit (signal to noise ratio = 3, injected amount 20 mL) of isovaleric acid and valeric acid was about 0.07 mM. Several parameters affecting the derivatization of isovaleric and valeric acids were discussed including amount of derivatization reagent, reaction temperature, reaction time, and base catalyst. Application of the method to the analysis of isovaleric and valeric acids in urine is in procession.

目 錄
中文摘要…………………………………………………………… i
英文摘要…………………………………………………………… ii
圖目錄……………………………………………………………… v
表目錄……………………………………………………………… vii
第一章、緒論……………………………………………………… 1
第二章、實驗部分………………………………………………. 7
一、試藥及材料……………………………………………… 7
二、儀器……………………………………………………… 9
三、衍生試劑之合成………………………………………… 10
四、試藥溶液之配製………………………………………… 13
五、高效能液相層析之分析條件…………………………… 15
六、Isovaleric acid與valeric acid衍生物之合成…… 16
七、Isovaleric acid與valeric acid衍生反應條件之探討
……………………………………………………………17
1. 衍生試劑需要量…………………………………… 18
2. 碳酸鉀需要量……………………………………… 19
3. 18-crown-6 ether需要量………………………… 20
4. 反應時間…………………………………………… 21
八、分析方法之選擇性……………………………………… 22
九、衍生物安定性…………………………………………… 23
十、應用分析………………………………………………… 24
1. 尿液中isovaleric acid與valeric acid之萃取… 24
2. 萃取所得isovaleric acid與valeric acid之衍生
反應與HPLC分析…………………………………… 25
第三章、結果與討論……………………………………………… 26
一、反應溶媒………………………………………………… 26
二、Isovaleric acid與valeric acid衍生物之HPLC螢光
層析圖與過量衍生試劑之酸處理……………………… 27
三、內部標準品的選擇……………………………………… 29
四、最適衍生反應條件之建立……………………………… 31
1. 衍生試劑需要量…………………………………… 31
2. 碳酸鉀需要量……………………………………… 33
3. 18-crown-6 ether需要量………………………… 35
4. 反應時間…………………………………………… 37
五、分析方法之選擇性……………………………………… 40
六、建立isovaleric acid與valeric acid檢量線……… 42
七、Isovaleric acid與valeric acid衍生物之安定性… 44
八、應用分析………………………………………………… 45
第四章、結論……………………………………………………… 51
第五章、參考文獻………………………………………………… 52
圖 目 錄
圖一 Isovaleric acid的代謝路徑………………………… 2
圖二 Isovaleric與valeric acids (RCOOH) 和NOEPES的
反應機轉示意………………………………………… 5
圖三 Isovaleric與valeric acids衍生物合成反應示意
圖…………………………………………………………16
圖四 Isovaleric acid與valeric acid (1 mM) 之代表
層析圖(a)未經過酸處理；(b)有進行酸處理。波峰1.
isovaleric acid 的衍生物; 波峰2. valeric acid
衍生物……………………………………………………28
圖五 NOEPES的濃度對isovaleric acid和valeric acid衍
生物形成的影響…………………………………………32
圖六 碳酸鉀的量對isovaleric acid和valeric acid衍生
物形成的影響……………………………………………34
圖七 18-crown-6 ether的濃度對isovaleric acid和
valeric acid衍生物形成的影響………………………36
圖八 在90℃下反應時間對isovaleric acid和valeric
acid衍生物形成的影響…………………………………37
圖九 Isovaleric acid與valeric acid (1 mM) 之代表層
析圖(a)待測物以NOEPES進行衍生反應；(b)為reagent
blank。波峰1. isovaleric acid 的衍生物; 波峰2.
valeric acid衍生物。…………………………………39
圖十 分析方法選擇性之層析圖………………………………41
圖十一 Isovaleric acid和valeric acid的檢量線………… 43
圖十二 Isovaleric acid 和valeric acid之衍生物的穩定性
……………………………………………………………44
圖十三 萃取溶媒體積對於isovaleric acid與valeric acid
(6 nmol)自尿液中萃取之萃取率的影響………………45
圖十四 尿液中isovaleric acid及valeric acid的HPLC層析
圖(a)為尿液檢品沒有添加isovaleric acid 與
valeric acid的層析圖 (b)為尿液檢品有添加
isovaleric acid 與valeric acid (各為12 nmol)的
層析圖。波峰1. isovaleric acid 的衍生物; 波峰2.
valeric acid衍生物。…………………………………46
圖十五 標準添加法檢量線之簡圖………………………………47
表 目 錄
表一 一些內部標準測試用化合物的滯留時間………………30
表二 三位健康者的尿液中所含待測物之分析 (n=7)………48
表三 Isovaleric acid分析所得檢量線之斜率 (slope)
與截距 (intercept) 的精密度……………………… 50
表四 Valeric acid分析所得檢量線之斜率 (slope) 與
截距(intercept) 的精密度……………………………50

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