臺灣博碩士論文加值系統

還原胺化法(Reductive amination)原本為醛酮轉化為胺基的反應，甲醛會先與含胺基化合物反應形成亞胺，再與還原劑氰基硼氫化鈉（NaBH3CN）反應，最後可形成雙甲基結構的化合物，此方法被使用於蛋白質體學研究人類相關疾病。還原胺化法的優點為使用易取得及便宜的試劑，合成步驟簡單且產率近乎100%。本論文研究是使用還原胺化法合成Oseltamivir phosphate (OP)、Oseltamivir carboxylate (OC) 及組織胺(Histamine)的同位素修飾物來當作標準品及內標準品並開發定量方法。OP的商品名為TamifluR，為目前最普遍使用的抗流感藥物，OP會經由肝的酵素代謝成OC，目前評估Tamiflu的毒性對於一些高危險族群是非常重要的，例如嬰兒與重症病人。OP和OC經人體代謝後，由尿液排出進到汙水處理廠，處理後會有殘留的現象。長期的累積下可能會產生流感病毒抗藥性病毒株並對環境造成污染。組織胺(Histamine)為過敏反應中的發炎因子，Histamine的釋放可能會引起人體皮膚紅腫、打噴嚏甚至過敏性休克。Histamine可作為紅肉魚類新鮮度的指標，不適當的食物處理過程及保存方式會使細菌滋生，造成魚體中的Histidine形成Histamine，高濃度的Histamine會產生過敏的症狀。此篇論文，還原胺化法被使用來合成OP、OC及Histamine的同位素類似物且使用液相層析串聯式質譜儀完成方法開發與確效，結果也發現經由還原胺化法修飾後，同位素修飾物質譜訊號比未修飾化合物的訊號高。最後，OP、OC及Histamine的定量平台成功地建立且還原胺化法也很適合應用到其他生物性化合物的研究。

Reductive amination is a synthetic method to generate amine and aldehyde into an intermediate compound “imine”. Eventually, the imine can be reduced by sodium borohydride to form two methyl groups. This method is utilized for comparative proteomic researches to study diseases of human. The advantages of reductive amination are using accessible and inexpensive reagents, simplified procedures and near 100% yield. In these researches, novel quantitative methods were developed by reductive amination to generate isotopic analogs of oseltamivir phosphate (OP), oseltamivir carboxylate (OC) and Histamine to be standards and internal standards. OP, trade name TamifluR, is the drug commonly used to treat influenza. OP would be metabolized by liver enzyme to generate its metabolite OC. However, it is important to evaluate the toxicity of Tamiflu in some high risk populations, including young infants and the severely ill patients. Moreover, OP and OC would be excreted from human via urine and remained in waste water by the sewage treatment plants after treatment. The risk mentioned above may produce the resistant strains of the viruses to OP and cause environmental pollution. Another research focused on Histamine. Histamine is well known as the inflammatory factor in anaphylactic reaction. The release of Histamine may cause rashes, sneeze, and even lead to serious complication. Furthermore, Histamine is an indicator of seafood freshness especially in scombroid fish. Inappropriately processed and stored method for food would lead to microbial contamination making Histidine become histamine by decarboxylation. High level of Histamine is poisonous and even life-threatening. Therefore, it is essential to establish a new research on TamifluR and Histamine. Reductive amination was used to synthesize isotopic analogs of OP, OC and Histamine. The method validations were achieved couple with liquid chromatography tandem mass spectrometry (LC-MS/MS). Furthermore, the modification of reductive amination showed the quality of signal enhancement compared with unmodified analogs. Finally, quantitative platforms of OP, OC and Histamine were successfully developed and reductive amination is appropriate to expand applications to other biological compounds.

中文摘要………………………………………………………………………………………………..i
英文摘要……………………………………………………………………………………………….ii
目錄……………………………………………………………………………………………………..iii
圖目錄…………………………………………………………………………………………….....vii
表目錄………………………………………………………………………………………………….ix

一、 緒論………………………………………………………………………………………..1
1.1研究動機………………………………………………………………………………………….1
1.2環境及藥物領域：克流感藥 物TamifluR及其代謝物的定量分析......5
1.2.1流行性感冒……………………………………………………5
1.2.2抗流感藥物…………………………………………………....6
1.2.3克流感藥物Tamiflu………………………………………………..7
1.2.4 Tamiflu對環境的影響………………………………………..8
1.2.5文獻回顧………………………………………………….......9
1.3生物醫學及食品安全領域：血清當中組織胺定量分析…………11
1.3.1 組織胺(Histamine)……………………………………….....11
1.3.2 組織胺中毒………………………………………...……….13
1.3.3文獻回顧……………………………………………………15

1.4層析管柱..................................................................................................18
1.4.1層析原理………………….………………………………….18
1.4.2逆相層析(Reverse phase chromatography)管柱……………19
1.4.3正相層析(Normal phase chromatography)管柱……………20
1.4.4親水性交互作用液相層析(Hydrophilic interaction liquid chromatography, HILIC)管柱....................................................................21
1.5質譜儀(Mass spectrometry)……………………………………23
1.5.1電灑游離法(Electrospray ionization, ESI)……….…….23
1.5.2三重四極桿串聯式質譜儀(Triple quadrupole mass spectrometry)…………………………………………………………..24
1.5.3多重反應監測(Multiple reaction monitoring, MRM)……..25

二、 實驗部分………………………………………………………………………………..26
2.1藥品與儀器……………………………………………………………………………………26
2.1.1藥品…………………………………………………………………………………….26
2.1.2儀器及軟體………………………………………………………………………..27
2.2實驗流程………………………………………………………………………………………..27
2.2.1還原胺化合成氫、氘取代化合物…………………………………..27
2.2.2還原胺化修飾後克流感藥物(Oseltamivir phosphate, OP)及其代謝物(Oseltamivir carboxylate, OC)檢量線及真實樣品配製……….28
2.2.2.1 H2-modified OP和H2-modified OC檢量線配製..….28
2.2.2.2 H2-modified OP和H2-modified OC訊號增加評估…28
2.2.2.3真實樣品方法確效配製……………………..……….29
2.2.2.4真實樣品基質干擾測試……………………………..31
2.2.2.5液相層析串聯式質譜儀分析條件…………………..31
2.2.3還原胺化修飾後組織胺(Histamine)檢量線及真實樣品配製
……………………………………………………………..32
2.2.3.1 H2-modified histamine檢量線配製……..………..32
2.2.3.2 H2-modified histamine訊號增加評估…….……….33
2.2.3.3 Histamine真實樣品方法確效：使用標準添加法..33
2.2.3.4 液相層析串聯式質譜儀分析條件………………...34

三、 結果………………………………………………………………………………………36
3.1 環境醫學：克流感藥物TamifluR及其代謝物的定量析…............36
3.1.1 Oseltamivir phosphate (OP)與oseltamivir carboxylate (OC)修飾後於三重四極桿質譜儀(triple quadrupole MS)中的斷片分析…….36
3.1.2 H2-modified OP與H2-modified OC方法確效…………….37
3.1.3 H2-modified OP與H2-modified OC的訊號增加評估…….37
3.1.4模擬真實樣品定量分析…………………………………….38
3.1.5真實樣品基質干擾測試.............................................................39
3.2生物醫學：血清中組織胺定量分析……………………………..40
3.2.1 Histamine修飾後於質譜儀中的斷片分析………………..40
3.2.2 H2-modified histamine方法確效…………………………..40
3.2.3 H2-modified histamine的訊號增加評估……………….….41
3.2.4模擬真實樣品定量分析：使用還原胺化法…………...…...41

四、 討論………………………………………………………………………………………53

五、 參考文獻………………………………………………………………………..55

圖目錄
一、 緒論
圖1.1還原胺化法反應機制…………………………………………………………….2
圖1.2還原胺化法應用於蛋白質體學示意圖…………………………………….3
圖1.3還原胺化法簡易化學反應式………………………………………………..4
圖1.4流感病毒感染示意圖……………………………………………………………5
圖1.5 2015年第一週到2017年第九週的流感死亡統計……………….6
圖1.6抗流感藥物作用示意圖……………………………………………………….7
圖1.7 OP代謝形成OC示意圖……………………………………………………..8
圖1.8組織胺結構…………………………………………………………………………..11
圖1.9 過敏反應機制………………………………………………..12
圖1.10 組胺酸產生組織胺反應式………………………………….13
圖1.11 正相、逆相層析與HILIC管柱的適用分析領域.................19
圖1.12 逆相層析管柱內的填充顆粒結構…………………………..20
圖1.13 逆相層析管柱內填充顆粒結構…………………………….20
圖1.14 正相層析管柱內顆粒結構…………………………………..21
圖1.15 HILIC管柱內填充顆粒結構…………………………………22
圖1.16 HILIC管柱內官能基類別……………………………………22
圖1.17 電灑游離法表示圖…………………………………………...23
圖1.18四極柱偵測器示意圖………………………………………..24
圖1.19三重四極桿串聯式質譜儀內部示意圖……………………..25

三、 結果
圖3.1 OP與OC使用還原胺化法合成氫取代修飾物示意圖…….42
圖3.2 OP與OC使用還原胺化法合成氘取代修飾物示意圖…….43
圖3.3 OP及其同位素修飾物質譜斷片圖…………………………44
圖3.4 OC及其同位素修飾物質譜斷片圖…………………………45
圖3.5 OP與OC氫氘取代同位素修飾物層析圖………………….46
圖3.6 H2-modified OP與H2-modified OC檢量線結果圖…………….46
圖3.7 OP與OC與氫取代修飾物訊號比較層析圖……………….47
圖3.8 Histamine使用還原胺化法合成氫、氘取代修飾物示意圖..49
圖3.9 Histamine及其同位素修飾物質譜斷片圖……………………………49
圖3.10 Histamine氫氘取代同位素修飾物層析圖………………….50
圖3.11 H2-modified histamine檢量線結果圖……………….……….51
圖3.12 Histamine及H2-modified histamine的訊號比較層析圖…..51
圖3.13模擬真實樣品標準添加法結果圖…………………………..52

表目錄
一、 緒論
表1.1 OP與OC的文獻回顧整理……………………………………..9
表1.2 組織胺中毒案件統計…………………………………………..15
表1.3 Histamine文獻回顧整理…………………………………………16

三、 結果
表3.1 OP與OC氫、氘同位素修飾物方法確效表………………….47
表3.2 OP、OC與氫取代修飾物訊號比較表………………………..48
表3.3 H2-modified OP與H2-modified OC模擬真實樣品定量結果表.48
表3.4 Histamine與氫取代修飾物訊號比較表……………………….52
表3.5所有Histamine未修飾與修飾後定量方法確效結果表………52

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