使用還原胺化法與液相層析串聯式質譜儀定量克流感藥物及組織胺

Quantification of Tamiflu and Histamine by Reductive Amination and Liquid Chromatography Tandem Mass Spectrometry

黃梅芳(Mei-Fang Huang)

指導教授 ： 梁世欣

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2017年)

摘要

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。

關鍵字

還原胺化法 ； Oseltamivir phosphate (OP) ； Oseltamivir carboxylate (OC) ； 組織胺(histamine) ； 液相層析串聯式質譜儀

並列摘要

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.

並列關鍵字

Reductive amination ； Oseltamivir phosphate (OP) ； Oseltamivir carboxylate (OC) ； Histamine ； Liquid chromatography tandem mass spectrometry (LC-MS/MS)

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