以同位素相似物修飾目標物的質譜定量方法

Development of Quantitative Methods for Target Molecules Based on Isotopic Analogs Modification

梁世欣(Shih-Shin Liang) ； 薛佑玲(Yow-Ling Shiue) ； 張育慈(Yu-Tzu Chang) ； 宋沛(Pei Sung)

《台灣法醫學誌》 9卷2期&10卷1期 (2018/06) Pp. 25-37

https://doi.org/10.6134/tjfm.201806_10(1).0003

摘要

定量方法的開發需要適當的標準品做為測定的參考，內標準品的選擇以同位素相似物最為合適。還原胺化法與碘乙醯胺的烷化法，可利用穩定同位素的化合物合成在具有胺基及硫醇官能基的目標分子上。因此，可以生成新穎的氫取代目標分子及同位素取代的目標分子作為標準品與內標準品。在還原胺化法中，甲醛與氘取代甲醛會分別與含胺基化合物反應形成亞胺，再與還原劑氰基硼氫化鈉（NaBH_3CN）作用，最後形成雙甲基結構的化合物。而烷基化法，則是透過碘乙醯胺與具同位素的碘乙醯胺與具有硫醇官能基的2-mercaptobenzothiazole（MBT）作用，分別合成出新式標準品與內標準品供定量分析使用。還原胺化法與碘乙醯胺的烷化法，二者的優點均具有合成步驟簡單且產率近乎100%的優點，甲醛與氘取代甲醛為便宜且容易取得的試劑。本報告以還原胺化法合成克流感（oseltamivir phosphate, OP）及其一階代謝物oseltamivir carboxylate（OC）為例，演示還原胺化法。碘乙醯胺的烷化法則取MBT為例，說明二者的應用。由質譜的結果也發現，經由還原胺化法及烷化法修飾之後，修飾物的質譜訊號均較未修飾的目標分子為高。

關鍵字

還原胺化法 ； 碘乙醯胺 ； 多重反應監控 ； Oseltamivir Phosphate（OP） ； Oseltamivir Carboxylate（OC）

並列摘要

In the development of quantification, it is suitable to utilize isotopic analogs as internal standards. Both methods such as reductive amination and alkylation are synthetic methods to react stable isotope compounds on target molecules which are being amine or thiol functional groups. Therefore, it will be generated novel hydrogenated target molecules to be standards and novel isotope modified target molecules to be internal standards. In reductive amination, formaldehyde and formaldehyde-d_2 are reacted with amine into compound ＂imine＂. And then, the imine functional group can be reduced by sodium cyanoborohydride to generate two methyl groups on amine group. Moreover, the method of alkylation is utilized isotopic iodoacetamide to generate with thiol group of 2-mercaptobenzothiazole (MBT) to produce a novel standard and an internal standard for quantification. Reductive amination and alkylation of iodoacetamide, both methods have some advantages such as simplified procedures and high yield, and formaldehyde and deuterated formaldehyde are accessible and inexpensive reagents. In this report, novel quantitative methods were developed by using oseltamivir phosphate (OP) and oseltamivir carboxylate (OC) to react with isotopic formaldehydes, and demonstrating alkylation of MBT with iodoacetamide. Besides, due to the modification of reductive amination and alkylation, spectra of mass spectrometry showed the quality of signal enhancement comparing with unmodified analogs.

並列關鍵字

Reductive Amination ； Iodoacetamide ； Multiple Reaction Monitoring ； Oseltamivir Phosphate (OP) ； Oseltamivir Carboxylate (OC)

參考文獻

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