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

10.6134/tjfm.201806_10(1).0003

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

還原胺化法 ； 碘乙醯胺 ； 多重反應監控 ； Oseltamivir Phosphate（OP） ； Oseltamivir Carboxylate（OC） ； Reductive Amination ； Iodoacetamide ； Multiple Reaction Monitoring ； Oseltamivir Phosphate (OP) ； Oseltamivir Carboxylate (OC)

台灣法醫學誌

9卷2期&10卷1期（2018 / 06 / 01）

25 - 37

繁體中文

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。

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.

1. Bahrami, G, ,Mohammadi, B, ,Kiani, A(2008).Determination of oseltamivir carboxylic acid in human serum by solid phase extraction and high performance liquid chromatography with UV detection.J Chromatogr B Analyt Technol Biomed Life Sci,846,38-42.
2. Concheiro, M,Simões, SM,Quintela, O(2007).Fast LC-MS/MS method for the determination of amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB and PMA in urine.Forensic Sci Int,171,44-51.
3. Ghosh, GC,Nakada, N,Yamashita, N,Tanaka, H(2010).Oseltamivir carboxylate, the active metabolite of oseltamivir phosphate (Tamiflu), detected in sewage discharge and river water in Japan.Environ Health Perspect,118,103-7.
4. Gold, LS,Manley, NB,Slone, TH,Ward, JM(2001).Compendium of chemical carcinogens by target organ: results of chronic bioassays in rats, mice, hamsters, dogs, and monkeys.Toxicol Pathol,29,639-52.
5. Gries, W,Kupper, K,Leng, G(2015).Rapid and sensitive LC-MS-MS determination of 2-mercaptobenzothiazole, a rubber additive, in human urine.Anal Bioanal Chem,407,3417-23.
6. Gries, W,Küpper, K,Leng, G(2015).Rapid and sensitive LC-MS-MS determination of 2-mercaptobenzothiazole, a rubber additive, in human urine.Anal Bioanal Chem,407,3417-23.
7. Heinig, K,Bucheli, F(2008).Sensitive determination of oseltamivir and oseltamivir carboxylate in plasma, urine, cerebrospinal fluid and brain by liquid chromatography-tandem mass spectrometry.J Chromatogr B Analyt Technol Biomed Life Sci,876,129-36.
8. Honjo, T,Makino, T(1986).The preconcentration and separation of a trace amount of copper(II) in water as its silica-immobilized salicylaldimine chelate by means of column chromatography.Bull Chem Soc Jpn,59,3273-4.
9. Hu, ZY,Laizure, SC,Meibohm, B,Herring, VL,Parker, RB(2013).Simple and sensitive assay for quantification of oseltamivir and its active metabolite oseltamivir carboxylate in human plasma using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry: improved applicability to pharmacokinetic study.J Pharm Biomed Anal,72,245-50.
10. Huang, SY,Tsai, ML,Wu, CJ,Hsu, JL,Ho, SH,Chen, SH(2006).Quantitation of protein phosphorylation in pregnant rat uteri using stable isotope dimethyl labeling coupled with IMAC.Proteomics,6,1722-34.
11. Ichinoki, S,Otani, S,Fujii, Y(2006).Selective determination of palladium ion in river water by solvent extraction with 5-chloro-2- mercaptobenzothiazole followed by reversedphase HPLC.J Liq Chromatogr Relat Technol,29,2457-69.
12. Ichinoki, S,Sasai, S,Sugai, H,Fujii, Y(2009).Determination of copper ion in river water by solvent extraction with 2-mercaptobenzothiazole followed by reversed phase HPLC.J Liq Chromatogr Relat Technol,32,3066-76.
13. Jabbaribar, F,Mortazavi, A,Jalali-Milani, R,Jouyban, A(2008).Analysis of oseltamivir in Tamiflu capsules using micellar electrokinetic chromatography.Chem Pharm Bull,56,1639-44.
14. Kanneti, R,Bhavesh, D,Paramar, D,R, S,Bhatt, PA(2011).Development and validation of a high-throughput and robust LC-MS/MS with electrospray ionization method for simultaneous quantitation of oseltamivir phosphate and its oseltamivir carboxylate metabolite in human plasma for pharmacokinetic studies.Biomed Chromatogr,25,727-33.
15. Kumihashi, M,Ameno, K,Shibayama, T(2007).Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography columnswitching method.J Chromatogr B Analyt Technol Biomed Life Sci,845,180-3.
16. Kvitvang, HF,Andreassen, T,Adam, T,Villas-Bôas, SG,Bruheim, P(2011).Highly sensitive GC/MS/MS method for quantitation of amino and nonamino organic acids.Anal Chem,83,2705-11.
17. Lin, YR,Wu, CE,Huang, MF(2017).Development of doxorubicin quantification by reductive amination.Curr Anal Chem,13,270-6.
18. Lindegårdh, N,Hanpithakpong, W,Wattanagoon, Y,Singhasivanon, P,White, NJ,Day, NP(2007).Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of oseltamivir and its metabolite oseltamivir carboxylate in plasma, saliva and urine.J Chromatogr B Analyt Technol Biomed Life Sci,859,74-83.
19. Liu, MC,Lin, YR,Huang, MF,Tsai, DC,Liang, SS(2015).Mass spectrometry signal enhancement by reductive amination.Int J Mass Spectrom,387,16-23.
20. Rodriguez, DM,Wrobel, K,Jiménez, MG,Wrobel, K(2004).Determination of 2-mercaptobenzothiazole (MBT) i n tannery wastewater by high performance liquid chromatography with amperometric detection.Bull Environ Contam Toxicol,73,818-24.
21. Simazaki, D,Kubota, R,Suzuki, T,Akiba, M,Nishimura, T,Kunikane, S(2015).Occurrence of selected pharmaceuticals at drinking water purification plants in Japan and implications for human health.Water Res,76,187-200.
22. So, PK,Ng, TT,Wang, H,Hu, B,Yao, ZP(2013).Rapid detection and quantitation of ketamine and norketamine in urine and oral fluid by woodentip electrospray ionization mass spectrometry.Analyst,138,2239-43.
23. Sorahan, T(2009).Cancer risks in chemical production workers exposed to 2-mercaptobenzothiazole.Occup Environ Med,66,269-73.
24. Sorahan, T,Hamilton, L,Jackson, JR(2000).A further cohort study of workers employed at a factory manufacturing chemicals for the rubber industry, with special reference to the chemicals 2-mercaptobenzothiazole (MBT), aniline, phenyl-β-naphthylamine and o-toluidine.Occup Environ Med,57,106-15.
25. Su, X,Wang, N,Chen, D(2016).Dansylation isotope labeling liquid chromatography mass spectrometry for parallel profiling of human urinary and fecal submetabolomes.Anal Chim Acta,903,100-9.
26. Suriyanarayanan, T,Qingsong, L,Kwang, LT,Mun, LY,Seneviratne, CJ(2018).Quantitative proteomics of strong and weak biofilm formers of Enterococcus faecalis reveals novel regulators of biofilm formation.Mol Cell Proteomics,17,643-54.
27. Terada, K,Morimoto, K,Kiba, T(1980).The chromatographic concentration of mercury in sea water with 2-mercaptobenzothiazole supported on silica-gel.Bull Chem Soc Jpn,53,1605-9.
28. Wu, Y,Wang, F,Liu, Z(2014).Five-plex isotope dimethyl labeling for quantitative proteomics.Chem Commun,50,1708-10.
29. Zhou, R,Li, L(2014).Quantitative metabolomic profiling using dansylation isotope labeling and liquid chromatography mass spectrometry.Methods Mol Biol,1198,127-36.