雌二醇及其代謝物之質譜分析研究

Simultaneous measurement of estradiol and estradiol metabolites by Mass Spectrometry

楊博甦(Bo-Su Yang)

指導教授 ： 張耀仁

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2007年)

摘要

雌激素具有廣泛的生理機能，除了在女性的生殖系統及第二性徵的發展扮演著重要的角色，目前已知有數種的雌激素代謝產物，被認為與癌症的發生有關，因此本研究首先建立E2及其代謝物(2OHE2、4OHE2、2MeOE2、4MeOE2)之氣相層析質譜儀(GC/NCI-MS)分析方法，並用於偵測女性頭髮中的E2及其代謝物。研究結果發現：方法偵測極限依序為2OHE2為1 pg， 4OHE2為0.6 pg， E2為5 pg， 2MeOE2和4MeOE2皆為4 pg。而在毛髮真實樣品分析中，我們偵測到女性毛髮中E2的濃度介於0.3~0.8 pg/mg hair，而E2代謝物4MeOE2濃度則介於0.7~1.3 pg/mg hair間；同時，我們發現男性毛髮中E2濃度介於0.1~0.17 pg/mg hair，而E2代謝物4MeOE2濃度則介於0.26 ~0.57 pg/mg hair間，男性約為女性的1/3。
隨後，我們進一步發展液相層析串聯質譜儀(LC/MS/MS)方法，我們發現將E2及其代謝物進行dansyl chloride的衍生化後，分析靈敏度約可大幅提升為未衍生化時的十倍到百倍之多，甚至略優於GC/NCI-MS的最低偵測極限結果。因此未來可利用本研究發展的LC/MS/MS和GC/NCI-MS作為分析工具，探討不同病症族群中的雌激素及其代謝物的差異性，瞭解其所代表的生理意義，並進一步作為疾病的生物指標。

關鍵字

雌二醇 ； 氣相層析負離子化學游離法質譜儀 ； 液相層析串聯式質譜儀

並列摘要

Estrogens have widespread biological actions, like development in sexual organs in women. The effect of estradiol (one kind of estrogens) in carcinogenesis may depend on individual variation of metabolites of estradiol. In this study, gas chromatography mass spectrometry method (GC/MS) and liquid chromatography tandem mass spectrometry method (LC/MS/MS) have been developed for measuring the estradiol and its metabolites, include 2-hydroxyestradiol、4-hydroxyestradiol、2-methoxy estradiol and 4-methoxyestradiol. For GC/MS with negative ion chemical ionization (GC/NCI-MS), the estradiol and four metabolites were derivatized with pentafluoropropionic anhydride (PFPA). The limit of detection was 5pg for estradiol, 1pg for 2-hydroxyestradiol, 0.6pg for 4-hydroxyestradiol, 4 pg for 2-methoxyestradiol and 4-methoxyestradiol. Then we applied to hair testing, we found the concentration of estradiol and 4-methoxyestradiol were 0.3~0.8, 0.7~1.3 pg/mg-hair for female, and 0.1~1.17, 0.26~0.57 pg/mg-hair for male.
For LC/MS/MS, we used the atmospheric pressure chemical ionization (APCI) mode with dansyl chloride derivate, and determined with multiple reaction monitor. The result show the sensitivity of APCI/MS/MS was slightly better than GC/NCI-MS. Since estrogen metabolism may influence the risk of illness, this sensitive, accurate, and precise method will facilitate future clinical and fundamental medical research for measuring estrogen metabolites in biological matrixes.

並列關鍵字

estradiol ； GC/NCI-MS ； LC/MS/MS

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