雌激素是對女性生殖器官和女性第二性徵的發展和維護的激素，並且關係到懷孕及女性月經週期。
近年來，許多新穎的分析技術，例如：LC-MS, GC-MS和其他技術發展偵測水、沉積物、組織和血漿中的雌激素。但是，較少研究有關於人體尿液中雌激素的定量分析。
HPLC-FL之靈敏度比HPLC-UV為佳，且因大部分雌激素並無螢光性，故將雌激素衍生化使其具有螢光性，再以HPLC-FL來分析，如此將可提高化學分析法的靈敏度。
以2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole及Dansyl chloride當衍生試劑與雌激素反應。以2-(4-carboxyphenyl)
-5,6-dimethylbenzimidazole當衍生試劑與雌激素反應時，反應條件為60 ℃下加入4-dimethylaminopyridine及N-(3-dimethylamino
propyl)-N’-ethylcarbodiimide hydrochloride，反應30分鐘後，經固相萃取後，過濾以HPLC-FL分析，螢光偵測器之激發波長設定為336 nm，放射波長設定為440 nm。CDB-E1在濃度範圍1000 ppb～10 ppb間可得一檢量線y = 29758008.1185 x + 367851.5823，CDB-E2在濃度範圍1000 ppb～10 ppb間可得一檢量線y = 22597313.25 x + 103941.95其R2=0.9979，CDB-E3在濃度範圍1000 ppb～10 ppb間可得一檢量線 y = 15449204.69 x + 35943.84其R2=0.9993。CDB-estrone分法偵測極限為12.9 ppb，因最後上機分析注射量為20 μL，因此換算成重量為0.26 ng；CDB-estradiol(β)分法偵測極限為12.0 ppb，因最後上機分析注射量為20 μL，因此換算成重量為0.24 ng；CDB-estriol分法偵測極限為11.7 ppb，因最後上機分析注射量為20 μL，因此換算成重量為0.23 ng。若利用微晶片進行分析，本實驗使用壓力進樣方式，各衍生物配製濃度為5000 ppb。在壓力驅動進樣時所設定的幫浦流速為0.5 μL/分鐘，進樣時間皆為0.05分鐘，因此換算成重量為0.125 ng。以Dansyl chloride當衍生試劑與雌激素反應時，反應條件為60 ℃下加入0.5M sodium carbonate，反應30分鐘後，經固相萃取後，過濾以HPLC-FLUO分析，螢光偵測器之激發波長設定為350 nm，放射波長設定為550 nm。Dansyl-Estrone在濃度範圍2000 ppb～30 ppb間可得一檢量線y = 157414x + 2309.3其R2=0.9994，Dansyl-17βestadiol在濃度範圍2000 ppb～30 ppb間可得一檢量線y =162008.4 x + 4820.8其R2=0.9993，Dansyl-17α estadiol在濃度範圍2000 ppb～30 ppb間可得一檢量線 y = 101809x + 2198其R2=0.9996，Dansyl-17αethylestadiol在濃度範圍2000 ppb～30 ppb間可得一檢量線 y = 108974x + 1803其R2=0.9990，Dansyl-estriol在濃度範圍2000 ppb～30 ppb間可得一檢量線 y =94285x + 2154.8其R2=0.9995。Dansyl-estrone分法偵測極限為40.5 ppb，因最後上機分析注射量為20 μL，因此換算成重量為0.81 ng；Dansyl-17αestradiol分法偵測極限為27.3 ppb，因最後上機分析注射量為20 μL，因此換算成重量為0.55 ng；Dansyl-17β estradiol分法偵測極限為32.1 ppb，因最後上機分析注射量為20 μL，因此換算成重量為0.64 ng；Dansyl-17αethylestradiol分法偵測極限為40.8 ppb，因最後上機分析注射量為20 μL，因此換算成重量為0.82 ng；Dansyl-estriol分法偵測極限30.0 ppb，因最後上機分析注射量為20 μL，因此換算成重量為0.60 ng。由HPLC-MS的結果發現Dansyl-Estrone 、Dansyl-estradiol(β) 、Dansyl-17α-ethylestradiol及Dansyl-estriol的訊號，因HPLC-MS具有高精準度及專一性，故可以利用HPLC-ESI-MS來偵測更多雌激素的種類及其機制。

Estrogens are hormones that are responsible for the development and maintenance of the female reproductive organs and female secondary gender characteristics. Estrogens also participate in the regulation of the menstrual cycle and in the maintenance of pregnancy.
In recent years, many novel analytical techniques, such as LC-MS, GC-MS and other techniques have been developed for the determination of estrogens in water, sediment, tissue and plasma. However, few studies are available for the quantitative analysis of estrogens in human urine.
To improve the detection sensitivity, we developed a simple fluorescence labeling method to detect estrogenic compounds in biological fluids.
Two fluorescence reagents were attempted in this study, 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole and Dansyl chloride. For 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole derivatization 4-dimethylaminopyridine and N-(3-dimethylaminopropyl)-N’-ethyl
-carbodiimide hydrochloride were mixed and the mixture was allowed to react for 30 minute at 60 ℃.The reaction mixture was then purified by SPE extraction and then filtered. The product was ready for HPLC-FL analysis using excitation (Ex) = 336 nm and emission (Em) = 440 nm. The standard curve ranging from 1000 ppb to 10 ppb was established for estrone was fitted into an equation of y =29758008.12x + 367851.58 with R2=0.9978. The detection limit of the method was determined to be 12.9 ppb at S/N = 3, which corresponds to 20 μL or 0.26 ng of the derivatized estrone . The standard curve ranging from 1000 ppb to 10 ppb was established for 17β-estradiol was fitted equation was y = 22597313.25 x + 103941.95 with R2=0.9979. The detection limit of the method was determined to be 12.0 ppb at S/N = 3, which corresponds to 20 μL or 0.24 ng of the derivatized 17β-estradiol. The standard curve ranging from 1000 ppb to 10 ppb was established for estriol was fitted equation was y = 15449204.69 x + 35943.84 with R2=0.9993. The detection limit of the method was determined to be 11.7 ppb at S/N = 3, which corresponds to 20 μL or 0.23 ng of the derivatized estriol. For the analysis by microchip electrophoresis under MEKC mode, using pressure injection, the detection limit was determined to be 5000 ppb or 0.125 ng and the analysis time was within 0.05 mins under the flow rate of 0.5 μL/min.
For Dansyl chloride derivatization, 0.5M sodium carbonate was mixed and the mixture was allowed to react for 30 minute at 60 ℃. The reaction mixture was then purified by SPE extraction and then filtered. The product was ready for HPLC-FL analysis using excitation (Ex) = 350 nm and emission (Em) = 550 nm. The standard curve ranging from 2000 ppb to 30 ppb was established for estrone was fitted equation was y = 157414x + 2309.3 with R2=0.9994. The detection limit of the method was determined to be 40.5 ppb at S/N = 3, which corresponds to 20 μL or 0.81 ng of the derivatized estrone. The standard curve ranging from 2000 ppb to 30 ppb was established for 17α-estradiol was fitted equation was y = 101809x + 2198 with R2=0.9996. The detection limit of the method was determined to be 27.3 ppb at S/N = 3, which corresponds to 20 μL or 0.55 ng of the derivatized 17α-estradiol. The standard curve ranging from 2000 ppb to 30 ppb was established for 17β-estradiol was fitted equation was y =162008.4 x + 4820.8 with R2=0.9993. The detection limit of the method was determined to be 32.1 ppb at S/N = 3, which corresponds to 20 μL or 0.64 ng of the derivatized 17β-estradiol. The standard curve ranging from 2000 ppb to 30 ppb was established for estriol was fitted equation was y =94285x + 2154.8 with R2=0.9995. The detection limit of the method was determined to be 30.0 ppb at S/N = 3, which corresponds to 20 μL or 0.60 ng of the derivatized estriol. The standard curve ranging from 2000 ppb to 30 ppb was established for 17α-ethylestradiol was fitted equation was y = 108974x + 1803 with R2=0.9990. The detection limit of the method was determined to be 27.3 ppb at S/N = 3, which corresponds to 20 μL or 0.55 ng of the derivatized 17α-ethylestradiol.
The method applied dansyl chloride as derivatization reagent for estrogenic compounds and combined with SPE column clean-up, followed by HPLC-ESI-MS detection. The detection limit was determined to be around 10 ppb for MS. The method was demonstrated to be a simple and useful method for the detection of Estrone, 17α-estradiol,17β-estradiol, Estriol and 17α-ethynylestradiol.
In the future, the combination of microfluidic chip and MS detection may offer as a high efficiency and ultra sensitive method for the quantitative analysis of estrogenic compounds.

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