結合態雌激素之液相層析分析研究
Determination of conjugated estrogens by high performance liquid chromatography
中文摘要
結合態雌性激素(conjugated estrogens)製劑的原料主要是從懷孕母馬尿中所提煉 出或由合成estrone 及 equilin 得來的一些有相互關連的雌性激素混合物，其製劑 主要用來預防及治療婦女更年期症狀。這些混合物已被科學家分離且定出分別的 結構；在美國藥典24 版所記載 conjugated estrogens 品目中包含了 estrone (1)、
equilin (2)、equilenin (3)、17α-estradiol (4)、17β-estradiol(5)、17α-dihydroequilin (6) 、17β-dihydroequilin (7)、17α-dihydroequilenin (8)、17β-dihydroequilenin (9) 及 Δ8,9-dehydroestrone (10) 等十種以 sodium sulfate 結合態存在成分之規格及範圍，
用來做為評估該原料品質的基準。但由於現行的公定方法為氣相層析法(gas chromatography, GC)，該法檢體配製須先以酵素水解為游離態雌激素成分後，
將其衍生化，然衍生化反應及氣相層析儀的操作、分析條件需經過相當繁複之調 整，方能得到良好的分析結果。
因此，本計畫擬開發出一種既通用、簡便而又精準的一般性高效液相層析法，用 來定性及定量有關 estrogens 的相關製劑。檢體之前處理依照美國藥典(USP) 24
版所收載方法，以硫酸酯酵素法先行水解後，加入標準品estriol 溶液溶解後，
直接加以分析，其分析條件為一般逆向層析管柱（18），移動相為氰甲烷：
0.22 M 硝酸銀-0.04 % 硝酸水溶液= 40：60 (v/v)混液，流速為 1.0 mL/min.，紫 外光偵測波長為 280 nm，分析結果良好，十種成分均能完全分離，系統適應性 之分離度R 值(resolution)均大於 1.2 以上，對稱性(symmetry)均在 0.9~1.1 之間。
同時，將本分析方法與氣相層析法在分離度及準確度上作一比較，並進一步使 用本分析方法應用於原廠及國內生產該製劑之品質比較及評估，以了解原料來 源及穩定性和均一性，作為行政機關及相關業界之參考，以提升該製劑之品質，
保障國人健康。
英文摘要
Conjugated estrogens are a mixture of at least ten estrogen sulfate conjugates from the urine of pregnant mares or chemical synthetically from estrone and equilin. The preparations of conjugated estrogens are now widely used for the menopausal women as estrogen replacement therapy. The mixture of close-related estrogenic compounds included: estrone (1); equilin (2); equilenin (3); 17a-estradiol (4); 17β-estradiol (5);
17a-dihydroequilin (6); 17b-dihydroequilin (7); 17a-dihydroequilin (8); 17β- dihydroequilenin (9) and D8,9-dehydroestrone (10). The test specifications of conjugated estrogens also have been regulated by USP XXIV. The official USP XXIV method for determination of conjugated estrogens is based on the capillary-GC
of trimethylsilyl ethers derivatization after enzyme hydrolysis. The GC condition requires an experienced technique to performe, otherwise, the satisfactory result is not easy to produce.
This study is to explore the use of simple HPLC method for determination of these ten conjugated estrogens. The test samples were hydrolyzed by sulfatase by USP XXIV method, and then dissolved in internal standard solution of estriol . Samples were chromatographed on a reverse-phase INERTSIL ODS-2 column (5um, 250 ´ 4.6 mm) and eluted by using a mobile phase of CH3CN：0.22 M AgNO3 in 0.04 % HNO3 aqueous solution = 40：60 (v/v). The flow rate was 1.0 mL/min and the effluent was monitored at UV of 280 nm. This HPLC method can satisfactorily make the baseline separation of these ten components. It can be provided to the determination of the ten conjugated estrogens in the bulk drug and their preparations.
