Determination of conjugated estrogens by high performance liquid chromatography

• Main Authors: Chih-ping Hong, 洪志平
• Other Authors: Chi-Ming Chen
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/76511548945007874077

碩士 === 台北醫學院 === 藥學研究所 === 90 === 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.