Amperometric Detectorを用いた高速液体クロマトグラフィーによる血清中ESTROGEN 4分画微量定量法

概要

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A simultaneous microdetermination of unconjugated estrone, estradiol, estriol and estetrol in serum or amniotic fluid by High Performance Liquid Chromatography with an Amperometric Detector is described. Steroids in serum or amniotic fluid were extracted with 10 volumes of ethylether, and then ether extract was evaporated to dryness under N2 gas. After defatting with a mixture of 50% methanol/n-hexane, the methanol phase was evaporated to dryness under N2 gas. The residue was applied to microcolumn packed with 2 ml volume of Sephadex LH-20 in the eluting soluvent benzene/methanol (85 : 15). Fractions contained estrone and estradiol; estriol and estetrol were collected and then evaporated to dryness under N2 gas. The sample solution was applied to HPLC using a reverse phase ODS column and acetonitrile : 0.1M KH2PO4 47 : 53 for estrone and estradiol fraction, and 30 : 70 for estriol and estetrol fraction as a mobile phase, respectively. The fraction of each estrogen was separated completely within a 20 minute period. The limit of detection of estrone, estradiol, estriol and estetrol was 50 pg, respectively.
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