Gas chromatography in the quantitative measurement of the classical estrogens and some newer metabolites
Chattoraj, Sati Charan
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The recent discovery of a number of metabolites of estrogens has necessitated the development of new methods for their analysis. The available methods of proven merit fall short of determining these newer fractions. Moreover, these methods are too time consuming to determine the daily excretion of estrogenic steroids. The development of a relatively rapid and highly sensitive methodfor the estimation of several estrogens was, therefore, undertaken. The procedure involves hydrolysis, extraction, preliminary purification and separation, acetylation and gas chromatography of the steroids. Simultaneous separation and quantification by gas-liquid chromatography (GLC) not only allows a more rapid analysis but sensitivity is also increased by the use of ionization detectors. Determination of the optimal flow rate of hydrogen, air and carrier gas was found necessary for obtaining maximum detection response. Detector linearity for seven estrogens over an adequate range was established. Since free estrogens suffer severe adsorption on the gas chromatographic column and generally do not separate well, the steroids were analyzed as suitable derivatives. After examining several such derivatives (acetates, formate, trifluoroacetate, trimethylsilyl ethers) the acetates were found to be most suitable for the specific purpose. Optimal conditions for the acetylation of steroids in submicrogram quantities were established. The effect of the solid support, stationary phase, priming of the column with estrogens and solvent impurity in quantitative analysis by gas chromatography was also investigated. Preliminary separation of estrogens into separate groups was necessary because of poor resolution and long retention times resulting in poor detector response. Moreover, in a crude urine extract, the large number of contaminants obscuring the peaks of the lesser estrogen components, mandated preliminary purification. Thin-layer chromatography crtc) was found to be a versatile tool for this purpose. Among several solvent systems developed, three were found to be eminently suitable. TLC in System I (benzene:ethyl acetate 1:1) separates the estrogens into four fractions: (a) estrone and 2-methoxyestrone, (b) ring-D-alpha-ketols and estradiol, (c) 16-epiestriol, and (d) estriol. Further TLC of fraction (b) in System II (pet. ether:dichloromethane:ethanol 10:9:1) was found necessary to separate these estrogens from the neutral 17-ketosteroid. An alternate method of preliminary purification and separation for the measurement of the three classical estrogens in low titer urine, involving alumina chromatography, has been developed. Following these preliminary procedures GLC permitted rapid separation and highly sensitive quantification of the individual fractions [TRUNCATED]
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