The Determination of Estrone, Estradiol-17β, and Estriol in Urine and Plasma with Column Partition Chromatography

Difficulties encountered in the chemical estimation of estrogens in urine and in plasma are caused principally by the low concentrations of estrogen frequently found in these fluids, by the presence of large amounts of interfering material, and by the lack of specificity of available methods for the quantitative estimation of the estrogens.In these circumstances, it is apparent that, unless adequate separation procedures are used, measurement of estrogens may involve considerable errors, particularly in the direction of overestimation, Morris and Williams (1) were able to obtain efficient separation of adrenal steroids by the use of column partition chromatography, and it appeared that the high degree of resolution afforded by this form of chromatography would be of value also in the separation of urinary and plasma estrogens.It was found that the three estrogens, estrone, estradiol-17/3, and estriol, could in fact be separated by column partition chromatography both from one another and from the interfering material present in extracts of biological fluids.A single partition column was used with one stationary and three successive mobile phases.A complete method based on this chromatographic procedure for the estimation of these three estrogens in urine and plasma is here reported.A preliminary report of the method was published by Preedy and Aitken in 1956 (2).Since then the method has been extensively used in studies of estrogen metabolism in the human (3-7).A modification to increase the sensitivity of the method was reported in 1958 (8). EXPERIMENTAL PROCEDURE PrincipleEstrone, estradiol-170, and estriol conjugates in urine or plasma are hydrolyzed by refluxing with concentrated acid, and the parent steroids are then extracted with ether.After partition between sodium hydroxide and toluene, the watery phase is adjusted to pH 9.0, and a further ether extraction carried out.The dried extract is then submitted to column partition chromatography.Fractions of eluate are collected, and the estrogen content of each determined by sulfuric acid fluorescence.