A chemical method for the quantitative determination of 2-methoxyoestrone, oestrone, ring D α-ketolic oestrogens, oestradiol-17β, 16-epioestriol and oestriol in human urine

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Abstract
A new chemical method which separates and quantitatively measures 2-methoxyestrone, estrone, ring D [alpha]-ketolic estrogens, estradiol- 17[beta], 16-epiestriol and estriol in human urine is described. The method involves: enzymic hydrolysis; ether extraction; separation of the ketonic from the non-ketonic estrogens by the Girard reaction; purification of the non-ketonic fraction by refluxing with alkali and alkali washes; separation and purification of the estradiol-17[beta], 16-epiestriol and estriol fractions by column-partition chromatography; purification of the estradiol-17[beta] fraction by refluxing with alkali; separation and purification of the 2-methoxyestrone, estrone and ring D [alpha]-ketolic estrogen fractions by column-partition chromatography; purification of the 2-methoxyestrone fraction by refluxing with alkali and the estrone fraction by alkali washes. The purified fractions are measured by the Kober-color reaction with a spectrophotometric correction for non-estrogen chromogenic substances. The efficiency of the different procedures is described. The method is evaluated for accuracy, specificity, adaptability and sources of error.