The preparation of pregnancy urine for an estrogen profile

A method is described for purifying the estrogen content of pregnancy urine with little loss of the labile estrogens. The procedure makes use of the initial 50-fold purification effected by their precipitation whith ammonium sulphate, with simultaneous elimination of most urinary corticosteroids and 50--60% of urinary ketosteroids. It also employs the antioxident ascorbic acid as an additive in most stages of the procedure. The mild organic-solvent-HIO partition system of Brown is used for separating the strongly polar, 2including all "labile" estrogens, and of the weakly polar estrogens, from neutral steroids. The remaining neutral steroid still interfering with the assays were removed by an ascorbic acid treated ion exchange resin (AG 1). The final residues were revealed by mass-spectroscopy to consist almost solely of estrogens. Gas-liquid chromatography in which just 2 chromatograms are required yields a total of 12 "estrogen" peaks (for 12 estrogens which are excreted in amounts greater than 0.1 mg/day) in normal pregnancy urine, including all the known labile estrogens. Identification as estrogen for all but a few minor peaks of the gas chromatogram was obtained by mass-spectroscopy. The practical significance of the method lies in the fact that some labile estrogens are much more important in the estrogen metabolism of pregnant and nonpregnant women than heretofore generally thought.