A Technic for Fractionation of 17-Ketosteroids from Urine

Abstract

An attempt has been made to shorten the extraction-hydrolysis procedure for fractionation of the urinary 17-ketosteroids to make this determination more attractive for the clinical laboratory use. The technic consists of extracting 20 ml of (NH₄)₂SO₄ saturated urine with tetrahydrofuran (THF), drying down the extract, and hydrolyzing according to the method of DePaoli, Nishizawa and Eik-Nes (1). The essential feature of this is the addition of 15 ml of THF containing 10^-2M perchloric acid with three hr incubation at 50 C. Ten percent aqueous KOH is added to neutralize the perchloric acid and remove phenols, the THF is evaporated, the solution is extracted three times with ether, and the ether pool washed with water. The ether extract is dried down and the residue chromatographed by TLC on a mixture of silica gel G and H (1:1). The solvent system is ethyl acetate, benzene (2:3). On a separate channel on the plate, dehydroepiandrosterone is chromatographed as a marker. When the solvent has reached the front of the chromatogram (approximately 14 cm), the plate is dried and exposed to iodine vapor. The area of sample chromatogram corresponding in mobility to the standard is marked and scraped off the plate and extracted with three, two, and one ml of ethanol. Fifty µg of estrone is added to this extract and the ethanol evaporated to dryness. The residue is then converted to TMS ether derivatives by adding hexamethyldisilazane and trimethylchlorosilane and allowed to stand overnight. The solution is then dried down.

Supported by NIH Grant No. B1363-08