A method for the quantitative fractionation of mixtures of 2-methoxyoestrone, oestrone, ring D α-ketolic oestrogens, oestradiol-17β, 16-epioestriol and oestriol by partition chromatography and the Girard reaction

Abstract

The object of this investigation was to develop a method for the quantitative fractionation of pure 2-methoxyoestrone, oestrone, ring D oc-ketolic oestrogens, oestradiol-17,B, 16-epioestriol and oestriol. This required, first, a technique for the Girard reaction which would result in quantitative recoveries, and, secondly, a method whereby the ketonic and non-ketonic oestrogen residues could be separated into fractions by partition chromatography. EXPERIMENTAL MaterialsReagents used were A.R. unless otherwise stated. Diethyl ether, benzene, methanol, ethanol (96 %, by wt.), Celite 535 and the Kober reagents were purified, prepared and tested as described by Bauld & Greenway (1957). The final distillation of solvents was done in all-glass apparatus.Absolute ethanol. Purified ethanol (96 %, by wt.) was left overnight with calcium oxide (300 g./l.), refluxed for 1 hr. and distilled under anhydrous conditions. Magnesium ethoxide was prepared with 75 ml. of dry ethanol, 5 g. of magnesium turnings and 0 5 g. of iodine. Dry ethanol (925 ml.) was then added, refluxed for 4 hr. and distilled (Vogel, 1951).Hexane. Hexane (Anachemia, Montreal, Canada, technical grade, b.p. 65-690) was washed with conc. H2SO4 (6 x 85 ml./l.) for 45 min. per washing, water (2 x 350 ml./l.), N-KMnO4 in 3.7 N-HiSO4 (4 x 85 ml./l.) and left overnight with the last KMnO4 portion. The hexane was then washed with water (4 x 170 ml./l.), 0.5 M-Na3CO3(1 x 35 ml./l.), water (4 x 170 ml./l.) and dried over Na2S04.The solvent (up to 31.) was poured through a column (48 in. x I in.) of 6-12-mesh silica gel, distilled and the 67.2-68.8°fraction collected.Stained glassware was cleaned with a solution of 0-4Noxalic acid in 3-7 N-H2SO4 .Girard P reagent (Girard & Sandule8co, 1936). Commercial material was refluxed with acid-washed charcoal in ethanol (96 %) for 15 min., filtered and left overnight at 5°. The crystals were washed with ethanol and stored in a brown bottle in a desiccator.

The Metabolism of Testosterone by Tissue From Normal and Neoplastic Rat Breast

“…The ether-soluble material underwent a Girard separation as described by Givner, Bauld & Vagi (1960) and this was repeated with the non-ketonic fraction.…”

The LOCALIZATION OF [6,7-3h]oestradiol-17β IN RAT UTERUS

“…Therefore, the following purification procedure was used routinely. Every 6-8 weeks an aliquot of the stock [6,7-3H]oestradiol solution in benzene was chromatographed on a Celite column (Givner, Bauld & Vagi, 1960) and the oestradiol fraction stored at 4°in benzene. Every 7-10 days an aliquot of this material was purified on paper in the system methanol : water : light petroleum : toluene (70:30:50:50), the oestradiol eluted with ethanol, evaporated to dryness in vacuo and dissolved in benzene.…”

An isotope dilution gas chromatographic–mass spectrometric method for the simultaneous assay of estrogens and phytoestrogens in urine

“…Looking at the history of estrogen profile method development the first attempts to measure more than the three "classical" estrogens was made by Givner et al [25,26], Hobkirk and Nilsen [27] and Breuer [28] utilizing column and/or paper chromatography and in some studies separating ketonic estrogens from non-ketonic ones by Girard separation. The final determination was made by the Kober reaction or modifications thereof.…”