The separation of the estrogens by gas chromatography
Martin, Horace Feleciano
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The prime purpose of this investigation was the development of a rapid and sensitive procedure for the assay of estrogenic hormones. The advent of gas chromatography offered a possible solution to this problem. A pre-requisite of any gas chromatographic method is that the compounds to be chromatographed be volatile and thermally stable at the temperatures of vaporization and separation in the presence of the eluting gas. Accordingly, the thermal stability of certain free and acetylated estrogens was studied. The results of these studies indicated that decomposition was minimal when small quantities were subjected to gas chromatographic separation. Estrogen acetates were found to be stable up to 340°C by infrared spectroscopy, ultraviolet spectroscopy and by an evaluation of the gas chromatographic peak symmetry. Once suitable derivatives had been found, the parameters of separation and detection were evaluated. The optimum conditions for separation were found to be: 1) a temperature of 240°-280°C, 2) column length of 3 feet, 3) a silicone grease liquid phase, 4) flow rates of 50-500 cc/min, and 5) non-polar solvents. The lower limits of detector sensitivity were found to be 0.2ug for a sr90 detector and lOOug for the thermal conductivity cell detector. The ionization detector responded linearly in the range of 0.2 to 25 ug. The thermal conductivity cell detector responded linearly in the range of 100 to 400 ug. In order to have a quantitative procedure, it was necessary to determine the optimal conditions for the preparation of the estrogen acetates. It was found by means of gas chromatography, infrared spectroscopy and ultraviolet spectroscopy that estrone was converted to its acetate in 86-90 per cent yield in 2 x 10-3 M solutions in one hour at 65C. Estradiol, under the same conditions was found to be converted in 92 percent yield to the diacetate. The remaining 8 per cent was found to be estradiol-3- acetate. By allowing this reaction to proceed overnight only two per cent of the monoacetate was found. Estriol, in reactions which were carried out under similar conditions, was found to be converted in 98 per cent yield to the triacetate. In order to develop a urinary assay procedure, recovery studies from water and acid hydrolyzed urine, were carried out. The results indicated a 75-80 percent recovery of estrone and estradiol and a 90-95 per cent recovery of estriol. The lowest concentration studied was the recovery of steroid from a solution containing 1 ug/ml. Studies at this lower concentration showed that the recoveries were independent of concentration. The method described above has the advantage of chemical specificity, rapidity and reproducibility. As yet, it has not been developed to its highest sensitivity, however, coupled with prepurification procedures and with advances in instrumentation, it appears that the method will become useful for low level estrogen determination in biological media.
Thesis (Ph.D.)--Boston University. Missing pages 78, 79.