phase. Estrone, the starting material in the synthesis of quinestrol, and ethinyl estradiol, a possible process contaminant, were not resolved from each other and had a retention time of 2 min. Estrone 3-cyclopentyl ether, the penultimate intermediate in quinestrol synthesis, eluted at 10.8 min and was well separated from the quinestrol peak at 8.5 min (Fig. 1).
Selectivity of Assay in Presence of Decomposition Products-A 1-g sample of quinestrol was heated in an open vial at 125' for 96 hr. This procedure was shown previously to produce a similar TLC pattern to that obtained with quinestrol tablets that had been exposed to air for several months. (Quinestrol in open containers is susceptible to autoxidation, which involved an induction period of over 1 year at room temperature. None of the decomposition products has the same mobility as the synthesis precursors and process contaminants. One of the two major decomposition products isolated by preparative TLC had an NMR spectrum consistent with the structure of 6/3-hydroxyquinestro110.) The brown material was pulverized, and a 50-mg portion was dissolved in acetonitrile and diluted to 50.0 ml with that solvent. A 5-ml aliquot was diluted further to 50.0 ml with the mobile phase and subjected to chromatography as described. The chromatogram (Fig. 2) exhibited unidentified peaks with retention times of -1.7,2.2,2.4,4.0,5.7,6.3, and 8.0 min, along with the peak for undegraded quinestrol at 8.5 min. The quantity of undelo Dr. R. C. Greenough, Oxford Management and Research Center, Uniroyal, Inc., Middlebury, CT 06749, personal communication.
COMMUNICA TIONS
graded quinestrol was estimated as 57.7% by this HPLC method and 58.9% by the colorimetric method (1).
Further evidence of the validity of the HPLC method and the colorimetric method as stability-indicating methods was obtained by dissolving the degraded quinestrol in the mobile phase to obtain a concentration of -10 mg/ml, injecting 50 p1 into the liquid chromatograph, and collecting the eluates corresponding to the decomposition products. These eluates were evaporated to dryness on the steam bath with a nitrogen stream, and the residue was dissolved in 2.0 ml of methanol. A 1-ml portion, corresponding to > 100 pg of quinestrol degradation products, was tested by the colorimetric method (1) and gave no color with the methanol-sulfuric acid chromogenic reagent.