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UBC Theses and Dissertations

High resolution gas chromatography of conjugated estrogens with glass capillary columns Pillai, Gopalakrishna


The pharmaceutical product, conjugated estrogens, is a mixture of ten or more equine estrogen steroid conjugates used as a replacement therapy to treat estrogen deficiency symptoms associated with menopause. This complex mixture, containing mainly sodium estrone sulphate and sodium equilin sulphate, is derived from pregnant mare's urine. The United States Pharmacopeia specifies a colorimetric method for the estimation of estrone, equilin and the total estrogen steroid content and includes a gas chromatographic identification test for the minor components. Many attempts have been made to resolve and quantitate each and every component in the mixture, however, most of these methods suffer drawbacks in terms of either incomplete resolution or long elution times. In order to achieve a better resolution, especially of the pair estrone/equilin and β-estradiol/α-dihydroequilin, capillary column gas chromatography was investigated. A variety of derivatives of estrogens, suitable for gas chromatography, such as trimethylsilyl ethers, methoxime-trimethyl-silyl ethers, tert.butyl dimethyl silyl ethers, heptafluorobutyrates, methoxime-heptafluorobutyrates, and oxime-trimethylsilyl ethers have been chromatographed on selected columns of varying polarity. A short OV-225 column (7 m x 0.25 mm) did resolve estrone and equilin as their trimethylsilyl derivatives but resolution of β-estradiol from α-dihydroequilin could not be achieved. Increasing the column length to 50 meters further improved the resolution of estrone and equilin but the two diols did not resolve. The latter pair was well resolved as their heptafluorobutyrates and tert.butyl dimethyl silyl ethers on an OV-225 column but estrone overlapped with equilin in the former and equilin overlapped with β-estradiol in the latter case. The oxime-trimethylsilyl or methoxime-trimethylsilyl derivatives of the ketosteroids have been noted to form syn- and anti-isomers as evidenced by two peaks on OV-225 as well as Silar 10 C capillary columns. However, a difference in the chromatographic characteristics of the oximes was observed when the two columns were compared. The resolution of the methoxime-trimethylsilyl isomers was far greater than the oxime-trimethylsilyl isomers on a 25 m Silar 10 C column. This led to the use of a shorter column (15 m) to further suppress the resolution of syn- and anti-isomers without affecting the resolution of other steroids. A baseline resolution of nine equine steroids as their oxime-trimethylsilyl ethers has been achieved on a 15 m Silar 10 C glass capillary column in a chromatographic time of 28 minutes. Employing the methodology developed, a variety of commercial formulations, such as preparations meant for intravenous administration, tablets of varying strength, vaginal cream and combination products with meprobamate and methyl testosterone have been analyzed. The estrogen conjugates present in these formulations were hydrolyzed to their free phenolic form by incubation with sulphatase enzyme under carefully controlled conditions. These steroids were then extracted into chloroform solution containing the internal standard, ethynyl estradiol. The mixture of keto and diol steroids were then derivatized to their oxime- trimethyl-silyl ethers by sequential treatment with hydroxylamine hydrochloride and N,O-bis-(trimethylsilyl) trifluoroacetamide in pyridine. For quantitative purposes, response factors and linearity were established using at least six data points for each of ten steroids. The high resolution achieved with the capillary column enabled precise quantitation of each steroid as well as a common impurity, equol. The method compares favourably with previously published methods and in addition, has the advantages that it is considerably more rapid, completely resolves all the known steroids and requires only a single injection of a dual derivative.

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