Clinical analysis on steroids. XXII Preparation and high-performance liquid chromatographic determination of guaiacol estrogen 17.BETA.-conjugates: The enzymatic O-methylation products of 2-hydroxyestradiol 17.BETA.-conjugates.

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Abstract
For the direct assay of the enzymatic O-methylation products of 2-hydroxyestradiol 17.beta.-sulfate (2) and 17.beta.-glucuronide (3), the corresponding guaiacol estrogens were prepared as authentic specimens and their high-performance liquid chromatography (HPLC) was investigated. The materials synthesized were: potassium 3-hydroxy-2-methoxyestra-1,3,5(10)-trien-17.beta.-yl sulfate, potassium 2-hydroxy-3-methoxyestra-1,3,5(10)trien-17.beta.-yl sulfate, potassium [3-hydroxy-2-methoxyestra-1,3,5(10)-trien-17.beta.-yl-.beta.-D-glucopyranosid]uronate, and potassium [2-hydroxy-3-methoxyestra-1,3,5(10)-trien-17.beta.-yl-.beta.-D-glucopyranosid]-uronate. These sulfates and glucuronides were separated quantitatively by reversed-phase HPLC. The separation was performed with a mixture of acetate buffer (50 mM, pH 5.0) and methanol (50:50) as the mobile phase on a column of ODS SIL. The eluates were monitored in terms of the absorbance at 280 nm. Calibration curves between the amounts of conjugated guaiacols and the peak heights on chromatograms were all linear. The results obtained by proposed HPLC method for the quantification of O-methylated products obtained by the incubation of 2 and/or 3 with purified rat liver catechol O-methyltransferase in the presence of (3H3C)-S-adenosyl-L-methionine were in good agreement with the results obtained by a different procedure, the reverse isotope dilution method.