ASSAY OF ENZYMATIC O-METHYLATION OF CATECHOL ESTROGENS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH COULOMETRIC DETECTION

A simple and sensitive method for the determination of guaiaicol oestrogens enzymically formed from 2- or 4-hydroxyoestradiol, by menas of high-performance liquid chromatography with coulometric detection, has been developed. Catechol and guaiacol oestrogens were efficiently separated on a reversed-phase column, using 0. 5% ammonium phosphate buffer (pH 3.0)-acetonitrile (59:41, v/v) as the mobile phase, and detected coulometrically in a screening-oxidation mode at +0.10 V and +0.35 V, respectively. The method was applied to the assay of in vitro enzymic 0-methylation of catechol oestrogens. After 2- or 4-hydroxyoestradiol had been incubated with rat red blood cells in the presence of S-adenosylmethionine, the resulting guaiacols and unchanged substrate were percolated through an Extrelut-3 cartridge. The dried eluate was redissolved and directly injected. This simple procedure was as sensitive as the previously reported method using gas chromatography-mass spectrometry in a selected ion monitoring mode.