HPLC-ECD procedure for the measurement of O-methylation of catechol estrogens by vascular tissue

A precolumn extraction procedure combined with high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed for the identification and quantitation of catechol estrogens, their immediate precursors (estrogens), and their 0-methylated metabolites. These compounds were isolated from Krebs solutions and from perchloric acid tissue extracts or whole tissues by simple solvent extraction prior to quantitation. The optimal conditions for detection by HPLC-ECD were those employing a radial compression module, a C18 Bondapak cartridge, and an isocratic mobile phase of ammonium phosphate (pH 3.0) combined with 30-70% acetonitrile. A biological application of this procedure includes the investigation of the 0-methylation of catechol estrogens by vascular tissue. In the present study, the 0-methylation of 2-hydroxyestradiol by rabbit aorta has been examined. The 0-methylation in segments of thoracic aorta was significantly less than that in abdominal aortic segments, but was reproducible along the length of the thoracic aorta.