High-performance liquid chromatographic methods for monitoring of isomers of 17-hydroxy-16-hydroxymethyl-3-methoxyestra-1,3,5(10)-triene.

Claisen condensation and consecutive reduction of 3-methoxyestra-1,3,5(10)-trien-17-one theoretically leads to four diastereomers of 17-hydroxy-16-hydroxymethyl-3-methoxyestra-1,3,5(10)-triene and their further transformations give different compounds with different biological activities. High-performance liquid chromatographic methods were developed for separation of the four isomers of 17-hydroxy-16-hydroxymethyl-3-methoxyestra-1,3,5(10)-triene: reversed-phase separation on a Nucleosil ODS C18 column with water-methanol as mobile phase; and normal-phase separation on an APEX Silica column with hexane-dichloromethane-2-propanol as mobile phase. The effects of eluent composition and flow-rate on the separation were investigated. This is the first chromatographic evidence for the formation of the 16alpha,17alpha isomer in the reduction of 16-hydroxymethylene-3-methoxyestra-1,3,5(10)-trien-17-one.