Chromatographic Separation of Synthetic Estrogen and Progesterone in Presence of Natural Congeners: Application to Saliva and Pharmaceutical Samples

Abstract

Estradiol valerate (EST) and Norethisterone acetate (NOR) are co-formulated as a hormone replacement therapy. Naturally occurring steroidal hormones like estradiol, estrone and estriol are also considered as impurities in drug substance. Chromatographic separation of these five steroidal hormones was developed by HPLC and TLC. Reversed-phase chromatography was applied using either acetonitrile–methanol containing 0.05% β-cyclodextrin as a mobile phase for HPLC with gradient elution and chloroform–propanol–formic acid (5:3:2, by volume) as a developing system in TLC. The methods have been applied for determination of EST and NOR in presence of other natural congeners in biological samples as well as in drug products as official impurities. Solid-phase extraction was applied for saliva sample cleaning using testosterone as internal standard whereas ampoules and tablets formulations were used in pharmaceutical applications. The methods have been validated for bioanalytical and pharmaceutical analysis following the respective FDA and ICH guidelines. The obtained results were statistically compared with the official methods for both drugs.