Systematic Selection of Impurities, Development, and Validation of Related Substance Methods for Estradiol and Progesterone in a Combination Drug Product.

This article focuses on the development and validation of reverse-phase high-performance liquid chromatography (HPLC) methods for identifying and quantifying related impurities of estradiol and progesterone in a combination soft gelatin capsule drug product used to treat moderate to severe menopause symptoms. The study addresses challenges posed by the large difference in drug ratios, similar molecular structures, and complex excipients, optimizing chromatographic conditions with UV/photo diode array and fluorescence detectors to achieve sensitive, specific, and stability-indicating impurity profiling. Validation according to International Conference on Harmonization (ICH) guidelines demonstrated the methods' accuracy, linearity, precision, and robustness, supporting their suitability for routine quality control of this combination hormone therapy. The work fills a gap in analytical methodology by enabling simultaneous impurity analysis of both hormones in a single dosage form.