Determination of estredox, a compound with sustained estradiol function, and its impurity profile by HPLC.

The HPLC methods described here for the assay and purity test of Estredox (E2CDS), a molecule with a redox-based, brain-targeted chemical delivery system for estradiol, allow reliable conclusions to be made on the potency and purity of API and E2CDS/HPCD complex samples. Extensive work was done to isolate and characterize the major, potential contaminants, and ensure the required stability of solutions of E2CDS, an inherently labile compound by design. Both the sample solvent and the eluent were thoroughly tested to avoid unwanted changes in sample solutions during analyses. The 12 minute isocratic assay method at 220 or 360 nm is simple, well-founded, highly precise and accurate. Purity profiling of E2CDS raised several problems in detection, stability and accuracy, owing to the fact that the pattern of the UV spectra and the stability of the compound and those of the potential contaminants often differed greatly. As a result of meticulous analysis of the UV spectra and the factors influencing the behaviour, in solution, of the compounds concerned, the 20 minute gradient method developed for the purity test, at 220 nm, of E2CDS and E2CDS/HPCD complex samples has proved to be a reliable means of adequately resolving 15-20 peaks of known and unknown compounds, and establishing the purity of various E2CDS samples. Sample impurity can be expressed as area % at 220 nm, and/or as approximate w/w % (if needed), since the relative response factors, at 220 nm, of the 6 major, potential contaminants have also been determined.