Characterization of In Vitro Metabolic Profiles of Elacestrant in Rat and Human Liver Microsomes Using HPLC–MS/MS and HPLC–Q‐Orbitrap–HRMS

Elacestrant is an orally administered selective estrogen receptor degrader designed for the treatment of ER-positive, HER2-negative breast cancer. In this study, a simple and sensitive HPLC-MS/MS method was developed and validated for the quantification of elacestrant in liver microsomes. Chromatographic separation was achieved on a Waters ACQUITY BEH C₁₈ column using a gradient of 0.1% formic acid in water and acetonitrile. Elacestrant and the internal standard (elacestrant-d₆) were detected in multiple reaction monitoring mode via the transitions of m/z 459.2 → 298.3 and m/z 465.3 → 304.4, respectively. The method exhibited excellent linearity over the range of 1.0-2000 nM (r > 0.995). Elacestrant was rapidly metabolized, showing half-lives of 22.45 ± 0.66 min in rat liver microsomes and 43.36 ± 2.48 min in human liver microsomes. Using HPLC-Q-Orbitrap-HRMS, seven metabolites were identified, with M5 (N-deethylation) being the most abundant. Key metabolic pathways involved O-demethylation, N-deethylation, N-dealkylation, and oxidative deamination, primarily mediated by cytochrome P450 3A4. This study establishes the first HPLC-MS/MS and HPLC-Q-Orbitrap-HRMS-based analytical strategy for in vitro metabolic profiling of elacestrant, supporting its future application in clinical pharmacokinetic and metabolism investigations.