Identification of related substances in letrozole using LC-Q-TOF/MS.

Letrozole, a nonsteroidal aromatase inhibitor of the triazole class, is widely used in breast cancer treatment. Its quality and stability directly influence therapeutic efficacy and safety. However, systematic characterization of its related substances (RSs) remains insufficiently documented. In this study, letrozole RSs were separated by an established HPLC-UV method and identified with LC-Q-TOF/MS determination following the ICH Q1A(R2) guidelines. 27 RSs were detected, including 11 process-related impurities and 16 previously unreported degradation products. Structural elucidation and degradation pathway analysis were performed for all the RSs by using their chromatographic retention behaviors, DAD spectral features, and high-resolution mass spectrometric data. Three main degradation pathways were proposed for them, including substitution on the triazole ring, hydrolysis of the cyano groups, and oxidation of the benzyl moiety. Furthermore, to support impurity risk assessment, the genotoxicities of all the RSs were evaluated using in-silico models according to the ICH M7 (R2) guideline. Most of the RSs were evaluated to be non-mutagenic, however four of them were identified as structurally alerting. These findings contribute to a better understanding of the stability behavior of letrozole and provide a scientific basis for the drug quality risk management from its related substances test and control point of view.