Derivative-Based Non-Target Identification of DNA-Reactive Impurities with Fragment Ion Filtering

DNA direct reactive impurities (DDRIs) can react with nucleophilic sites of DNA, leading to mutations. The control strategies outlined in International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) M7 are based on the known compound structure of DDRIs. Non-target screening of DDRIs in drugs is still challenging due to the diversity of the species and the poor stability. In this study, a derivatization reagent including a reactive group and report group was designed to screen DDRIs. Based on the electrophilic theory of chemical carcinogenesis, an amine reagent was used as a reactive group to interact with DDRIs. Two derivatization reagents, p-methoxyaniline and p-methoxybenzoyl-β-alaninamide, were employed, each containing different chromatographic modification groups to mitigate matrix effects. The derivatization products were analyzed by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS). Non-target screening for DDRIs was achieved by product ions filtering of the report group.