Mitigation of ionization‐induced quantitative bias in <scp>LC</scp> – <scp>MS</scp> / <scp>MS</scp> analysis of <i>N</i> ‐nitrosamine drug‐substance‐related impurities in <scp>Rasagiline</scp> pharmaceutical products

Abstract Accurate quantification of N ‐nitrosamine drug‐substance–related impurities (NDSRIs) in pharmaceutical products requires careful control of ionization‐induced artifacts during LC–MS/MS analysis. In pharmaceutical matrices where the active pharmaceutical ingredient (API) is present at concentrations several orders of magnitude higher than the target impurity, in‐source fragmentation can generate overlapping product ions, leading to significant quantitative bias. In this study, we demonstrate that certain atmospheric‐pressure ionization conditions induce in‐source decomposition of both rasagiline and its corresponding nitroso impurity, producing a common fragment ion at m / z 117. This overlap can compromise analytical specificity and potentially result in overestimation of impurity levels. To mitigate this ionization‐induced quantitative bias, Heated Electrospray Ionization (H‐ESI) parameters were systematically optimized to preserve the intact protonated molecular ion ( m / z 201) while minimizing thermally induced fragmentation. In addition, a gradient LC method incorporating a high‐organic wash step and flow diversion strategy was established to enhance analytical robustness under routine quality control conditions. The optimized LC–MS/MS method demonstrated excellent linearity ( R 2 &gt; 0.998), acceptable precision (RSD ≤ 11.5%), and sufficient sensitivity (LOQ 30 ng/mL) to meet current regulatory requirements. These findings highlight the critical importance of ion‐source selection in nitrosamine impurity analysis and provide a practical strategy to reduce the risk of ionization‐induced quantitative bias in pharmaceutical LC–MS/MS applications.