Background/Objectives: Nitrosamine drug substance-related impurities (NDSRIs) are a class of potent genotoxic impurities that pose a critical risk to patient safety, thereby necessitating the stringent control of pharmaceutical products. Nortriptyline (NORT) and sertraline (SERT) are two widely prescribed antidepressants that form highly potent NDSRIs, N-nitroso-nortriptyline (NNORT) and N-nitroso-sertraline (NSERT), respectively. Despite these risks, a substantial gap exists in terms of the validated analytical methods for surveillance. Accordingly, this study addressed this need by developing a liquid chromatography–tandem mass spectrometry method for the quantification of NNORT and NSERT in drug products. Methods: A comparative evaluation of two reversedphase columns (general-purpose C18 column and phenyl-hexyl column) was performed to achieve optimal chromatographic resolution of the parent active pharmaceutical ingredients (APIs). Results: The phenyl-hexyl column demonstrated superior separation for both NDSRI/API pairs by leveraging π-π interactions to markedly enhance the resolution. This was particularly critical for SERT. The method was fully validated according to the International Council for Harmonization guideline Q2(R1) and demonstrated excellent linearity (r2 = 0.998 for both NNORT and NSERT) with limits of quantitation of 20 ng/g for NNORT and 125 ng/g for NSERT. Accuracy was confirmed with recoveries of 96.6–99.4% for NNORT and 98.6–99.4% for NSERT, and precision was acceptable, with relative standard deviation below 3.9% and 1.9%, respectively. The application of this method to commercial products subjected to accelerated stress testing revealed NNORT formation in NORT products with an average concentration of 190 ng/g, as well as NSERT formation in SERT products resulted in an average concentration of 172 ng/g. Conclusions: This validated method provides a reliable tool for routine quality control, thereby enabling pharmaceutical manufacturers and regulatory agencies to ensure safety and compliance with widely used antidepressant medications.
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