An analytical quality by design-guided method development for the quantification of N-nitroso-fluoxetine to support mitigation strategies for a fluoxetine drug product.

This work focused on N-nitroso-fluoxetine, a N-nitrosamine drug substance-related impurity (NDSRI) formed in fluoxetine-containing products via nitrosation pathways. N-nitroso-fluoxetine was previously detected above the established safety threshold of 100 ng/day, leading to temporary withdrawal of a marketed product in Belgium. An Analytical Quality by Design (AQbD) approach supported the development of a reversed-phase LC-(ESI+)-MS/MS method. Design of experiments identified aqueous mobile phase pH and gradient time as critical method parameters affecting detection or quantification limits and chromatographic separation performance, respectively. Adequate separation was achieved using Acquity® HSS T3 guard-column and column with a run time of 22 min. The method was validated according to ICH Q2(R2) guideline using a combined approach based on total error. It enables accurate and precise quantification of N-nitroso-fluoxetine from 0.09 to 4.75 ppm (injected concentration in ng/mL) while simultaneously screening for other small-molecule N-nitrosamines in both drug substance and finished products. The validated method was applied to assess risk mitigation measures implemented by the manufacturer, including the use of low-nitrite excipients, desiccant-integrated aluminum/aluminum blister packaging, and reduced shelf-life. In the updated formulation, N-nitroso-fluoxetine contents were 0.58 ppm at release and increased to 1.15 ppm after 21 months of storage at 25 °C/60% RH, remaining below the safety threshold of 1.67 ppm calculated according to the EMA "Call for Review" procedure. Together, these modifications have effectively controlled N-nitroso-fluoxetine formation. Therefore, the present study demonstrates a robust AQbD-guided LC-MS/MS method, contributes to NDSRI analysis, and supports science- and risk-based control strategies for these emerging impurities.