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

Zhang, Yue; Sacre, Pierre-Yves; Streel, Bruno; Ziemons, Eric; Hubert, Philippe; Hubert, Cédric

doi:10.1016/j.jpba.2026.117692

Article (Scientific journals)

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

Zhang, Yue; Sacre, Pierre-Yves; Streel, Bruno et al.

2026 • In Journal of Pharmaceutical and Biomedical Analysis, 282, p. 117692

Peer Reviewed verified by ORBi

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https://hdl.handle.net/2268/347961

DOI
10.1016/j.jpba.2026.117692

PubMed
42561806

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Keywords :

Analytical Quality by Design; Analytical procedure validation; LC-MS/MS; N-nitrosamine; N-nitroso-fluoxetine; NDSRI; Quality control

Abstract :

[en] 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.

Disciplines :

Pharmacy, pharmacology & toxicology

Author, co-author :

Zhang, Yue ; Université de Liège - ULiège > Unités de recherche interfacultaires > Centre Interdisciplinaire de Recherche sur le Médicament (CIRM)

Sacre, Pierre-Yves ; Université de Liège - ULiège > Département de pharmacie > Chimie analytique

Streel, Bruno ; Université de Liège - ULiège > Département de pharmacie ; Galephar M/F, Marche-en-Famenne, Belgium

Ziemons, Eric ; Université de Liège - ULiège > Département de pharmacie > Chimie analytique

Hubert, Philippe ; Université de Liège - ULiège > Département de pharmacie

Hubert, Cédric ; Université de Liège - ULiège > Département de pharmacie > Chimie analytique

Language :

English

Title :

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

Publication date :

04 August 2026

Journal title :

Journal of Pharmaceutical and Biomedical Analysis

ISSN :

0731-7085

eISSN :

1873-264X

Publisher :

Elsevier BV, England

Volume :

282

Pages :

117692

Peer reviewed :

Peer Reviewed verified by ORBi

Available on ORBi :

since 18 August 2026

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