Method development and validation for determination of N-Nitrosamines in pharmaceutical preparations by LC-MS/MS: Application to extractables and leachables studies

The use of highly sensitive and reliable analytical methods is essential for Extractables & Leachables studies. Especially the determination of N-Nitrosamines in drugs, which have carcinogenic properties and may contaminate drugs at trace levels, is quite important. In this study, a new, sensitive, short-time and reliable liquid chromatography with tandem mass spectrometry method was developed for the analysis of 15 N-Nitrosamines defined in the European Pharmacopoeia within the scope of Extractables & Leachables studies and validated according to the International Council for Harmonization (ICH Q2 (R2)). The analysis of N-Nitrosamines was carried out in positive mode using an Atmospheric Pressure Chemical Ionization source in the dynamic multiple reaction monitoring scanning mode. In the chromatographic separation, gradient elution was applied using a reverse phase Phenyl column and the mobile phase (A: 0.1 % formic acid in ultrapure water, B: 0.1 % formic acid in methanol); total analysis time was 16 mins and the flow rate was optimized as 0.6 mL/min. N-Nitroso-dimethylamine-d6 was used as an internal standard. The developed method was used in extractables studies to control the potential presence of N-Nitrosamines that may be caused by interactions between the product and primary packaging materials (e.g. polypropylene bag, LDPE container, disposable eye drop packaging and bromobutyl stopper). It was also successfully applied to pharmaceutical preparations containing sugammadex, metformin, gliclazide and paracetamol in the leachables studies. © 2025