Determining N-Nitrosamines in Pharmaceutical Preparations Using LC–MS/MS

A multi-institutional study has developed a new method for determining N-nitrosamines (NAs) in pharmaceutical preparations. Based in Turkey, the team have published their findings in the Journal of Chromatography A (1).

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During storage of pharmaceutical preparations, various impurities may occur. These can possess potentially serious risks to patient health depending on toxicity levels and concentrations. To minimize these risks, comprehensive pharmacovigilance procedures are used, focusing on monitoring pharmaceutical products’ stability and impurity levels. As such, it should be ensured that toxicological limits of impurities are kept at acceptable levels based on the risk tolerability approach. Further, these control and evaluation processes are critical for maintaining the overall quality and safety of active pharmaceutical ingredients (APIs).

N-nitrosamines (NAs) are chemical compounds notorious for their carcinogenicity and for their widespread occurrence throughout the human environment, from air and water to drugs (2). Many efforts have been made to understand NAs as contaminants, and methods for their prevention, remediation, and detection have been developed. NAs are viewed as unwanted impurities in the pharmaceutical industry, which has led the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) to determine acceptable daily intake (AI) limits of 183 and 276 NAs. FDA guidelines divide nitrosamine impurities into two categories: small-molecule NAs, such as N-Nitrosodimethylamine (N-DMA), and nitrosamine drug substance-related impurities (NDSRI). Small-molecule NAs were the focus of this study, since they are commonly found in pharmaceutical products.

With this study, scientists created a new, sensitive, short-time, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method for analyzing 15 N-nitrosamines defined in the European Pharmacopoeia within the scope of Extractables & Leachables (E&L) studies. Analysis was carried out in positive mode using an atmospheric pressure chemical ionization source in the dynamic multiple reaction monitoring scanning mode.

The presence of low-molecular-weight and structurally very similar NAs in this study made the analytical process more complicated. However, the scientists’ method offered a unique advantage because it could simultaneously determine 15NAs with high sensitivity and selectivity within an analysis time of only 16 min. The developed method allowed NA quantitative analysis by offering a wide dynamic range covering daily AI limits. According to the scientists, their procedures are some of “the most comprehensive studies evaluating the stability of NAs depending on temperature and time” (1). E&L studies detected NA contamination arising from potential interactions between products and primary packaging materials containing leachables.

The method was successfully validated and applied to various packaging materials, such as polypropylene bags and disposable eye drop packaging. They also successfully used the method in commercial pharmaceutical preparations. Overall, the proposed analytical method is believed to offer significant innovation in pharmaceutical and packaging safety studies by enabling simultaneous, rapid, reliable, highly sensitive, accurate, and precise analysis of 15 NAs. According to the scientists, their study is supposedly the first in the scientific literature to combine a sensitive, rapid, and reliable method with E&L analyses to quantify NAs. This approach could, however, pave the way for further research utilizing these techniques in a similar manner.

References

(1) Dalkılıç, O.; Demircioğlu, İ. H.; Çelik, S.; et al. Method Development and Validation for Determination of N-Nitrosamines in Pharmaceutical Preparations by LC-MS/MS: Application to Extractables and Leachables Studies. J. Chromatogr. A 2025, 1745, 465741. DOI: 10.1016/j.chroma.2025.465741

(2) Beard, J. C.; Swager, T. M. An Organic Chemist’s Guide to N-Nitrosamines: Their Structure, Reactivity, and Role as Contaminants. J. Org. Chem. 2021, 86 (3), 2037–2057. DOI: 10.1021/acs.joc.0c02774