Something in the air–Unveiling the role of atmospheric nitrogen oxides in nitrosamine formation from amine-containing APIs: Mechanistic insights and risk assessment

Abstract

Atmospheric nitrogen oxides (NO_x) have emerged as a potential contributor to nitrosamine formation in active pharmaceutical ingredients (APIs) containing vulnerable amines. This study investigates the extent and mechanisms of NO_x-mediated nitrosation under controlled conditions and evaluates its relevance to real-world scenarios during API manufacturing, storage and analysis. Five structurally diverse APIs were exposed to NO, NO₂, and their mixtures at concentrations representative of urban air and forcing conditions. Nitrosamine formation was confirmed for all free base APIs, whereas corresponding salts exhibited significantly lower reactivity. Overall conversion was poor, with only ∼3% of available NO_x consumed, even under high humidity and elevated NOx levels. Mechanistic analysis suggests two pathways: solution-phase nitrosation in residual moisture and gas-solid surface reactions, with NO/NO₂ mixtures forming N₂O₃ as the most potent nitrosating species. Ambient air experiments revealed that small, open samples are most susceptible, while bulk storage poses minimal risk due to NO_x depletion and limited replenishment. These findings highlight the importance of considering NO_x exposure during analytical handling and certain manufacturing steps involving forced airflow, while supporting a low risk for typical API storage.

Introduction

The presence of nitrosamines (term refers to N-nitrosamines in this manuscript) in active pharmaceutical ingredients and drug products has been a focus for the pharmaceutical industry since mid-2018, when N-nitroso-dimethylamine (NDMA) and other low molecular weight nitrosamines were observed in some active pharmaceutical ingredient (API) and drug product (DP) batches of the Sartans drug family.1,2 It was then identified that several other APIs and/or respective drug products, such as acyclovir,3 amitriptyline,4 metformin,5-8 nizatidine,9 pioglitazone10,11 and ranitidine,12-14 were also observed to have NDMA present, even though the underlying root causes were different. Soon thereafter it became increasingly evident that low molecular weight nitrosamines, such as NDMA, may not be the biggest challenge to control to appropriate levels, but rather those derived from APIs and their impurities, so-called nitrosamine drug substance related impurities (NDSRIs).15

The predominant source of nitrosamines is when susceptible (also referred to as vulnerable) amine groups are exposed to nitrosating agents under promoting conditions, sometimes referred to as the “nitrosamine triangle”.16 Vulnerable amines are most commonly secondary amines, though exceptions exist.17 A substantial proportion of the current APIs and their associated impurities have been shown to contain such vulnerable amine functionalities.18,19 Arguably in most reported cases to date, the nitrosating species originated from inorganic nitrite, either intentionally introduced as a reagent or present at low levels in solid reagents or excipients. For this reason, the pharmaceutical industry continues to collect and share analytical data on nitrite levels in these materials to better understand and control nitrosamine formation.20,21 Other sources, such as nitroalkanes, hydroxylamines, hydrazines, hydrazides, hydrazones or chloramines in combination with certain conditions (typically oxidative) are known,16,22 but their relevance as risk factors is lower due to their limited application in pharmaceutical manufacturing processes under the specific conditions potentially conducive towards nitrosation.

The most common pathway for inorganic nitrite (NO2−) to lead to nitrosamine formation involves protonation to nitrous acid (HNO2) which then forms various nitrosating agents, including N2O3.23 Since atmospheric NOx (NO and NO2) can form N2O3 (Fig. 1), it is theoretically possible that exposure of a vulnerable amine to atmospheric NOx could lead to nitrosamine formation. The potential for atmospheric NOx to lead to nitrosamine formation during forced air operations such as jet-milling or fluid-bed drying has been highlighted by the FDA24 and in recent investigations into the varying levels of the nitrosamine NDMA in different metformin manufacturing sites.25 The potential for these forced air operations in the presence of a vulnerable amine to lead to nitrosamine formation is not entirely unexpected, given the large volumes of air involved and, in the case of fluid-bed drying, the favorable conditions for nitrosation – namely heat and humidity.

In addition to forced air operations, a vulnerable amine in an API or drug product could be exposed to atmospheric NOx at a significantly lower rate through air ingress during storage. Studies on sitagliptin API suggest that better air-protective packaging reduces NTTP (N-Nitroso Sitagliptin Amine) formation in small-scale API stability tests. However, the link to NOx-mediated nitrosation was not confirmed by the authors, and the NTTP precursor has a very low pKa, making it highly susceptible to nitrosation.26

The above examples indicate that, under certain conditions, exposure of a vulnerable amine to atmospheric NOx could result in nitrosamine formation. However, there is limited understanding of which scenarios relevant to API or DP processing or storage might lead to nitrosation, or the extent to which nitrosation could occur.

To aid fundamental understanding of NOx-mediated nitrosation of APIs, a structurally diverse series of vulnerable amines were exposed to measured levels of NO and NO2 under controlled conditions and monitored for nitrosamine formation. The results are discussed and a reaction mechanism is proposed in which nitrosation can occur in solution and at gas-solid interfaces. In addition, the experimental conditions are compared and contrasted to the manufacturing processes, storage and analysis conditions of an API.

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Rok Grahek, Renat Selimov,  Miha Drev, Holger Bauer, Paul Garbe, Brunhilde Guessregen, Joerg Schlingemann, Olivier Dirat, Krishna Veerubhotla, Ian W. Ashworth, Paula Tomlin, Something in the air–Unveiling the role of atmospheric nitrogen oxides in nitrosamine formation from amine-containing APIs: Mechanistic insights and risk assessment, Journal of Pharmaceutical Sciences, 2026; 115

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