Analysis of Nitrite in Pharmaceutical Excipients: A Multi‑Laboratory Comparative Study

Abstract

Rigorous control of N-nitrosamine impurities within drug products is essential due to their potential carcinogenicity. Nitrosamines are often formed through the reaction of vulnerable amines present in the drug substance with nitrite from excipients, where nitrite is often the limiting reagent. Therefore, it is important to understand the level of nitrite in excipients to assess the risk of nitrosamine formation in a drug product. For this purpose, the Lhasa Nitrites database was created to collate and curate nitrite data in excipients, reagents and solvents contributed by member companies worldwide.

This data is generated using different methods by different companies, it is therefore necessary to understand the accuracy and variability of this data. A comparative testing study was conducted, by the Lhasa Nitrites consortium, involving six independent labs using their analysis procedure including ion chromatography with conductivity detection (IC-CD), pre-column derivatisation with Griess reagent followed by LC-UV analysis (Griess/LC-UV), or pre-column derivatisation with 2,3-diaminonaphthalene followed by LC-FLR analysis (DAN/LC-FLR). Three common excipients were selected including microcrystalline cellulose, magnesium stearate and lactose. The results were evaluated with respect to detection method, sample extraction procedure, and clarification method. Consistent results were obtained across different laboratories for lactose. However, no quantifiable nitrite levels were detected in the MCC lots used due to matrix interference during sample selection. Highest variability was observed for magnesium stearate, primarily due to the use of sonication as part of the sample preparation to overcome the challenges to extract nitrite from the matrix.

This study reveals the potential variability for the analysis of trace level nitrite in excipients, underscoring the need for more rigorous method characterisation including specificity, accuracy and precision. As an initial response, the Lhasa Nitrites consortium developed and disseminated updated guidance for method validation, reflecting the collective knowledge gained through comparative testing and data sharing.

1. Introduction

N-Nitrosamines in pharmaceutical products pose potential safety risks to patients due to their classification as probable human carcinogens by the International Agency for Research on Cancer (IARC) (World Health Organisation International Agency for Research on Cancer 2025). Several products have been recalled due to the presence of nitrosamines above acceptable intake limits including Valsartan, Metformin, Ranitidine, etc (World Health Organisation 2025). As a result, nitrosamine risk assessments have become a critical component of pharmaceutical development to identify and control nitrosamine impurities. Mitigating risks of nitrosamine formation may require careful selection of raw materials, optimisation of manufacturing processes, and stringent storage conditions (Dirat et al., 2025).

More recently, attention has expanded to include nitrosamine drug substance-related impurities (NDSRIs) (Nudelman et al., 2023). The structure of the drug substance itself, particularly vulnerable structures like secondary amines, play a crucial role in NDSRI formation, with up to 40% of common active pharmaceutical ingredients (APIs) containing potential nitrosamine precursors (Schlingemann et al., 2023). The presence of nitrosating agents during drug substance synthesis, within the drug product excipients, or introduced during manufacturing can lead to nitrosamine formation. In such scenarios, nitrosating agents may predominantly react with the drug substance’s vulnerable amine moiety (Schlingemann et al., 2023). However, this should be thoroughly assessed as part of the risk assessment process as the likelihood for nitrosation depends on a variety of factors, for example but not limited to concentration, pKa, solubility, and availability of nitrosating agent (Dirat et al., 2025, Cioc et al., 2023).

Nitrosating agents derived from nitrite are often the limiting reagent in nitrosamine formation in drug products. Therefore, limiting the amount of available nitrite can reduce the potential for nitrosamine formation. Excipients have been identified as a major source of nitrosating agents within drug products, often containing low levels of nitrite (EFPIA 2025). While nitrites are a primary concern, other nitrosating agents including but not limited to, nitric oxides (mainly N2O3 and N2O4), and nitrosyl halides, may be introduced through manufacturing inputs like reagents, water or air and therefore should be considered as part of the risk assessment process (EFPIA 2025, Fukuda et al., 2023, Dirat et al., 2025).

Nitrite levels in excipients are well documented in the Lhasa Nitrites database through the contributions from member companies (Nitrites data sharing 2025). Since establishment of the database and consortium in 2020, over 100 different excipients have been analysed, raising awareness of the typical nitrite levels present in different excipients, and the importance of controlling nitrite content. Advances in excipient manufacturing have resulted in some low nitrite grade excipients becoming available, reflecting ongoing research and commitment to product safety and quality. Adoption of these low nitrite excipient grades might enable marketing authorisation holders to further reduce the risk of nitrosamine formation.

In recognition of the importance of excipient‑derived nitrite in nitrosamine risk assessments and the challenges of confidently quantifying trace nitrite levels, a multi‑laboratory comparative assessment was performed on common excipients (microcrystalline cellulose, magnesium stearate, and lactose) using multiple detection approaches alongside varying extraction and clarification procedures. The objective is to understand the quality and variability of the data in Lhasa Nitrites database and identify areas to improve the data quality for future data generation. These insights will be translated into practical guidance to strengthen method validation and improve the reliability and comparability of nitrite measurements, supporting nitrosamine risk assessments.

2. Summary of Analytical Methods

Understanding the nitrite content in pharmaceutical excipients and reagents is critical to nitrosamine risk assessment and control. The industry continues to push the boundaries of nitrite detection and quantitation, continuously developing more sensitive and selective analytical methods (Hickert et al., 2025). However, method selection should balance three key criteria; ease of use, sensitivity and quality (encompasses accuracy, precision, robustness and control of false positives and negatives) (Figure 1). For example, ion chromatography using supressed conductivity detection (IC-CD) is relatively easy to use but offers lower sensitivity and specificity than more advanced approaches (Hickert et al., 2025).

Figure 1. Three key criteria for nitrite quantification.

Accurate trace-level quantitation requires rigorous contamination control. Nitrite is ubiquitous in water, reagents and laboratory consumables. Additionally, ambient NOX, can form nitric and nitrous acid upon contact with water. The use of blank solutions prepared using identical procedures as the test samples are required to ensure reliable low-level nitrite quantitation. This practice is indispensable for maintaining the integrity of analytical results and validating the accuracy of nitrite determinations at low concentrations. Additionally, challenging matrices with low recovery may necessitate quantitation using standard addition. The choice of analytical technique often hinges on its ability to provide accurate and reliable data for these challenging matrices. Figure 2 presents an overall summary of the most commonly used analytical procedures for the top 10 excipients in the Lhasa Nitrites database 2025.1 version.

Figure 2. Summary of the most common procedures for nitrite quantitation for the top 10 excipients, expressed as percentage of the number of entries in the Lhasa Nitrites database.

Historically, most of the nitrite analysis was performed using IC-CD or Griess-based LC-UV but complementary analytical procedures are increasingly employed to address matrix-specific limitations and LOQ needs (Hickert et al., 2025). Several generic industry methods have emerged as a result:

1. IC-UV with post-column derivatisation using Griess reagent (Managing nitrite impurities: A combined supplier-manufacturer view to nitrosamine risks 2023).
2. Precolumn derivatisation using Griess reagent or DAN, coupled with: a. LC-FLR b. Liquid chromatography mass spectrometry (LC-MS) (Managing nitrite impurities: A combined supplier-manufacturer view to nitrosamine risks 2023, Damiani, 1986, Jireš and Douša, 2022, International Agency for Research on Cancer 2008).
3. Cyclamate derivatisation with headspace gas chromatography and flame ionisation detection (HS-GC-FID) (Zhang et al., 2018) or mass spectrometry detection (HS-GC-MS) (Baumann and Näf, 2024).

Download the full article as PDF here Analysis of Nitrite in Pharmaceutical Excipients

or continue reading here

Giorgio Blom, Tamas Balogh, Petra Loos, Jinjian Zheng, Grace Kocks, Gemma Packer, Analysis of Nitrite in Pharmaceutical Excipients: A Multi‑Laboratory Comparative Study, European Journal of Pharmaceutical Sciences, 2026, 107516, ISSN 0928-0987, https://doi.org/10.1016/j.ejps.2026.107516.

Enjoy our online event on Nitrosamine analytics (view on demand) here:

Nitrites/ Nitrosamines The impacts on analytics, but not only