Managing Nitrite Impurities: A Supplier-Manufacturer Approach to Mitigate Nitrosamine Risk

N-Nitrosodimethylamine (NDMA) and other nitrosamines, are categorized as probable human carcinogens, and were first identified in drug products containing valsartan in 2018. Small, potent, alkylnitrosamines such as NDMA, commonly arise from secondary amine precursors used as starting materials, reagents, solvents or from impurities of these during active pharmaceutical ingredient (API) synthesis or drug product manufacturing and storage. A second type of nitrosamines are the so-called NDSRIs (Nitrosamine Drug Substance Related Impurities), which originate from APIs or API impurities that contain vulnerable amines. Since first identified, both nitrosamines and NDSRIs have been found in other pharmaceuticals. An assessment of active pharmaceutical ingredients (APIs), formulated drug products, and packaging revealed both nitrosamines and NDSRIs are more widespread than previously believed. Given their potential safety risk, drug manufacturers must assess the levels of nitrosamine impurities in pharmaceutical products and identify appropriate mitigation actions.

This page describes how nitrosamines are formed, the prevalence of nitrosamines and chemical precursors in pharmaceuticals, quantification methods, and strategies for managing nitrite impurities.

Section Overview:

• The Formation of Nitrosamines
• Prevalence of Nitrosamine Precursors in Pharmaceuticals
• Methods for Nitrite Quantification
• Sources of Nitrites in Excipients
• Categorizing Emprove® Products Based on Nitrite Levels

The Formation of Nitrosamines

Nitrosamine impurities are most frequently derived from secondary amines, which can be starting materials or impurities in the raw materials used in the synthesis of APIs, APIs themselves or API-related impurities. Primary amines cannot form nitrosamines, while tertiary amines require nitrosative de-alkylation or nitrosative cleavage, which are much slower processes. Quaternary ammonium ions may degrade to secondary and tertiary amines or contain those as impurities.

Prevalence of Nitrosamine Precursors in Pharmaceuticals

The potential prevalence of nitrosamine precursors in pharmaceuticals was explored in a recent publication by Schlingemann, et al. and by 2021 it was clear that many APIs contain precursors with the potential to form NDSRIs4. Several databases were reviewed for the presence of secondary and tertiary amines in drugs and drug impurities; dependent on the database, between 25-40% of entries featured secondary or tertiary amines with secondary amines accounting for 10-19% of entries.

A comprehensive review of nitrosamine potency published in 2023 by the Journal of Phamaceutical Sciences revealed nitrosamines derived from secondary amines primarily fall into the less potent categories, with only a small fraction classified as the most potent. Nitrosamines derived from tertiary amines are distributed relatively evenly among the potency categories while potent NDSRIs are more likely to be derived from less reactive tertiary amines than from secondary amines.

Methods for Nitrite Quantification

Several methods are available to detect the presence of nitrite, but not all are applicable for identifying trace amounts at the ng/g level.

Griess derivatization is an indirect quantitation method based on a simple reaction that transfers nitrites to a pink azo dye. This derivatization increases retention in RP-HPLC. However, sample matrix can inhibit the chemical reaction and if the full conversion does not occur, the nitrite content in a sample may be underestimated. Use of HPLC-MS/MS following Griess derivatization compensates for varying yields by stable isotope dilution analysis and offers high sensitivity and specificity. However, mathematical correction for spectral isotope overlap is required when using this method.

Anion exchange chromatography with conductivity detection is a direct method for nitrite detection but offers lower sensitivity and specificity compared to other approaches due to frequent interferences. A combination of ion chromatography (IC) with a post-column Griess derivatization enables UV detection and removes interfering compounds that might suppress the Griess reaction.

Challenging sample matrices drive the need for orthogonal methods of nitrite detection as UV-active matrices can lead to co-elution in HPLC-UV preventing accurate integration. In this situation, higher specificity techniques such as LC-MS/MS or a different separation mechanism such as IC with post-column derivatization (PCD) to separate and then derivatize the nitrite should be used.

Salts or other materials that bring high ion loads into the ion chromatography process can saturate the column, preventing integration of nitrite.

When sample matrix components inhibit the Griess reaction, nitrite quantification can be performed by direct detection (IC-CD) or matrix removal (IC-PCD-UV).

Table 1 summarizes the various nitrite detection methods and the typical limits of quantification (LOQ) of each method.

Sources of Nitrites in Excipients

The presence of nitrites in excipients poses the risk of nitrosamine formation in drug products; these arise from raw materials or steps such as spray drying used in excipient production, Table 2. Nitrosamines can also form during drug product formulation and storage. The combination of a nitrosating agent and a vulnerable amine from solvents, reagents, reaction byproducts, raw and starting materials or degradation products, can lead to low molecular weight nitrosamine formation under suitable conditions.

The FDA highlights the risk of formation of nitrosamine impurities due to the presence of nitrites in excipients in their 2021 guidance for industry. Similary, the EMA states that nitrites have been identified as impurities and in many common excipients and marketing authorization holders or applicants should be aware that nitrosamine impurities can form at levels exceeding acceptable intake levels due to nitrites in excipients.

Categorizing Emprove® Products Based on Nitrite Levels

More than 350 products contained within the Emprove® Essential, the Emprove® Expert and the Emprove® API portfolio, have been assessed for the presence of nitrites and grouped into three categories based on their level of nitrite.

• Nitrite concentrations less than 200 ng/g or below the LOQ:  are low level of risk for introducing a relevant level of nitrites into a process.
• Nitrite levels between 200 and 500 ng/g: the presence of nitrites has been detected in at least one batch and these products are periodically tested to monitor nitrite levels. These products are of moderate concern.
• Nitrite levels  >500 ng/g in at least one batch. While this is mostly in the sub-ppm, range additional control steps are being applied and more batch data are being collected to derive a feasible limit and method for regular control.

Information related to the presence of nitrite is provided in the Emprove® Material Qualification and Operational Excellence dossiers that accompany these products. This information supports risk assessments of nitrosamine formation in drug products and provides drug manufacturers with valuable information to mitigate risk from nitrosamines and improve drug safety for patients. Download free Emprove® chemicals demo dossiers.

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Source: Merck, website https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/pharmaceutical-and-biopharmaceutical-manufacturing/managing-nitrite-impurities

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