Five Questions to Ask When Choosing Low Nitrite Excipients as Part of Your Nitrosamine Mitigation Strategy

ƒ

Low nitrite excipients have emerged as a potential nitrosamine risk mitigation strategy for some formulations, especially when a vulnerable amine is present in the drug product.^1-4 If deemed suitable, low nitrite excipients can offer drug manufacturers an effective route to minimize nitrosamine risk – without having to jump through regulatory hoops or compromise the efficacy of the final drug product. What’s more, they can be used alongside other mitigation strategies, forming part of a holistic approach to nitrosamine control. If you’ve not leveraged low nitrite excipients yet – but you’re curious – you may have some questions about their effectiveness and how they might fit into your formulation process.

Here, IFF Pharma Solutions’ Elizabeth Tocce and Koudi Zhu explore the role of nitrite in excipients in nitrosamine formation, shed light on how low nitrite excipients can support the creation of safer pharmaceuticals, and address some of the most frequently asked questions that their team encounters.

Understanding the Role of Nitrite in Excipients and Nitrosamine Formation

Pharmaceutical excipients make up a large portion of the end drug product, accounting for up to 90% of the total volume in some cases.^5,6 These excipients are fundamental to the functionality of a drug product, providing different roles from manufacturability to stability, dose uniformity, and effective delivery of the active pharmaceutical ingredient (API).⁷ However, despite their many advantages for formulations, they may play a critical role in nitrosamine formation.

Nitrosamines can form during API manufacturing, drug product manufacturing, and storage, often through the reaction of nitrosating agents with secondary amines. One of the growing concerns is for nitrosamine drug substance-related impurities (NDSRI), which can form from secondary amine-containing APIs reacting with nitrosating agents used in API production and/ or in the final formulated drug product.⁸ Typically, the main source of nitrosating agent within a formulated drug product is nitrite – which may be present at trace levels in excipients. One study suggests that 40% of APIs could be potential nitrosamine precursors, which means many drug products could be at risk for NDSRI formation.⁸ It is important to note that in the reaction between nitrites in excipients and secondary amines in an API, it is the trace elements of nitrites that usually determine the rate of nitrosamine formation, and therefore the level of contamination.¹ As such, excipients can have a substantial impact on the risk of nitrosamine formation. Many excipients contain nitrites on the order of parts per billion to parts per million¹⁹ leading some drug developers to choose low nitrite excipients as part of their nitrosamine mitigation strategy. It should be noted that the definition of low nitrite can be different for any given drug product and is not a regulated set level.

Selecting low nitrite excipients can be an effective way to minimize the risk of nitrosamine contamination without compromising the therapeutic efficacy of the final drug product, as well as carrying minimal risks and regulatory requirements. For those interested in switching to low nitrite excipients, here’s what you need to know…

1. What is the best approach for measuring nitrites in excipients?

To understand the potential risk of nitrosamine formation, drug manufacturers need to know the extent of nitrite content in the excipients they intend to use. Being able to accurately measure nitrite levels in excipients, even low nitrite excipients, is critical for assessing nitrosamine risk in drug products. There are two approaches – indirect or direct nitrite analysis.

Indirect methods involve derivatization, which is the process of chemically altering an analyte, in this case – nitrite. For example, gas chromatography-mass spectrometry (GC-MS) can be used to analyze nitrite that has been derivatized using pentafluorobenzyl bromide reagent.⁹ This process, however, is very labor intensive.¹⁰ Similar methods, such as using 2,3-diaminonaphthalene to derivatize nitrite, followed by liquid chromatography-mass spectrometry (LC-MS) analysis, give poor sensitivity to trace levels of nitrite (0.2 ppm in sample).¹¹ The most common derivatization method uses the Griess reagent to convert nitrite into an azo compound with strong ultra-violet (UV)-absorption.^12,13 These derivatives can then be analyzed using methods such as LC-UV or LC-MS.¹⁰ Yet, research reveals that factors like the pH can affect the kinetics of the Griess reaction, and potential side reactions involving nitrites can create uncertainties for low-level nitrite quantification.^10,14 In some cases, the reaction can be completely inhibited by the sample matrix.¹⁵ Another drawback is that a high background signal can form in the blank sample with some commercially available Griess reagents, which limits the quantitative ability of the test.¹⁶

In contrast, direct analysis methods can measure nitrite levels without the need for derivatization or additional chemical reactions. Traditional methods like ion chromatography with conductivity detection (IC-CD), however, can still face challenges.¹⁷ When using IC-CD to measure nitrites in the commonly used excipient, microcrystalline cellulose (MCC), other anions present in MCC can interfere with nitrite, leading to peak interference, as shown in Figure 1.¹⁰ This is known as co-elution, as two or more compounds elute from the chromatographic column at the same time, making it challenging to distinguish between them, possibly causing falsely high or low nitrite results.

One way to address peak interference is to measure nitrites in MCC using ion chromatography coupled with mass spectrometry (IC-MS).¹⁰ This approach uses selective ion monitoring (SIM) which means that MS will only measure nitrites, eliminating interference from other compounds present in the excipient, as shown in Figure 2. While the interfering compounds no longer show up on the MS chromatogram, they can still enter the MS detector and affect the ionization of nitrite, which can lead to ion suppression of nitrite and low recovery. This challenge can be addressed by using an isotope-labeled N-15 sodium nitrite internal standard (IS).¹⁰ The isotope-labeled N-15 nitrite elutes at the same retention time as nitrite and experiences the same degree of ion suppression in the MS detector. Therefore, the ion suppression effect of nitrite can be corrected by using isotope-labeled nitrite as the IS, which results in accurate nitrite measurement across various MCC samples.⁹

Drug product manufacturers can, and should, discuss possible nitrite measurement challenges with their excipient suppliers as they develop their methods. Moreover, they can consider choosing excipient suppliers that utilize scientifically validated and up-to-date techniques to assess nitrate content, which may alleviate strain on quality and analytical resources that are tasked with nitrosamine risk assessment and mitigation strategies.

2. How do formulation conditions impact nitrite conversion to nitrosamines?

While the presence of nitrites in excipients is a potential contributing factor to nitrosamine formation, various process conditions within the drug product and during manufacture can also influence the rate of conversion of nitrites into nitrosamines. For instance, if there is no vulnerable amine, like a secondary or tertiary amine, present in the formulation, nitrosamine formation cannot occur. If a vulnerable amine is present, the higher the levels of nitrites in a formulation, the greater the potential rate of conversion.

Additional factors, such as conditions within the drug product, like pH level and storage processes can also influence the rate of conversion. Research shows that the most favorable conditions for maximum conversion are large quantities of secondary amine API, API in the salt form, and storage in high-humidity conditions. Drug products developed under these circumstances are deemed at high risk for nitrosamine contamination. Important findings demonstrate that – even for these high-risk products – the formation of nitrosamine impurities in formulations using low nitrite excipients remained under the acceptable daily intake levels proposed by regulatory agencies.¹⁸ This evidence highlights that nitrite content in excipients can be one means of controlling nitrosamine formation.

3. Does the drug product manufacturing technique impact nitrosamine formation?

When it comes to nitrosamine formation from a secondary amine and nitrites in excipients, certain manufacturing techniques can increase the risk of contamination. For instance, formulations that use wet granulation manufacturing processes increase the risk of nitrosamine formation, compared to dry granulation formulations. This is because wet granulation promotes the distribution and mobility of both nitrites and secondary amines, increasing the chance of contact and reactivity.¹⁸

If wet granulation presents a nitrosamine contamination risk for formulators, they might want to consider switching from a wet to a dry granulation process if possible. If this is not feasible or does not alone obtain the desired reduction, low nitrite excipients could be a viable strategy to limit nitrite levels in the formulation, thereby reducing the risk of nitrosamine formulation and contamination.

4. Does the excipient supplier matter?

When it comes to low nitrite excipients – yes. Nitrite levels in excipients vary considerably from supplier to supplier and even between different batches from the same supplier.¹⁹ In one study, the median nitrite levels in MCC from different suppliers varied from 0.1 ug/g to 1.6 ug/g.¹⁹ Research has also demonstrated that choosing an excipient with low levels of nitrites can substantially reduce nitrosamine formation, compared to using excipients with high nitrite levels.² These findings illustrate the significant impact of selecting an excipient supplier that provides low nitrite levels as part of a holistic approach to nitrosamine risk mitigation. It should be noted that what is deemed as low or high is a relative term that must be evaluated and determined on a case-by-case basis.

Nitrite levels are not the only consideration when selecting an excipient supplier. Good communication, alignment on desired goals, and agreement on how to achieve these goals are also important in successfully implementing low nitrite excipients into a nitrosamine risk mitigation strategy. For example, who takes ownership of confirming nitrite content within the excipient? There is no right answer to this question, but it should be addressed and both parties should agree on the answer. Effective communication between the excipient user and supplier is critically important as each nitrosamine mitigation strategy is unique.

5. What are the regulatory implications of switching to low nitrite excipients?

In the evolving landscape of nitrosamine mitigation, staying on top of the latest guidance can be challenging. As such, formulators might not be aware that switching to low nitrite excipients is relatively straightforward if the excipient chemistry is the same (e.g. a lower nitrite MCC for a higher nitrite MCC). Low levels of nitrites in an excipient are not expected to impact the performance of a drug product, such as the physical properties or drug release mechanisms, if the rest of the formulation is relatively equal. However, any change in supplier or ingredient should go through a risk assessment per normal management of change processes. Using low nitrite excipients is one of the U.S. Food and Drug Administration's (FDA’s) recommended mitigation strategies.²⁰ The FDA’s guidance encompasses a supplier qualification program designed to mitigate potential nitrite impurities across excipient suppliers and lots if the introduction of nitrites from excipients is deemed to play a role in nitrosamine formation.

In summary, low nitrite excipients can be an effective tool for mitigating nitrosamine formation in drug products, particularly in high-risk formulations containing secondary amines. It is essential to consider factors such as how to accurately measure nitrite levels in excipients, supplier variations, and formulation conditions, before implementing this approach. The most important factor for successful implementation is effective communication between pharmaceutical developers and excipient providers.

References

1. Berardi, A., Jaspers, M., & Dickhoff, B. H. J. (2023). Modeling the Impact of Excipient Selection on Nitrosamine Formation Towards Risk Mitigation. Pharmaceutics, 15(8), 2015.
2. Schlingemann, J, Boucley, C, Hickert, S, et al. (2022) Avoiding N-nitrosodimethylamine formation in metformin pharmaceuticals by limiting dimethylamine and nitrite. Int. J. Pharm. 620, 121740.
3. Horne S, Vera MD, Nagavelli LR, Sayeed VA, Heckman L, Johnson D, Berger D, Yip YY, Krahn CL, Sizukusa LO, Rocha NFM, Bream RN, Ludwig J, Keire DA, Condran G. (2023) Regulatory Experiences with Root Causes and Risk Factors for Nitrosamine Impurities in Pharmaceuticals. J Pharm Sci. 112(5):1166-1182.
4. Răzvan C. Cioc, Ciarán Joyce, Monika Mayr, and Robert N. Bream. (2023) Formation of N-Nitrosamine Drug Substance Related Impurities in Medicines: A Regulatory Perspective on Risk Factors and Mitigation Strategies. Organic Process Research & Development 27 (10), 1736-1750.
5. Abrantes C.G., Duarte D., Reis C.P. (2016) An Overview of Pharmaceutical Excipients: Safe or Not Safe. J. Pharm. Sci. 105:2019–2026.
6. Haywood A., Glass B.D. (2011) Pharmaceutical excipients—Where do we begin? Aust. Prescr. 34:112–114.
7. van der Merwe, J., Steenekamp, J., Steyn, D., & Hamman, J. (2020). The Role of Functional Excipients in Solid Oral Dosage Forms to Overcome Poor Drug Dissolution and Bioavailability. Pharmaceutics, 12(5), 393.
8. Schlingemann J, Burns MJ, Ponting DJ, et al. (2023) The Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals. J Pharm Sci. 112(5):1287-1304.
9. D. Tsikas, R.H. Boeger, S.M. Bode-Boeger, F.M. Gutzki, J.C. (1994) Froelich, Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography-mass spectrometry using their N-labeled analogs, J. Chromatogr. B. Biomed. Appl. 661, 185–191.
10. Zhu, K., Kerry, M., Serr, B., & Mintert, M. (2023). Parts per billion of nitrite in microcrystalline cellulose by ion chromatography-mass spectrometry with isotope-labeled internal standard. Journal of pharmaceutical and biomedical analysis, 235, 115648.
11. J. Jakub, D. (2022) Nitrites as precursors of N-nitrosation in pharmaceutical samples – A trace level analysis, J. Pharm. Biomed. Anal. 213, 114677.
12. Q. Wen, D. Paik. (2012) Using the Griess colorimetric nitrite assay for measuring aliphatic β-nitroalcohols. Exp. Eye Res. 98:52-57.
13. S.S. Shin, H. Fung. (2011) Evaluation of an LC-MS/MS assay for 15N-nitrite for cellular studies of L-arginine action, J. Pharm. Biomed. Anal. 56: 1127-1131.
14. J.B. (1979) Fox Jr, Kinetics and mechanisms of the Griess reaction, Anal. Chem. 51 1493–1502.
15. S. Hickert, U. Reichert, J. Schlingemann, Managing nitrite impurities: A combined supplier-manufacturer view to nitrosamine risks. Webinar by sigma Aldrich, Darmstadt, Germany, 28-Sep-2023.
16. M.D. Croitoru. (2012) Nitrite and nitrate can be accurately measured in samples of vegetal and animal origin using an HPLC UV/VIS technique, J. Chromatogr. B 911:54-161.
17. M. Helaleh, T. Korenaga. (2000) Ion chromatographic method for simultaneous determination of nitrate and nitrite in human saliva, J. Chromatogr. B 744: 433–437.
18. Moser, J, Ashworth, I.W, Harris, L, Hillier, M.C, Nanda, K.K, Scrivens, G. (2023) N-Nitrosamine Formation in Pharmaceutical Solid Drug Products: Experimental Observations. J. Pharm. Sci. 112, 1255 1267.
19. Boetzel, R, Schlingemann, J, Hickert, S, et al. (2022) A Nitrite Excipient Database: A Useful Tool to Support N-Nitrosamine Risk Assessments for Drug Products. J. Pharm. Sci. 112, 1615–1624.
20. Updates on possible mitigation strategies to reduce the risk of nitrosamine drug substance-related impurities in drug products. Drug Safety and Availability. U.S. Food and Drug Administration.

Author Details

Elizabeth Tocce, Lead Application Scientist- IFF Pharma Solutions; Koudi Zhu, Senior Scientist- IFF Pharma Solutions

Publication Details