Nitrosamine Impurity Analysis

Small-molecule nitrosamine impurities are a class of compounds that have gained significant attention from regulatory agencies worldwide due to their potential to be genotoxic and carcinogenic. These impurities can inadvertently be formed in a variety of drug substances and drug products through reactions between nitrosating agents and susceptible amines. Given the toxicological potency of these impurities, even trace amounts pose considerable health risks for patients. As a result, regulatory agencies such as the FDA and EMA have implemented strict guidelines and requirements for manufacturers to identify, quantify, and control these impurities. Comprehensive and sensitive analytical testing for nitrosamines is therefore not just a best practice, but essential for ensuring product safety and maintaining regulatory compliance. Novatia has extensive understanding of these regulatory requirements and the analytical methods necessary to perform these analyses.

Novatia offers services to help you meet and exceed regulatory expectations. Our expertise in this area includes:

• Nitrosamine Risk Assessment: Identifying potential pathways for introduction of small-molecule nitrosamine impurities in your drug substance manufacturing processes.
• Advanced Confirmatory Testing: Utilizing state-of-the-art analytical techniques, including highly sensitive LC-MS/MS and HRMS, for accurate and trace-level quantification of nitrosamines.
• Method Development & Validation: Developing and validating fit-for-purpose analytical methods tailored to your specific needs.
• Regulatory Compliance Support: Providing expert guidance on FDA and other global nitrosamine guidelines, ensuring your testing and reporting meet all requirements.

Example Nitrosamine Analysis: 

Regulatory agencies have specifically highlighted certain small-molecule nitrosamine impurities that are commonly encountered and issues Recommended Acceptable Intake Limits (RAILs) for each. These are summarized in the table below.

*As per FDA Final Guidances “Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs)” issued in August 2023 and “Control of Nitrosamine Impurities in Human Drugs” issued in September 2024 and European Medicines Agency (EMA) Guidance “Nitrosamine impurities: guidance for marketing authorisation holders”. Recommended Acceptable Intake Limits (RAIL) is available for the FDA using the CDER NDSRI AI limits Document Search and for the EMA by using Appendix I of the Guidance.
**Followed guidance recommendation for calculation of RAIL.

Novatia has experience using GC-MS to identify the presence of these common nitrosamine impurities. Using GC-MS and GC-MS/MS, Novatia can profile a sample containing a complex mixture of small-molecule nitrosamine impurities with the resolution shown in the figure below.

GC chromatograph of a sample containing a complex mixture of nitrosamines.

Nitrosamine Drug Substance-Related Impurities (NDSRIs) are a class of nitrosamine impurities that are structurally similar to an active pharmaceutical ingredient (API) or one of its intermediates. Unlike small-molecular nitrosamine impurities, NDSRIs often form during formulation and storage of drug products, primarily through reactions between APIs having susceptible amine groups and trace levels of nitrites in excipients. As these impurities are unique to their respective APIs, prediction and control of their formation can be quite challenging. As these impurities represent a risk to patient health, regulatory agencies have issues stringent guidelines and methods for establishing acceptable intake (AI) limits for NDSRIs. In these guidelines, they have also laid out a looming deadline: manufacturers must ensure that all NDSRIs in their drug products adhere to these limits by August 1, 2025. Non-compliance could lead to significant regulatory consequences, including potential fines, market delays, and costly product recalls.

At Novatia, we are at the forefront of this critical area, offering a comprehensive suite of services to help you meet and exceed regulatory expectations. Our expertise in this area includes:

• Robust NDSRI Risk Assessment: Identifying potential NDSRI formation pathways in your drug substances, excipients, and manufacturing processes.
• Assessing Potential for NDSRI Formation: Implementing Nitrosamine Assay Procedure (NAP) screening methods to assess your specific drug substance’s potential for NDSRI formation
• Procurement of NDSRI Standards: Sourcing or synthesizing analytical standards of NDSRIs of concern and determining purity by quantitative ¹H NMR so that standards are fit for method development and accurate testing
• Advanced Confirmatory Testing: Utilizing state-of-the-art analytical techniques, including highly sensitive LC-MS/MS and HRMS, for accurate and trace-level quantification of NDSRIs.
• Method Development & Validation: Developing and validating fit-for-purpose analytical methods tailored to your specific drug products and NDSRI profiles.
• Regulatory Compliance Support: Providing expert guidance on FDA and other global nitrosamine guidelines, ensuring your testing and reporting meet all requirements.

Don’t let the August 2025 deadline catch you unprepared. Partner with Novatia to ensure your drug products are safe, compliant, and ready for market.