How the Nitrosamine Guidance Shift is Changing LC-MS Workflows for APIs and Peptides
The author would like to acknowledge and thank Matthew Webberley, Associate Director, Analytical R&D, SK pharmteco, for his guidance.
For chromatography and mass spectrometry groups, the most important recent change in the nitrosamine field is not simply that the limits got tighter.1-3 It is that the analytical target has become less generic. The field has moved away from a narrow focus on a short list of classic, small nitrosamines and toward API-specific nitrosamines, nitrosamine drug substance-related impurities, packaging-linked risks, and matrix-dependent confirmation problems. This change has direct consequences for how methods are selected, developed, and validated.1-3,8
In that sense, the current nitrosamine regime is best understood as a shift from contamination response to lifecycle analytical control, consistent with modern analytical lifecycle principles outlined in ICH Q14.2,3,10 FDA's newer framework expects risk assessment, confirmatory testing, control, and mitigation to work together. For analytical scientists, that means a method is increasingly judged not only by sensitivity, but by how well it fits the underlying formation hypothesis, the matrix, and the broader control strategy.2,3
What Changed in the Lab After the Newer Guidance
The 2023 FDA NDSRI guidance changed the problem definition by introducing a formal framework for assigning acceptable intake limits to API-related nitrosamines. The practical consequence is that laboratories can no longer assume a fixed universal target list. Instead, they now need a route-aware, molecule-specific analytical plan that starts with a plausible impurity hypothesis and then narrows toward confirmatory targets.1,3
The 2024 FDA control guidance widened the analytical field again by explicitly covering both small-molecule nitrosamines and NDSRIs in APIs and drug products. That pushes nitrosamine work upstream and downstream at the same time: upstream into process chemistry and excipient selection, and downstream into packaging, stability, and finished-product control.2,4
The June 23, 2025, FDA implementation update did not relax the need for confirmatory testing. Instead, it made documented analytical progress more important, because sponsors that could not complete all implementation steps were expected to provide evidence on methods, batches tested, detected NDSRIs, and root-cause status. For the analytical lab, that means development work must be defensible even before the final control strategy is fully locked.3
Why LC-MS/MS Remains Central, but HRMS is Gaining Ground
Targeted LC-MS/MS remains the workhorse for routine quantitation because it offers the sensitivity needed for low acceptable-intake thresholds and can be deployed as a validated confirmatory tool once the analyte list is known. That is still the backbone of most commercial nitrosamine control programs.2,3,8
High-Resolution Mass Spectrometry (HRMS) is becoming more important for a different reason: the problem increasingly involves unknowns, near-isobars, route-specific structures, and complex matrices. When the putative nitrosamine is API-related or when a matrix produces ambiguous peaks, exact-mass measurement and structurally informative fragmentation become central to proving what a peak is and what it is not. In practice, the emerging pattern is HRMS for discovery and structural confirmation, followed by a simpler quantitative assay (LC-MS/MS) where feasible.5,6,8
This is one reason the newer nitrosamine problem feels different from the earlier one. The hard part is often no longer only sensitivity. It is selectivity, attribution, and mechanistic fit.2,8
Why Peptides Raise the Bar
Peptides complicate nitrosamine work especially when direct nitrosamine literature is sparse. They bring structurally rich matrices, adsorption concerns, degradation pathways that can expand under formulation stress, and a higher premium on orthogonal confirmation. The analytical literature from 2025 illustrates this through peptide mapping and degradation studies rather than through large numbers of peptide-specific nitrosamine case reports.5,6
A recent peptide mapping study used UPLC-HRMS to establish primary-structure sameness for synthetic peptide APIs, while another study used LC-HRMS to examine how pH, buffer choice, molarity, and temperature shape peptide degradation. Those workflows are highly relevant to peptide nitrosamine readiness because they solve the same practical problems: discriminating low-level species, understanding degradation context, and separating product-related change from route-related impurities.5,6
For peptide programs, then, nitrosamine strategy is likely to succeed only when it is embedded inside broader analytical development: forced degradation, stability-indicating purity methods, impurity characterization, and targeted confirmatory testing once plausible species have been defined.2,4-6
What This Means for Process Development and Analytical Support
The analytical consequences of the new nitrosamine regime line up closely with the kinds of integrated support that modern development groups often seek: route-aware risk assessment, forced-degradation design, stability studies, orthogonal characterization, nitrosamine and genotoxic impurity testing, extractables and leachables work, and scalable confirmatory methods. Those needs also align with the analytical-development, peptide, and impurity-control capabilities that SK pharmteco has described publicly in its own materials. But the larger message for LCGC readers is broader than any one provider: nitrosamine control now rewards laboratories that can connect chromatography, mass spectrometry, process understanding, and CMC context rather than treating each as a separate silo.2-4,8
Final Thoughts
The obvious takeaway is that nitrosamine analysis has become a more chromatographically and structurally demanding problem. The regulatory shift matters because it is forcing the analytical workflow to evolve from generic screening toward matrix-aware, API-specific, orthogonally confirmed strategies that are tightly linked to process development and stability science. Peptides make that evolution easier to see, because they expose the limits of one-method-fits-all thinking earlier than simpler APIs do.1-3,5,6,8
References
- U.S. Food and Drug Administration. Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities. August 2023.
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/recommended-acceptable-intake-limits-nitrosamine-drug-substance-related-impurities - U.S. Food and Drug Administration. Control of Nitrosamine Impurities in Human Drugs. September 2024.
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/control-nitrosamine-impurities-human-drugs - U.S. Food and Drug Administration. CDER Nitrosamine Impurity Acceptable Intake Limits. Updated June 23, 2025 and later revision history.
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cder-nitrosamine-impurity-acceptable-intake-limits - World Health Organization. WHO Good Practice Considerations for the Prevention and Control of Nitrosamines in Pharmaceutical Products. TRS 1060, Annex 2. 2025.
https://www.who.int/publications/m/item/trs-1060---annex-2--who-good-practice-considerations-for-the-prevention-and-control-of-nitrosamines-in-pharmaceutical-products - A. Malgave, et al., Effect of PH, Buffers, Molarity, and Temperature on Solution State Degradation of Semaglutide Using LC-HRMS: A Preformulation Protocol for Peptide Drug Delivery, Eur. J. Pharm. Biopharm., (2025).
https://www.sciencedirect.com/science/article/pii/S0939641125001572 - S.H. Kim, et al., Peptide Mapping Analysis of Synthetic Semaglutide and Liraglutide for Generic Development of Drugs Originating from Recombinant DNA Technology, J. Pharm. Biomed. Anal., (2025).
https://www.sciencedirect.com/science/article/pii/S0731708525000238 - U.S. Food and Drug Administration. Emerging Scientific and Technical Information on Leachable NDBA and Other Small-Molecule Nitrosamines in Infusion Bags, August 18, 2025.
https://www.fda.gov/media/188238/download - Y. Zhang, et al., N-Nitrosamine Risk Assessment in Pharmaceuticals: Where Are We From a Regulatory Point of View in 2025?, Trends in Chemistry, (2025).
https://www.sciencedirect.com/science/article/pii/S2949771X25000350 - European Medicines Agency. Nitrosamine Impurities: Guidance for Marketing Authorization Holders. Current Guidance Page.
https://www.ema.europa.eu/en/human-regulatory-overview/post-authorisation/pharmacovigilance-post-authorisation/referral-procedures-human-medicines/nitrosamine-impurities/nitrosamine-impurities-guidance-marketing-authorisation-holders - International Council for Harmonisation (ICH). ICH Q14: Analytical Procedure Development. 2023.
https://database.ich.org/sites/default/files/ICH_Q14_Guideline_2023_1116.pdf