PHARMACEUTICALS
Figure 5- Condition-dependent pathways for NTTP formation from residual TTP
Note: Nitrosation proceeds via nitrosating species( e. g. acidified nitrite / N2O3 and NO +) under suitable conditions. The specific nitrosating agent has not been conclusively identified and may vary with pH, nitrite activity, formulation matrix and excipients
( weighing, grinding, sonication, filtration, etc.) introduce significant variability, requiring strict control.
The standardisation of extraction protocols, use of nitrosamine-free consumables and implementation of stabilised extraction buffers were critical. The transition was made from conventional solid-liquid extraction to microextraction approaches, achieving improved sensitivity and efficiency for the thirteen small nitrosamines.
The analytical strategy employed orthogonal methods: LC-MS / MS for less volatile nitrosamines( NDBA and NEPA) and GC-MS / MS for highly volatile compounds( NDMA and NDEA). Tandem mass spectrometry with sub-ppb LOQ(< 0.5 ppb) was essential for tracelevel compliance.
Phase-appropriate validation was implemented per USP < 1469 > and ICH Q2( R1), with emphasis on LOQ rather than LOD. Confirmatory testing across twelve batches demonstrated all nitrosamines remained below 20 % of acceptable intake, supporting omission of routine release testing.
Six-month stability studies at 25 ° C and 40 ° C confirmed no nitrosamine formation over time. This case demonstrates that robust sample preparation is the foundation of data reliability, regulatory compliance and patient safety in nitrosamine control. Effective control requires an integrated lifecycle approach combining mechanistic understanding, quantitative purge assessment, robust sample preparation, sensitive detection and risk-based regulatory decision-making.
Overall, regulatory expectations recognise that analytical control strategies must evolve across the development lifecycle. Early qualification or partial validation supports preclinical and clinical studies, while progressively more comprehensive validation is required as products approach commercialisation.
Conclusion
Nitrosamine control is a central component of pharmaceutical development and manufacturing, driven by stricter regulatory expectations and a growing emphasis on proactive risk management. Current strategies focus on preventing nitrosamine and NDSRI formation through early assessment of APIs, excipients, formulations and manufacturing processes, supported
by computational prediction tools and highly sensitive analytical techniques for trace-level detection.
Regulatory agencies have strengthened oversight by issuing guidance, risk-assessment frameworks, and acceptable intake limits. In particular, the EMA / CMDh nitrosamine Q & A document is regularly updated to include newly identified nitrosamines and their corresponding acceptable intake limits. 10
Early-stage evaluations should consider API structure, nitrosationprone functional groups, degradation pathways, polymorphism, moisture sensitivity, formulation composition and process conditions. Once established, mitigation strategies must be supported by process validation, inprocess controls, analytical monitoring, specification setting, and ongoing quality management.
Effective mitigation requires a lifecycle approach combining process validation, analytical monitoring, in-process controls, specification setting and continuous quality management to ensure product safety and compliance. CDMOs like Seqens can provide expertise and cuttingedge instruments to support the drug substance’ s lifecycle, including risk assessment to nitrosamines screening and control of routine production, within cGMP compliance.
You should evaluate during preformulation studies, potential solid-state vulnerabilities, such as polymorphic forms that may accelerate degradation or moisture uptake, leading to potential nitrosamine impurities. Finally, tackle challenges to identify and quantify at ppb levels NDSRIs in APIs. ●
References: 1: A. Tarafder et al., Org. Process Res. Dev., 2025, 29( 11), 2602 – 2624. 2: M. Sulc et al., Gen. Physiol. Biophys., 2010, 29, 175 – 185. 3: J. Schlingemann et al., J. Pharm. Sci., 2023, 112( 5), 1287 – 1304. 4: W. Wichitnithad et al., ACS Chem. Health Saf., 2026, 33( 3), 350 – 370. 5: G. Eisenbrand et al., Nitrosation of Bromhexine: The Significance of N-Nitrosation of Drugs, Gustav Fischer Verlag, Stuttgart, 1990, pp. 181 – 196. |
6: European Medicines Agency( EMA), Nitrosamine Impurities: Guidance for Marketing Authorisation Holders. https:// www. ema. europa. eu / en / human-regulatory-overview / post-authorisation / pharmacovigilance-post-authorisation / referralprocedures-human-medicines / nitrosamineimpurities / nitrosamine-impurities-guidancemarketing-authorisation-holders 7: European Medicines Agency( EMA), ICH Guideline M7( R1): Assessment and Control of DNA Reactive( Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. https:// www. ema. europa. eu / en / documents / |
scientific-guideline / ich-guideline-m7r1- assessment-controldna-reactive-mutagenicimpurities-pharmaceuticals-limit _ en. pdf 8: K. Moorthy Manchuri et al., Chem. Res. Toxicol., 2024, 37( 9), 1456 – 1483. 9: W. Wichitnithad et al., Saudi Pharm. J., 2024, 31( 2), 295 – 311. 10: EMA / CMDh, Questions & Answers for Marketing Authorisation Holders / Applicants on the CHMP Opinion for the Article 5( 3) of Regulation( EC) No. 726 / 2004 Referral on Nitrosamine Impurities in Human Medicinal Products, including Annexes 1 – 3, current version, January 2024. |
Juliette Martin
SCIENTIFIC LIAISON MANAGER
SEQENS
k + 33( 0) 6 78 28 62 76 J juliette. martin @ seqens. com j www. seqens. com
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