Analysis of N-Nitroso-Sertraline in Drug Substance and Tablet Using LC-MS/MS

Importance of the Topic

The presence of N-nitrosamines in pharmaceuticals represents a critical safety and regulatory challenge due to their potent carcinogenic potential even at trace levels. N-nitrosamine impurities can form during drug substance synthesis, formulation, storage or from contaminated excipients. Regulatory agencies worldwide have implemented stringent guidelines to limit patient exposure, requiring analytical methods capable of sensitive, accurate quantification of these genotoxic impurities in both active pharmaceutical ingredients (APIs) and finished dosage forms.

Objectives and Study Overview

This study aimed to develop and validate a robust UPLC-MS/MS assay for the quantitation of N-nitroso-sertraline, a nitrosated derivative of the widely prescribed antidepressant sertraline. Key objectives included:

• Chromatographic separation of sertraline and its nitrosamine impurity to avoid matrix interference.
• Establishment of linearity, limit of quantitation (LOQ), accuracy, precision and carry-over in compliance with FDA and EMA guidelines.
• Application of the method to drug substance and tablet formulations to demonstrate workflow efficiency and robustness.

Methodology and Instrumentation

A Waters ACQUITY UPLC I-Class system equipped with an Atlantis Premier BEH C18 AX column (2.1×100 mm, 2.5 µm) was coupled to a Xevo TQ-S Micro tandem quadrupole mass spectrometer operating in positive ESI mode. Mobile phases were 0.1% formic acid in water (A) and in acetonitrile (B), using a gradient to achieve baseline separation of the API (RT ~3.3 min) and N-nitroso-sertraline (RT ~6.8 min). MRM transitions were initially sourced from the Quanpedia Nitrosamine Impurity Assay database and further optimized using MassLynx Intellistart. Sample preparation involved methanolic extraction of sertraline hydrochloride or tablet matrices, followed by centrifugation and direct injection.

Main Results and Discussion

The method demonstrated:

• LOQ of 0.25 ppb (10% of the regulatory threshold), with signal-to-noise > 10:1.
• Linear dynamic range of 0.25–100 ppb (R² > 0.99, 1/X weighting).
• Recovery of 85–93% at threshold and 10% threshold levels (n=6), with %RSD < 20%.
• No detectable carry-over following a 100 ppb standard injection.
• Quantification in tablet samples yielded 0.49 ppb N-nitroso-sertraline (%RSD < 12%, n=9), above the 10% threshold but below the limit derived from the FDA acceptable intake.
• Robustness over 360 continuous injections across four days (%RSD ~9% for tablet samples), with observed in-vial nitrosation after ~250 injections, highlighting the need for quenching strategies.

Benefits and Practical Applications

The developed UPLC-MS/MS workflow provides:

• Trace-level detection meeting AI-based regulatory limits.
• Efficient method development via Quanpedia database integration.
• High accuracy, precision, and robustness compatible with QC release testing.
• Applicability to both drug substance and finished dosage forms without additional derivatization.

Future Trends and Applications

Further advances may include:

• Integration of in-vial quenching agents (e.g., ascorbic acid) to prevent on-line nitrosation.
• Expansion of MRM libraries to cover emerging nitrosamine impurities across diverse APIs.
• Automation of sample preparation and data processing workflows for high-throughput screening.
• Implementation of high-resolution MS techniques for structural elucidation of unknown N-nitrosamines.

Conclusion

A sensitive and robust UPLC-MS/MS method for N-nitroso-sertraline quantitation was established, fulfilling stringent regulatory requirements. The approach ensures accurate impurity profiling in both API and tablet matrices, supporting pharmaceutical quality control and patient safety.

Reference

1. U.S. Food and Drug Administration. Nitrosamine Impurities in Human Drugs. 2019.
2. FDA Updates and Press Announcements on Angiotensin II Receptor Blocker Recalls: Valsartan, Losartan and Irbesartan. FDA Safety Communications.
3. Cioc R.C., Joyce C., Mayr M., Bream R.N. Formation of N-Nitrosamine Drug Substance Related Impurities in Medicines: A Regulatory Perspective on Risk Factors and Mitigation Strategies.
4. European Medicines Agency. Questions and Answers on Nitrosamine Impurities. 2020.
5. EMA. Updated Information on Acceptable Intake Limits for Nitrosamines. Quality Guidance. October 2023.
6. U.S. Food and Drug Administration. Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities. FDA Guidance. October 2023.
7. Burns M.J., Ponting D.J., Foster R.S., et al. Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals. Journal of Pharmaceutical Sciences. 2023.