Quantitation of N-Nitroso Carvedilol in Drug Substance Using LC-MS/MS

Importance of the topic

N-nitrosamines are recognized carcinogens that can inadvertently form as impurities or degradation products in pharmaceutical substances, posing critical health risks and regulatory challenges. The detection and quantification of trace levels of these compounds in drug substances are essential for patient safety, compliance with international guidelines, and minimizing the need for batch‐release delays in quality control operations.

Objectives and study overview

This study aimed to develop and validate a robust, sensitive UPLC-MS/MS method for quantifying N-nitroso carvedilol in carvedilol drug substance. Key goals included:

• Achieving chromatographic separation of carvedilol API and its two N-nitroso isomers
• Meeting a lower limit of quantitation (LOQ) at 10 % of regulatory acceptable intake (AI) under the CPCA framework
• Demonstrating linearity, accuracy, precision, and recovery within established pharmaceutical impurity guidelines

Methodology and instrumentation

Samples and standards were prepared by dissolving carvedilol and N-nitroso carvedilol in appropriate solvents, followed by matrix spiking at threshold (0.4 ppm), 10 % threshold (4 ppm), and ten-times threshold (40 ppm) levels. Calibration curves ranged from 0.4 ng/mL to 100 ng/mL using both neat and matrix-matched approaches. Chromatographic separation employed a C18 column with a gradient of 0.1 % acetic acid in water and methanol, diverting the high-concentration API fraction to waste for the initial 9.9 minutes.

Instrumentation

• UPLC System: Waters ACQUITY Arc Premier
• Detector: ACQUITY PDA at 220 nm
• Mass Spectrometer: Xevo TQ-S cronos Triple Quadrupole with ESI source
• Software: MassLynx with Nitrosamine Impurity Quanpedia database and TargetLynx processing

Main results and discussion

Chromatographic conditions successfully resolved carvedilol (RT 2.38 min) from two N-nitroso carvedilol isomers (RT 10.17 min and 10.52 min). The LOQ at 0.4 ng/mL (10 % AI threshold) exhibited S/N ratios above 40 for both isomers. Linearity was demonstrated across 0.4–100 ng/mL (R² > 0.995) with residuals < 15 % under 1/x weighting. Recoveries, corrected for endogenous API levels, ranged 85–110 % with %RSD < 18 %. Matrix effects were below 5 %, and no carry-over was detected after high-level injections.

Benefits and practical applications

• Meets stringent regulatory requirements for N-nitrosamine impurities at trace levels
• Efficient method development leveraging the Quanpedia database for MRM transitions
• Chromatographic resolution of API and isomers reduces matrix interferences in QC workflows
• Total isomer concentrations easily reported via TargetLynx “Totals” function

Future trends and possibilities

Expanding this platform approach to other nitrosamine impurities will accelerate method deployment across diverse APIs. Future enhancements may include automated high-throughput screening, integration of AI-guided method optimization, and broader adoption of CPCA categorization to refine acceptable intake thresholds.

Conclusion

A validated UPLC-MS/MS assay for N-nitroso carvedilol in drug substance satisfies regulatory criteria for sensitivity, accuracy, and precision. The method’s chromatographic selectivity, use of a dedicated nitrosamine database, and seamless data processing provide a robust solution for routine QC monitoring and impurity risk management.

References

1. U.S. Food and Drug Administration. Nitrosamine Impurities in Human Drugs. 2019.
2. FDA. Updates and Press Announcements on ARB Recalls: Valsartan, Losartan. 2019.
3. Cioc R.C., Joyce C., Mayr M., Bream R.N. Formation of N-Nitrosamine Drug Substance Related Impurities in Medicines: A Regulatory Perspective. Regulatory Toxicol. Pharmacol. 2023.
4. European Medicines Agency. Nitrosamine Impurities: Questions and Answers. 2020.
5. EMA. Q&A Document on Nitrosamine Impurities (EMEA/H/A5(3)1490). Oct. 2023.
6. FDA. Updated Information on Acceptable Intake Limits for N-nitrosamine Drug Substance-Related Impurities. Oct. 2023.
7. Burns M.J. et al. Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals. J. Pharm. Sci. 2023; DOI:10.1016/j.xphs.2023.10.001.
8. Journal Article. Carvedilol (Coreg) Nitrosation Study. DOI:10.1080/00397910903531839.