Analysis of N-nitroso-nebivolol, Nitroso Drug Substance Related impurity in Nebivolol 20 mg tablet formulation using LC-MS/MS system

Significance of the Topic

The quantification of N-nitroso-nebivolol, a genotoxic impurity classified by the FDA with a stringent intake limit of 1,500 ng/day, is essential for ensuring patient safety and meeting regulatory standards in hypertension medication quality control.

Objectives and Study Overview

This study aimed to develop and validate a highly specific, sensitive, and reproducible LC-MS/MS method for the determination of N-nitroso-nebivolol in a 20 mg nebivolol tablet formulation. Key goals included achieving sub-picogram detection limits, robust quantitation across a wide calibration range, and clear separation of the impurity from the active pharmaceutical ingredient (API).

Employed Methodology and Instrumentation

• Chromatographic system: Agilent 1290 Infinity III UHPLC coupled to an Agilent 6495D triple quadrupole mass spectrometer.
• Column: Agilent Pursuit XRs 3 µm Diphenyl, 150 mm × 3 mm.
• Mobile phases: A) 1 mM ammonium trifluoroacetate + 0.004 % formic acid in water; B) Methanol; flow rate 0.5 mL/min; injection volume 10 µL.
• MS conditions: Jet Stream ESI source in positive mode; MRM transition m/z 435.2 → 254.0 selected for impurity detection.
• Sample preparation: Tablets ground to powder, dispersed to 0.6 mg/mL nebivolol in diluent, vortexed, shaken, and centrifuged. API, placebo, and spiked QC samples processed similarly.

Main Results and Discussion

• Chromatographic separation of impurity and API achieved in approximately 10 minutes using a software-controlled diverter to prevent high-concentration API interference.
• Calibration curve: 13 concentration levels spanning 5 pg/mL to 50 ng/mL with 1/x weighting, yielding R² = 0.997.
• Detection limits: LOD = 2 pg/mL (S/N > 20:1), LOQ = 5 pg/mL (S/N > 50:1).
• Reproducibility: %RSD ~3.1 % for six replicate injections at the LOQ level.
• Spike recovery at 200 pg/mL: 116.8 % in API, 108.3 % in placebo, and 111.8 % in tablet matrix, demonstrating efficient extraction and accuracy.

Benefits and Practical Applications

The validated method supports routine quality control of nebivolol tablets by enabling trace-level determination of a critical genotoxic impurity, ensuring compliance with international safety guidelines and enhancing laboratory throughput.

Future Trends and Potential Applications

• Adaptation for analysis of other nitrosamine impurities across various pharmaceutical formulations.
• Integration with automated sample preparation systems to further improve throughput and consistency.
• Employment of high-resolution mass spectrometry for comprehensive impurity profiling in complex matrices.

Conclusion

A robust and sensitive LC-MS/MS method was successfully established for N-nitroso-nebivolol quantitation in nebivolol tablets, achieving sub-picogram sensitivity, excellent linearity, reproducibility, and recovery, thus meeting stringent regulatory requirements for impurity control.

Reference

• Prasanth J., Banerjee S., Vyas S. Determination of Genotoxic Nitrosamine Impurity in Bumetanide API and Tablets Using the Agilent 6470 Triple Quadrupole LC/MS. Agilent Technologies application note, 5994-2967EN, 2020.