Quantitation of N-Nitroso Nebivolol in Nebivolol Tablet Formulation

Importance of the Topic

N-Nitroso Nebivolol is classified as a Class 4 nitrosamine impurity by the FDA, with a strict intake limit of 1,500 ng/day. As Nebivolol is widely prescribed for hypertension and heart failure, accurate quantitation of this genotoxic impurity at sub-ppb levels is critical for patient safety and regulatory compliance.

Study Objectives and Overview

This study aimed to develop and validate a highly sensitive, specific, and reproducible analytical method to quantify N-Nitroso Nebivolol in 20 mg Nebivolol tablets. Utilizing an Agilent 1290 Infinity II LC coupled to a 6495D triple quadrupole MS, the method targets a specification limit of 22.5 ng/mL at 0.6 mg/mL sample concentration, in alignment with FDA guidelines.

Methodology

Chromatography was performed on an Agilent Pursuit XRs 3 µm Diphenyl column (150 × 3 mm) using a water mobile phase containing 1 mM ammonium trifluoroacetate and 0.004% formic acid (A) and methanol (B). A gradient elution (55 % A to 15 % A over 16 min, then to 0 % A by 21 min) at 0.5 mL/min and 40 °C provided ~10 min separation between the API and impurity. Sample preparation involved extraction of tablet powder at 0.6 mg/mL in acetonitrile:water (1:1), vortex mixing, rotational shaking, and centrifugation. A diverter valve directed the high-concentration API to waste, minimizing matrix effects during impurity quantitation.

Applied Instrumentation

• Agilent 1290 Infinity II LC system with high-speed pump (G7120A), multisampler (G7167B), multicolumn thermostat (G7116B), and diode array detector (G7117A).
• Agilent 6495D triple quadrupole LC/MS with Jet Stream AJS ion source (G1958B).
• Agilent MassHunter Acquisition 12.0; Qualitative Analysis 12.0; Quantitative Analysis 12.1 with Optimizer tool.

Main Results and Discussion

The method achieved an LOD of 2 pg/mL (S/N > 20) and LOQ of 5 pg/mL (S/N > 50) for N-Nitroso Nebivolol. Calibration linearity from 5 pg/mL to 50 ng/mL demonstrated R² > 0.997 (1/x weighting). Spike recovery at 200 pg/mL yielded 116.8 % in API, 108.3 % in placebo, and 111.8 % in tablet samples. Six replicate injections at LOQ showed %RSD ≈ 3.1 %, confirming method reproducibility.

Benefits and Practical Applications

• Enables sub-ppb detection of nitrosamine impurities to meet stringent regulatory expectations.
• Diverter valve integration reduces API interference, improving assay specificity.
• Robust precision and recovery support routine quality control and batch release testing.

Future Trends and Possibilities

• Adaptation of this workflow for other nitrosamine impurities across diverse pharmaceutical formulations.
• Automation of sample preparation and data processing for high-throughput laboratories.
• Potential integration of high-resolution MS techniques to further enhance selectivity and structural confirmation.

Conclusion

The validated triple quadrupole LC/MS method on the Agilent 6495D system delivers rapid, sensitive, and reproducible quantitation of N-Nitroso Nebivolol in tablet formulations, ensuring compliance with safety thresholds for nitrosamine impurities.

Reference

• Prasanth Joseph, Saikat Banerjee et al. Quantitation of N-Nitroso Nebivolol in Nebivolol Tablet Formulation Using Agilent 6495D Triple Quadrupole LC/MS. Agilent Technologies Application Note 5994-8053EN, 2025.
• Wu L., Bertram L., Chan S.-A. Determination of Nitrosamine Impurities Using the Agilent 6475 Triple Quadrupole LC/MS System. Agilent Technologies Application Note 5994-5919EN, 2023.