Highly Sensitive Quantification of Mutagenic NDSRI N-Nitroso Propranolol in Propranolol API and 40 mg Tablets Using LC/MS/MS

Significance of the Topic

Nitrosamine impurities, classified as mutagenic and genotoxic “cohorts of concern” under ICH M7 guidelines, have triggered numerous global pharmaceutical recalls since 2018. Among these, nitrosamine drug substance related impurities (NDSRIs) posed a recent safety issue in propranolol products, necessitating extremely sensitive analytical methods to ensure patient safety and regulatory compliance.

Objectives and Study Overview

This study aimed to develop and validate a highly sensitive LC/MS/MS method for quantifying N-Nitroso Propranolol in propranolol API and 40 mg tablet formulations. The target was a limit of quantitation (LOQ) of 25 pg/mL (0.005 ppm relative to 5 mg/mL API), meeting or exceeding emerging regulatory expectations.

Applied Methodology and Instrumentation

Sample Preparation:

• API: 25 mg propranolol API dissolved in 5 mL methanol diluent, vortexed and sonicated, then analyzed directly.
• Tablets: Crush tablets to equivalent API mass, extract in methanol with extended shaking, centrifuge, and filter.

Chromatography:

• UHPLC system: Agilent 1290 Infinity II (pump, autosampler at 5 °C, column thermostat at 40 °C).
• Column: InfinityLab Pursuit Diphenyl XRS 3.0 × 150 mm, 2.7 µm.
• Mobile phases: A = 5 mM ammonium acetate; B = 0.1% formic acid in methanol; gradient 90:10 to 10:90 over 15 min; flow 0.4 mL/min.

Mass Spectrometry:

• Instrument: Agilent 6470 Triple Quadrupole with Agilent Jet Stream ESI source.
• Mode: Positive MRM; quantifier transition m/z 289.1 → 259.2, qualifier with adjusted collision energy.
• Source settings: Gas temp 200 °C, gas flow 10 L/min, sheath gas temp 350 °C, sheath gas flow 12 L/min, capillary 5,500 V.

Main Results and Discussion

Limit of Detection (LOD) and Quantitation (LOQ): Achieved LOD of 7.5 pg/mL (0.0015 ppm) and LOQ of 25 pg/mL (0.005 ppm) with signal-to-noise ratios >30 and >100, respectively.

Linearity and Precision:

• Calibration range: 0.025–10 ng/mL (0.005–2 ppm); R² > 0.995 with 1/x² weighting.
• Reproducibility: RSD ≤ 3% at LOQ and ≤ 2.6% at 0.03 ppm levels across replicates and bracketing standards.

Accuracy and Recovery:

• API matrix: 100.6% recovery at 0.15 ng/mL (0.03 ppm).
• Tablet matrix: 99.4% recovery at 0.6 ng/mL (0.12 ppm).

Specificity: Demonstrated baseline separation from a forced-degradation N-formyl propranolol impurity (m/z 288), confirming no coelution or MRM interference.

Benefits and Practical Application

This method delivers sub-picogram sensitivity for N-Nitroso Propranolol, supporting robust QA/QC workflows in API release and tablet stability testing. Its high specificity and throughput enable routine screening to meet stringent regulatory limits for genotoxic impurities.

Future Trends and Opportunities

Potential developments include automated high-throughput sample preparation, extension of the workflow to other NDSRIs across diverse pharmaceutical matrices, and integration with online diversion systems to protect MS detectors from high-concentration API eluents.

Conclusion

A validated LC/MS/MS approach using Agilent 6470 LC/TQ achieves exceptional sensitivity, precision, and specificity for N-Nitroso Propranolol in propranolol API and tablets. The method aligns with ICH M7 safety guidelines and supports ongoing efforts to control genotoxic impurities in drug products.

Used Instrumentation

• UHPLC: Agilent 1290 Infinity II (pump G7120A, multisampler G7167B, column thermostat G7116B).
• Column: InfinityLab Pursuit Diphenyl XRS, 3.0 × 150 mm, 2.7 µm.
• Mass Spectrometer: Agilent 6470 Triple Quadrupole LC/MS/MS with Agilent Jet Stream ESI.
• Software: Agilent MassHunter Acquisition and Quantitative Analysis v10.1.

References

1. ICH Harmonised Guideline M7 (2018) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals.
2. US FDA (2018–2022) Updates and Press Announcements on Angiotensin II Receptor Blocker Recalls.
3. US FDA (2021) NDMA in Metformin Safety Updates and Recalls.
4. US FDA (2020) Nitrosamine Contamination in Ranitidine (Zantac) Guidance and Recalls.
5. US FDA (2021) Nitrosamine Impurities in Varenicline (Chantix) Press Announcements.
6. EDQM (2021) Nitrosamine Contamination Overview. European Directorate for the Quality of Medicines.
7. US FDA (2021) Updates on Nitrosamine Drug Substance Related Impurities Recalls.
8. Government of Canada (2021) Recall Notice: Inderal (Propranolol) Tablets Due to N-Nitroso Propranolol Impurity.