Quantitation of N-Nitroso-Propranolol in Drug Substance using LC-MS/MS

Significance of the Topic

Since nitrosamine impurities are known mutagens and carcinogens, their presence in pharmaceuticals poses serious safety and regulatory challenges. The discovery of N-nitrosamines in marketed drugs has driven the need for highly sensitive analytical methods to ensure patient protection and compliance with updated EMA and FDA guidelines.

Objectives and Study Overview

This study aimed to develop and validate an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for quantifying N-nitroso-propranolol in propranolol drug substance. The goal was to achieve a limit of quantitation that meets or exceeds the revised acceptable intake limits established by regulatory authorities.

Methodology and Instrumentation

The chromatographic separation employed a reversed-phase C8 column with a formic acid–water/methanol gradient. Samples were injected in high-organic solvent with an added 50 µL extension loop to preserve peak shape. Detection was performed by electrospray ionization (positive mode) and multiple reaction monitoring transitions were optimized for quantifier and confirmatory ions.

Used Instrumentation

• ACQUITY Premier UPLC Binary System with Flow-Through Needle sample manager
• Xevo TQ-S micro Tandem Quadrupole Mass Spectrometer
• 2998 Photodiode Array Detector
• MassLynx Software with Intellistart autotune and quantitation modules

Main Results and Discussion

The method achieved an authentic standard limit of detection (LOD) of 0.005 ng/mL and limit of quantitation (LOQ) of 0.01 ng/mL. In a 1 mg/mL propranolol matrix, calibration was linear from 0.01 to 100 ng/mL (R2 > 0.998) with <15% deviation. Recoveries ranged from 89.3% to 104.6%, precision was <2% RSD, and accuracy within ±10%. Critical baseline separation of N-formylpropranolol from N-nitroso-propranolol prevented isotope interference in quantifier MRM channels.

Benefits and Practical Applications

• Trace-level detection beyond regulatory thresholds defined by the Carcinogenic Potency Categorization Approach (CPCA).
• Robust quantitation with a broad dynamic range suitable for routine quality control.
• Selective MRM transitions and chromatographic resolution ensure reliable impurity profiling in drug substance release testing.

Future Trends and Opportunities

Future work will extend this workflow to other nitrosamine impurities across diverse APIs. Advances may include high-throughput sample processing, integration of alternative ionization strategies, and in silico potency predictions to streamline safety assessments and regulatory submissions.

Conclusion

A UPLC-MS/MS assay has been successfully established for quantifying N-nitroso-propranolol in propranolol drug substance at sub-ng/mL levels. The method fulfills current EMA and FDA guidelines, offering the sensitivity, selectivity, and robustness required for routine analytical and quality control laboratories.

References

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