Quantitation of N-nitroso propranolol (NOP) in Propranolol formulation and its placebo using LC-MS/MS

Significance of the Topic

The presence of N-nitrosamine impurities in pharmaceutical products poses a critical safety concern due to their potent carcinogenic potential. Regulatory agencies have recalled several drug products after detecting low-molecular-weight N,N-dialkylnitrosamines. N-nitroso propranolol (NOP), a genotoxic impurity related to propranolol API, requires highly sensitive analytical methods for trace-level detection to ensure drug safety and compliance.

Objectives and Study Overview

This study aims to develop and validate a robust LC-MS/MS assay for quantifying NOP in propranolol formulations and placebo matrices. The method targets trace concentrations down to single-digit parts per billion, offering reliable quantitation for both quality control and regulatory monitoring.

Methodology

A targeted multiple reaction monitoring (MRM) approach was optimized for NOP on a Shimadzu UHPLC coupled to a triple quadrupole mass spectrometer. Chromatographic separation employed a C8 column with a gradient of 0.1 % formic acid in water and methanol at 0.5 mL/min. Linearity was established over 10–4000 ppb with a coefficient of determination (r2) of 0.999. Detection and quantitation limits were determined as 2 ppb and 10 ppb, respectively, based on signal-to-noise criteria. Sample preparation involved weighing 25 mg of API, formulation or placebo, spiking with NOP standard, dilution, centrifugation and filtration prior to a 30 µL injection.

Použitá instrumentace

• UHPLC system: Shimadzu Nexera X3
• Mass spectrometer: Shimadzu LCMS-8060NX triple quadrupole
• Ionization: APCI; Interface 350 °C; Desolvation line 200 °C; Heater block 200 °C
• Column: Shim-pack Scepter C8-120, 3 × 150 mm, 5 µm
• Mobile phases: 0.1 % formic acid in water (A) and methanol (B)
• Gradient: 50–100 % B over 3–8 min, hold at 100 % until 13 min, re-equilibration to 50 % by 16 min
• Injection volume: 30 µL

Main Results and Discussion

Calibration yielded a linear equation y = 36769.33 x + 428.53 (r2 = 0.999). Method precision at LOQ showed %RSD below 8 %. Analysis of two formulation lots revealed high NOP levels (3967.9 and 3640.6 ppb), while placebo samples contained 9.0 and 16.3 ppb. Recovery experiments in placebo at 10 ppb returned 104 % and at 20 ppb 119 %, demonstrating method accuracy and robustness.

Benefits and Practical Applications

• Enables trace-level monitoring of carcinogenic nitrosamines in pharmaceuticals
• Supports quality assurance and regulatory compliance
• Applicable to both formulation and placebo matrices for routine screening

Future Trends and Potential Uses

Advancements in detector sensitivity and noise reduction will further lower detection limits. Integration with high-throughput and in-line process analytical technologies is expected. Expansion of this approach to other nitrosamine-related impurities and diverse drug matrices will broaden its applicability.

Conclusion

The validated LC-MS/MS method provides a sensitive, precise and accurate tool for quantifying N-nitroso propranolol at trace levels. Its performance characteristics meet stringent regulatory requirements, making it ideal for pharmaceutical safety surveillance.

Reference

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