Determination of Genotoxic Nitrosamine Impurity in Bumetanide API and Tablets Using the Agilent 6470 Triple Quadrupole LC/MS

Significance of the Topic

The presence of genotoxic nitrosamine impurities in pharmaceuticals poses a significant health risk and is subject to strict regulatory limits to protect patients from potential carcinogenic effects. Effective detection and quantitation of N-nitroso Bumetanide impurity at trace levels is essential for ensuring drug safety and regulatory compliance.

Objectives and Study Overview

This application note describes the development and validation of a highly sensitive LC–MS/MS assay using multiple reaction monitoring (MRM) and triggered MRM (tMRM) to quantify N-nitroso Bumetanide in both API and low-dose tablet formulations. Critical challenges included chromatographic separation from high-excipient matrices, mitigation of matrix effects, and avoidance of false-positive confirmation.

Methodology and Instrumentation

• Sample Preparation: Protocols for API, placebo, and tablet matrices involving sonication, centrifugation, and PVDF filtration to extract analyte.
• Chromatography Conditions: Agilent 1290 Infinity II LC with Poroshell HPH-C18 column (3×150 mm, 4 μm), mobile phases 0.5 % formic acid in water (A) and methanol:0.5 % formic acid (95:5) (B), flow rate 0.5 mL/min, injection volume 20 μL, column temperature 30 °C, gradient elution program.
• Mass Spectrometry: Agilent 6470 Triple Quadrupole LC/MS with AJS ESI source in positive mode. MRM transitions 394.1→321.0 and 394.1→240.0 m/z, optimized fragmentor voltages and collision energies, source gas temperature 325 °C, gas flows of 12 L/min.
• Data Acquisition: Agilent MassHunter software for MRM transition optimization, 1/X^2 weighted calibration over 0.1–100 ng/mL, and confirmatory tMRM spectra compared against impurity standards.

Key Results and Discussion

• Chromatographic separation achieved impurity retention at ~13.9 min and effective diversion of the API to waste at ~15.5 min, minimizing matrix interferences.
• Calibration curve exhibited linearity across three orders of magnitude (0.1–100 ng/mL) with R²=0.9957 and signal-to-noise ratio >120:1 at the 0.1 ng/mL LOQ.
• Recovery studies in API and tablet matrices showed recoveries between 89.8 % and 118.5 %, demonstrating accuracy in complex formulations.
• Reproducibility was confirmed with a %CV of ~5 % for eight injections at 1 ng/mL and consistent retention times across batches.
• Triggered MRM provided matching product ion spectra (>99.9 % library match) in both API and tablet samples, ensuring reliable qualitative confirmation.

Benefits and Practical Applications

This method delivers rapid, robust, and high-throughput quantitation of genotoxic nitrosamine impurities in drug substances and formulations. The combination of MRM and tMRM enables sensitive detection, reliable confirmation, and compliance with regulatory guidelines for impurity control in pharmaceutical quality assurance.

Future Trends and Potential Applications

Advances in high-resolution mass spectrometry, automated sample preparation, and integrated data analytics will further improve sensitivity, throughput, and multi-analyte screening capabilities. Real-time monitoring and expanded impurity libraries may streamline QC workflows and strengthen regulatory compliance.

Conclusion

The Agilent 6470 triple quadrupole LC/MS method employing MRM and tMRM offers a powerful solution for the quantitation and confirmation of N-nitroso Bumetanide impurity in API and tablet formulations. It combines excellent sensitivity, accuracy, and reproducibility to meet stringent regulatory standards.

References

• Ward A, Heel RC. Bumetanide: A Review of Its Pharmacodynamic and Pharmacokinetic Properties and Therapeutic Use. Drugs. 1984;28(5):426–464.
• Tata NPV et al. Analytical Profiles of Drug Substances and Excipients. Anal Profiles Drug Subst. 1993;22:107–144.
• Gupta A et al. Genotoxic Impurities: An Important Regulatory Aspect. Asian J Pharm Clin Res. 2020;13(6):10–25.
• FDA Guidance. Development and Validation of a RapidFire-MS/MS Method for Screening of Nitrosamine Impurities.
• USFDA. Liquid Chromatography-High Resolution Mass Spectrometry Method for NDMA in Ranitidine Drug Substance and Product.
• Mani C, Banerjee S. Determination of Nitrosamine Impurities Using the Ultivo Triple Quadrupole LC/MS. Agilent Technologies App Note. 2019;5994-1383EN.
• Covert K. How to Catch a Potential Mutagenic Impurity. Agilent Technologies App Note. 2019;5994-0864EN.