Quantitation of N-Nitroso Moxifloxacin in 400 mg Moxifloxacin Tablet Formulation

Significance of the Topic

The presence of N-nitroso moxifloxacin in pharmaceutical products is regulated due to its classification as a potential genotoxic impurity by the FDA, setting a maximum intake of 1,500 ng/day. Achieving reliable detection at trace levels is crucial for patient safety and regulatory compliance.

Objectives and Study Overview

This study aims to develop a highly sensitive and reproducible LC/MS/MS method for quantifying N-nitroso moxifloxacin in 400 mg moxifloxacin tablets. Key goals include low detection limits, robust separation from the active drug, and suitability for routine quality control.

Methodology and Instrumentation

Sample preparation involved crushing tablets, extracting the analyte with acetonitrile–water, and preparing calibration standards from 2.5 pg/mL to 10 ng/mL. The analytical system comprised an Agilent 1290 Infinity II LC with high-speed pump, multisampler, multicolumn thermostat, and diode array detector, coupled to an Agilent 6495D triple quadrupole mass spectrometer. Chromatographic separation used an Agilent Pursuit XRS3 Diphenyl column with an ammonium formate/ammonium fluoride/formic acid buffer and methanol gradient at 0.4 mL/min. MS detection employed positive electrospray ionization targeting the 431.2→232.0 m/z transition with optimized source parameters.

Main Results and Discussion

• Baseline separation of N-nitroso impurity and moxifloxacin APIs with a >1.5 min retention gap and use of a diverter valve to route high-concentration API peaks to waste.
• Limit of detection of 1 pg/mL and limit of quantification of 2.5 pg/mL, corresponding to 0.0006 ppm and 0.0017 ppm in the test matrix.
• Signal-to-noise ratios of 102:1 at LOD and 232:1 at LOQ levels, ensuring confident trace-level detection.
• Calibration demonstrated linearity over four orders of magnitude (2.5 pg/mL–10 ng/mL) with R² > 0.991 (1/x² weighting).
• Average recovery in placebo at the LOQ level was 86.3%, and reproducibility at the LOQ yielded %RSD of 4.7% across seven injections.

Benefits and Practical Applications

The validated method enables precise monitoring of N-nitroso moxifloxacin impurities in tablet formulations, fulfilling regulatory requirements and supporting quality control in pharmaceutical manufacturing. Its high sensitivity and reproducibility make it suitable for routine batch release testing.

Future Trends and Opportunities

Advances may include automation of sample preparation, extension to other nitrosamine impurities, integration with high-resolution mass spectrometry, and application of artificial intelligence for data processing. Miniaturized and high-throughput workflows will further streamline impurity profiling in drug development and production.

Conclusion

The developed LC/MS/MS approach on the Agilent 6495D platform provides a robust, sensitive, and reproducible solution for quantifying N-nitroso moxifloxacin in tablet formulations, meeting stringent regulatory thresholds and ensuring patient safety.

Reference

• Joseph P., Banerjee S., Vyas S. Determination of Genotoxic Nitrosamine Impurity in Bumetanide API and Tablets Using the Agilent 6470 Triple Quadrupole LC/MS; Agilent Technologies Application Note 5994-2967EN, 2020.
• Wu L. et al. Determination of Nitrosamine Impurities Using the Agilent 6475 Triple Quadrupole LC/MS System; Agilent Technologies Application Note 5994-5919EN, 2023.