A Robust and Sensitive Instrument for Quantification of N-nitroso Morpholine impurity in Mycophenolate Mofetil HCl Drug Product

Importance of the Topic

Mycophenolate mofetil is widely used as an immunosuppressive agent to prevent organ rejection in transplant patients. Monitoring trace-level genotoxic impurities such as N-nitroso morpholine is critical to ensure patient safety, as these compounds may form during synthesis or storage and pose carcinogenic risks when exceeding regulatory thresholds.

Objectives and Study Overview

This study aimed to develop a sensitive, selective, and robust analytical method for quantifying N-nitroso morpholine impurity in Mycophenolate mofetil HCl drug product. Key challenges included the close chromatographic elution of the impurity and the active pharmaceutical ingredient (API), necessitating optimized separation and detection conditions.

Methodology and Instrumentation

The method combined ultrahigh-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) using the following setup:

• Waters Xevo TQ-S Cronos triple quadrupole mass spectrometer
• Waters Acquity UPLC H-Class Plus system
• Acquity UPLC BEH C18 analytical column
• RADAR acquisition mode to collect both full-scan and multiple reaction monitoring (MRM) data simultaneously

Chromatographic parameters were fine-tuned to achieve baseline separation. A RADAR scan facilitated rapid method development by revealing matrix effects and confirming the elution order of API and impurity.

Key Results and Discussion

The method achieved:

• Method limit of quantification (LOQ): 0.03 ppm
• Instrument LOQ: 0.003 ppm
• Signal-to-noise ratio > 100 at 0.015 ppm
• Linear dynamic range: 0.015–0.75 ppm
• Spiked recovery: 85 %

RADAR-enabled chromatograms demonstrated clear separation of N-nitroso morpholine from the API, and diverting the API peak prevented MS contamination, enhancing robustness.

Benefits and Practical Applications

This approach provides pharmaceutical quality control laboratories with a reliable tool for trace impurity profiling. It ensures compliance with regulatory guidelines, minimizes false negatives due to coelution, and supports accelerated method development workflows.

Future Trends and Opportunities

Emerging directions include integrating high-resolution mass spectrometry and machine learning for automated peak deconvolution, adopting green solvent systems to reduce environmental impact, and expanding RADAR-like strategies for broader impurity screening across diverse drug matrices.

Conclusion

A robust UHPLC-MS/MS method leveraging the Xevo TQ-S Cronos and RADAR acquisition mode successfully quantifies N-nitroso morpholine at sub-ppm levels in Mycophenolate mofetil HCl. The validated protocol offers exceptional sensitivity, selectivity, and operational efficiency for pharmaceutical impurity analysis.