Analysis of Genotoxic Impurities of Imatinib Mesylate by LC-MS from Early Development to Routine Monitoring

Significance of the Topic

The accurate detection and control of genotoxic impurities in pharmaceutical drug substances is critical to patient safety and regulatory compliance. Genotoxic impurities can damage DNA, leading to mutations and potential cancer risk. Imatinib mesylate, a key anti cancer therapy, requires stringent monitoring of trace process byproducts with structural alerts for mutagenicity. This study presents sensitive and rapid LC MS approaches to support early stage development and routine quality control of imatinib mesylate.

Objectives and Study Overview

This work aims to develop and compare two mass detection methods for quantifying two genotoxic impurities of imatinib mesylate, MPBA and PNMP. The study focuses on achieving ultra low level detection in early development using a tandem quadrupole Xevo TQ S micro in MRM mode, and demonstrating a robust workflow for late stage and QC environments using a single quadrupole ACQUITY QDa detector. Key performance metrics include limits of detection and quantification, linearity, precision, recovery, and assessment of matrix effects.

Methodology and Instrumentation

Sample Preparation

• Stock solutions of MPBA and PNMP prepared at 1 mg/mL in methanol and mixed to create calibration standards ranging from low ng/mL levels upward.
• Imatinib mesylate API from three vendors diluted to 0.1 mg/mL in a 20 to 80 water methanol diluent.

Chromatographic Conditions

• ACQUITY UPLC H Class system with HSS PFP column (2.1 x 50 mm, 1.8 µm) at 40 deg C.
• Mobile phases: 2.5 mM ammonium formate pH 3.0 and acetonitrile; gradient elution in five steps.
• Flow rate of 0.7 mL/min and 8 µL injection volume.

Detection Mode

• Tandem MS: Xevo TQ S micro in ESI positive mode with MRM transitions 235 134 for MPBA and 278 106 for PNMP, using RADAR to acquire full scan simultaneously.
• Single Quad: ACQUITY QDa detector in ESI positive mode with selective ion recording at m/z 235.2 and 278.2.

Used Instrumentation

• Xevo TQ S micro tandem quadrupole mass spectrometer for ultra sensitive analysis in MRM mode with RADAR acquisition.
• ACQUITY QDa single quadrupole mass detector for routine monitoring in SIR mode.
• ACQUITY UPLC H Class system and HSS PFP column.
• Empower 3 CDS for QDa and MassLynx with TargetLynx for Xevo TQ S micro control and data processing.

Main Results and Discussion

Tandem MS Performance

• Limits of quantification of 0.015 ng/mL for MPBA and 0.1 ng/mL for PNMP, with signal to noise criteria of 10:1 and RSD of peak areas under 9% at LOQ.
• Excellent linearity from LOQ to 10 ng/mL (r2 > 0.999) and recoveries between 90 and 115% in spiked API samples.
• RADAR full scan data demonstrated absence of co elution with background ions, enabling confident quantification.

Single Quadrupole Performance

• LOQ values of 0.26 ng/mL for MPBA and 1.0 ng/mL for PNMP, with RSD of peak areas below 6%.
• Linearity from LOQ to 100 ng/mL (r2 > 0.997) and recoveries between 87 and 115% across three spike levels.
• Method proves suitable for later stage routine QC applications.

Benefits and Practical Applications of the Method

• Early stage ultra sensitive detection supports ALARP reporting as per ICH M7, guiding impurity control strategy.
• Simultaneous full scan and MRM acquisition accelerates identification of unexpected substances and matrix evaluation.
• Integrated UPLC MS workflows reduce solvent consumption and analysis time compared to traditional HPLC UV methods.
• Compliant ready software environments ensure traceable and robust data handling for regulatory submissions.

Future Trends and Potential Applications

The deployment of MS friendly assays across the drug lifecycle will continue to expand as molecules become more complex and present lower UV absorbance. Emerging high throughput sample introduction techniques and advanced data processing algorithms will further enhance trace analysis capabilities. Integration of real time monitoring and continuous purification feedback loops is anticipated, delivering tighter control of genotoxic impurities and improved manufacturing efficiency.

Conclusion

The described UPLC MS approaches deliver sensitive, selective, and robust quantification of two genotoxic impurities in imatinib mesylate. The tandem quadrupole method achieves ultra low level detection for early development and impurity profiling, while the single quadrupole platform provides a streamlined route for routine QC monitoring. Together, these workflows support comprehensive impurity control strategies in compliance with ICH M7 guidelines and contribute to ensuring product safety.

References

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2. Arava V R, Bethi M R, Cherukuri K R, Thota G, Cherukupally S R LC MS MS Method for Determination of Potential Genotoxic Impurities in Imatinib Mesylate Scholars Res Lib 2013 5(6) 47 52
3. Madireddy V, Babu K S, Narayanareddy P Stability indicating Fast HPLC Method for Quantification of Two Genotoxic Impurities in Imatinib Mesylate Global J Anal Chem 2011 2(5) 198 206
4. ICH M7 Assessment and Control of DNA Reactive Mutagenic Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk International Conference on Harmonization 2014