HRAM LC-MS method for the determination of nitrosamine impurities in drugs

Significance of the Topic

Nitrosamines are low molecular weight chemicals recognized as probable human carcinogens and have been detected as impurities in various pharmaceuticals. Regulatory agencies, including the US FDA and EMA, have set strict limits for daily intake of nitrosamines and initiated product recalls, driving the need for reliable analytical methods. Accurate quantification of these genotoxic impurities in drug substances, products, and excipients is essential for patient safety and regulatory compliance.

Goals and Study Overview

This application note describes the development and validation of a high resolution accurate mass (HRAM) LC-MS method to detect and quantify nine nitrosamines in ranitidine drug substance and product. The approach uses a single analytical run on an Orbitrap Exploris 120 mass spectrometer combined with a Vanquish Horizon UHPLC system and compliance-ready Chromeleon CDS software to achieve sensitivity below acceptable daily intake limits.

Methodology and Instrumentation

• Sample Preparation: Calibration standards prepared in methanol and excipient matrix mimicking ranitidine formulation; ranitidine substance and tablet extracts spiked with internal standards.
• Extraction Protocol: Shaking, centrifugation, and filtration steps optimized to maximize recovery from complex matrices.
• Chromatography: Acclaim Polar Advantage II column (100 × 2.1 mm, 2.2 μm), water and methanol mobile phases with 0.1% formic acid, 0.5 mL/min flow rate, 12-minute gradient.
• Mass Spectrometry: APCI source in positive mode, targeted SIM and MS2 acquisition at 120,000 resolution, fast scan speed, mass tolerance 3 ppm.
• Data System: Thermo Scientific Chromeleon 7.2.10 CDS for acquisition and processing, compliant with 21 CFR Part 11 and EU Annex 11.

Main Results and Discussion

• Recovery and Precision: Extraction recoveries between 95% and 105% for all nine nitrosamines with RSD below 10%.
• Sensitivity: Limits of detection (LOD) from 0.1 to 0.5 ng/mL and quantitation (LLOQ) as low as 0.1 ng/mL, corresponding to ppb levels in drug extracts.
• Linearity: Calibration curves linear (R² > 0.99) across 0.1–50 ng/mL in both neat and excipient matrices.
• Matrix Effects: Comparable performance in excipient and neat solutions, demonstrating method robustness.
• Product Analysis: NDMA levels in ranitidine substance and tablets exceeded regulatory limits (ppb to ppm range), highlighting the need for routine monitoring.
• Interference Avoidance: High mass resolution and tight mass tolerance essential to distinguish NDMA from co-eluting DMF isotopes and prevent overestimation.

Benefits and Practical Applications of the Method

• Single-run detection and quantitation of nine nitrosamines.
• Compliance-ready workflow for cGMP and regulated laboratories.
• Sufficient sensitivity to meet current and anticipated regulatory limits.
• Adaptable to other drug products and excipients for broader impurity control.

Used Instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific Acclaim Polar Advantage II column
• Thermo Scientific Orbitrap Exploris 120 mass spectrometer with APCI source
• Thermo Scientific Chromeleon 7.2.10 Chromatography Data System

Future Trends and Opportunities

As regulatory scrutiny of nitrosamine impurities expands, future developments may include:

• Extension to additional nitrosamine species and other drug classes.
• Automation and high-throughput workflows for large-scale screening.
• Integration of real-time monitoring and advanced data analytics.
• Enhanced software tools for automated interference detection and quantitation.

Conclusion

The described HRAM LC-MS method on an Orbitrap Exploris 120 platform offers rapid, sensitive, and selective quantitation of nine nitrosamines in pharmaceutical matrices. With robust extraction, high resolution, and compliance-ready data handling, it meets stringent regulatory requirements and supports quality control efforts to ensure drug safety.

Reference

1. ICH M7(R1): Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk. 2017.
2. EMA/369136/2020: Nitrosamine impurities in human medicinal products, EMA assessment report.
3. US FDA FY19-177-DPA-S: LC-HRMS method for NDMA in ranitidine drug substance and product.
4. US FDA FY19-107-DPA-S: LC-HRMS method for six nitroso impurities in ARBs.
5. US FDA FY20-106-DPA-S: LC-ESI-HRMS method for nitrosamines in metformin.
6. US FDA Guidance for Industry: Control of Nitrosamine Impurities in Human Drugs. 2020.
7. Yang H, et al. A cautionary tale: quantitative LC-HRMS procedures for NDMA in metformin, AAPS Journal, 2020, 22:89.