High-resolution accurate-mass liquid chromatography mass spectrometry (HRAM LC-MS) methodology for the determination and quantitation of nitrosamine impurities in drug products

Importance of the Topic

Nitrosamine impurities in pharmaceuticals are a major safety concern due to their genotoxic and carcinogenic potential. Regulatory bodies such as the FDA and EMA have set strict limits based on the threshold of toxicological concern (TTC) of 1.5 µg per day. Reliable, sensitive, and high-throughput analytical methods are essential for ensuring drug safety and compliance with guidelines.

Objectives and Study Overview

This study aimed to develop a fast, simple, and cost-effective high-resolution accurate-mass liquid chromatography–mass spectrometry (HRAM LC-MS) method, in line with FDA recommendations, for the determination and quantitation of a panel of 11 common nitrosamines in finished drug products and drug substances within a single analytical run.

Methodology and Instrumentation

• Sample Preparation: Drug substances and formulations were dissolved or extracted in methanol to a concentration equivalent to 30 mg/mL of API, followed by vortex mixing or shaking and centrifugation.
• Liquid Chromatography: Separation used an Acclaim PolarAdvantage II column (100 × 2.1 mm, 2.2 µm) at 40 °C with a water/formic acid and methanol/formic acid gradient over a 12-minute run, 0.5 mL/min flow rate, and 2 µL injection volume.
• Mass Spectrometry: Detection employed a Q Exactive Plus quadrupole–Orbitrap instrument in PRM mode at 35 000 resolution, with a 4000 V spray voltage, optimized gas flows, and temperatures for sensitive and selective analysis.

Key Results and Discussion

• Linearity: Calibration curves for all 11 nitrosamines (NDMA, NDEA, NDPA, NMOR, NDBA, NMEA, NEIPA, NDIPA, NPIP, NPYR, NDPhA) were linear (r² > 0.99) over 0.5–100 ng/mL with 1/x weighting.
• Sensitivity and Detection Limits: All compounds were detectable at 0.5 ng/mL (limit of quantitation), with instrument detection limits ranging from 0.05 to 0.50 ng/mL, satisfying FDA reporting thresholds.
• Precision and Accuracy: Intra-batch precision (n=10) showed RSDs between 0.32% and 3.15%, with bias within ±4% at 5 ng/mL quality-control level.
• Real-Sample Testing: Commercial valsartan and aged ranitidine standards revealed NDMA levels of 1.23 ppm and 0.83 ppm respectively; an expired ranitidine sample exhibited NDMA well above the calibration range, confirming method robustness for high-level detection.

Benefits and Practical Applications

• Single-run profiling of multiple nitrosamines improves laboratory efficiency and reduces analysis time.
• High-resolution mass detection ensures confident identification and confirmation of impurities.
• Method aligns with GMP data integrity and 21 CFR Part 11 compliance for regulated environments.

Future Trends and Opportunities

Advances in HRAM technology and data processing are expected to further enhance sensitivity and throughput. Integration with automated sample preparation and multiplexed workflows can support routine screening in quality control laboratories. Expanding the nitrosamine panel and exploring new ionization strategies may address emerging impurities and regulatory requirements.

Conclusion

The developed HRAM LC-MS method offers a rapid, sensitive, and accurate approach for quantifying 11 nitrosamine impurities in drug substances and finished products. It meets regulatory guidelines, demonstrates excellent linearity, precision, and detection limits, and is suitable for routine pharmaceutical quality control.

References

• FDA and EMA guidelines on genotoxic impurities and threshold of toxicological concern.
• USFDA application note on LC-HRMS method for NDMA determination in ranitidine.