High Sensitivity Quantitation of Nitrosamine Genotoxic Impurities: LC-MS Analysis of Ranitidine Drug Product using the Waters ACQUITY UPLC I-Class/Xevo TQ-XS Tandem Quadrupole Mass Spectrometer

Significance of the topic

Nitrosamine impurities, such as NDMA, pose significant genotoxic and carcinogenic risks even at trace levels. Regulatory agencies have set stringent limits for daily intake of these impurities in pharmaceuticals, prompting the need for highly sensitive, accurate, and reproducible analytical methods. This study addresses the challenge of quantifying low-parts-per-billion levels of multiple nitrosamines in a widely used drug product, ranitidine.

Objectives and Study Overview

The primary goal was to develop and validate an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous detection and quantification of six N-nitrosamine impurities (NDMA, NDEA, NEIPA, NDIPA, NDBA, NMBA) in ranitidine drug substance and product. Key objectives included:

• Achieving limits of quantification (LLOQs) below 0.1 ng/mL.
• Ensuring chromatographic separation of polar nitrosamines from the API.
• Demonstrating method robustness, linearity, accuracy, and sensitivity to meet regulatory guidelines.

Methodology and Instrumentation

Sample Preparation:

• Individual nitrosamine stock solutions (5 mg/mL) in methanol; combined working solution at 250 µg/mL.
• Ranitidine drug substance and product prepared at 30 mg/mL in water.
• Calibration standards spiked across 0.025–100 ng/mL range in ranitidine matrices.

Chromatography and Mass Spectrometry:

• Instrument: Waters ACQUITY UPLC I-Class PLUS with a 2.1×100 mm HSS T3 column, 1.8 µm particle size.
• Mobile phase: gradient with ammonium formate buffer to enhance peak shape and reduce noise.
• Flow rate: 0.35 mL/min, benefiting sensitivity over higher flow rates.
• MS: Waters Xevo TQ-XS triple quadrupole with APCI probe (250 °C probe, 130 °C source).
• Ionization: positive APCI provided ~10× sensitivity improvement over ESI.
• Detection: Multiple reaction monitoring (MRM) optimized for each nitrosamine with short dwell times.

Key Results and Discussion

The developed method delivered LLOQs of 0.025–0.1 ng/mL (0.75–3 pg on column) for all six nitrosamines in drug substance, and 0.1 ng/mL in drug product, corresponding to <0.003 ppm relative to a 30 mg/mL dose. Calibration curves exhibited excellent linearity (R²≥0.99, 1/x weighting) and accuracy (85–115% recovery). Chromatographic separation effectively resolved NDMA from ranitidine and co-eluting NMBA. APCI optimization and low-temperature settings minimized in-source fragmentation, boosting signal-to-noise ratios. Analysis of a ranitidine tablet revealed endogenous NDMA levels of approximately 28 ng/mL (~1 ppm), confirmed by dual MRM transitions and standard addition correction.

Benefits and Practical Applications

This UPLC-MS/MS assay offers:

1. Sub-ppt sensitivity for rapid screening of genotoxic nitrosamines.
2. High specificity through optimized MRM transitions and APCI ionization.
3. Reproducible quantification meeting ICH M7 and regulatory safety thresholds.
4. Simplified workflow suitable for routine QA/QC laboratories.

Future Trends and Potential Applications

Future developments may include expanding the scope to additional nitrosamines and other drug matrices, integrating high-resolution mass spectrometry for non-targeted screening, and automating sample preparation in 96-well formats. Continuous refinement of chromatographic and ionization strategies will further lower detection limits and improve throughput.

Conclusion

The presented UPLC-MS/MS method using Waters ACQUITY UPLC HSS T3 and Xevo TQ-XS with APCI enables ultra-sensitive, accurate detection of multiple genotoxic nitrosamine impurities in ranitidine. It satisfies stringent regulatory requirements and can be readily adopted for routine impurity control in pharmaceutical development and manufacturing.

Reference

• FDA safety communications and recalls for ranitidine due to NDMA detection.
• ICH M7(R1) guideline on control of DNA-reactive impurities.
• Application notes and technical resources from Waters Corporation on UPLC and Xevo TQ-XS performance.