Nitrosamine Impurities Application Guide - Confidently Detect and Quantify Mutagenic Impurities in APIs and Drug Products

Importance of the Topic

N-Nitrosamine impurities are potent mutagens and carcinogens that may form at trace levels during the manufacturing, storage, or handling of active pharmaceutical ingredients (APIs) and finished drug products. Regulatory bodies worldwide have issued guidance and recalls for sartan-based antihypertensives, ranitidine, and metformin following the detection of NDMA, NDEA, and other nitrosamines. Sensitive, accurate analytical methods are critical to ensure patient safety and regulatory compliance.

Study Objectives and Overview

This application guide demonstrates robust workflows for the confident detection and quantification of major nitrosamines—NDMA, NDEA, NEIPA, NDIPA, NDBA, and NMBA—in APIs and drug products. It covers both gas chromatography–mass spectrometry (GC/MS and GC/MS/MS) and liquid chromatography–tandem mass spectrometry (LC/MS/MS and LC/Q-TOF) approaches, tailored to sartan drugs, ranitidine formulations, and metformin matrices. The aim is to meet or exceed FDA, EMA, and other global regulatory limits (ppb level).

Methodology and Instrumentation

Two complementary platforms are described:

• GC/MS and GC/MS/MS Workflows
  • Instruments: 8890 GC with 7697A Headspace Sampler and 5977B GC/MSD or 7693 LS with 7010B GC/TQ
  • Column: J&W VF-WAXms or DB-WAX, 30 m × 0.25 mm
  • Headspace or direct injection, pulsed splitless mode, helium carrier
  • MRM transitions optimized for each nitrosamine with Retention Time Locking
• LC/MS/MS and LC/Q-TOF Workflows
  • Instruments: 1290 Infinity II LC coupled to 6470 or Ultivo Triple Quadrupole, or 6546 Q-TOF
  • Columns: InfinityLab Poroshell HPH-C18 or Poroshell HPH C18
  • Gradient and isocratic conditions adjusted to resolve nitrosamines ahead or after the API peak
  • MRM or high-resolution accurate mass for confirmatory analysis

Sample preparation typically consists of:

• API spiking with isotopically labeled internal standards (e.g., NDMA-C13-D6)
• Extraction with dichloromethane (GC workflows) or dilute aqueous/organic mixtures (LC workflows)
• Vortexing, centrifugation, and filtration through 0.2–0.45 µm syringe filters

Main Results and Discussion

Limit of quantitation (LOQ) for each nitrosamine was established at or below regulatory thresholds (0.005–0.025 ppm for GC/MS; 0.05 ppm to 0.4 ppm for LC/MS). Calibration curves exhibited excellent linearity (R2 ≥ 0.990–0.998). Signal-to-noise ratios at LOQ exceeded 10, and precision (%RSD) across six injections was ≤ 5–10%. Recoveries in spiked API samples ranged from 86% to 114% across all analytes and platforms, demonstrating method accuracy and ruggedness. Chromatographic runs were completed within 12–20 minutes per sample, enabling medium-throughput QC testing.

Benefits and Practical Applications

These validated workflows offer:

• Regulatory alignment: Methods adhere to FDA, EMA, EDQM, and other guidance.
• Flexibility: A single GC/MS/MS or LC/MS/MS method can analyze multiple nitrosamines without modification for different sartan APIs or ranitidine/metformin matrices.
• High sensitivity: Achieve low-ppb detection consistent with stringent health-based limits.
• Ease of implementation: Standardized sample prep, automated tuning, and mass spectrometer operation streamline adoption in QC laboratories.
• Data integrity: MassHunter software supports efficient quantitation and audit trails.

Future Trends and Opportunities

Emerging developments include:

• High-resolution MS workflows (LC/Q-TOF) for broader screening of unknown nitrosamine variants.
• Automation of sample preparation and online extraction to boost throughput.
• Integration with data-driven quality systems and real-time monitoring for continuous process verification.
• Expansion of methods to additional drug classes and combination formulations.

Conclusion

Robust GC/MS and LC/MS/MS workflows are now available to detect and quantify mutagenic nitrosamine impurities at trace levels in APIs and drug products. These methods fulfill regulatory requirements and help pharmaceutical manufacturers maintain patient safety and compliance. The versatility and sensitivity of the described Agilent solutions support current needs and can adapt to future analytical challenges.

Reference

• FDA Press Releases on nitrosamine impurities
• EMA Press Releases and EDQM guidance documents