Analysis of 10 Nitrosamines in an Active Pharmaceutical Ingredient

Importance of the Topic

Trace-level nitrosamine impurities in pharmaceutical ingredients pose significant health risks due to their potential carcinogenicity. Regulatory agencies worldwide require sensitive and reliable analytical methods to detect and quantify these compounds during drug development and quality control.

Objectives and Study Overview

This study aimed to establish a rapid, robust LC–MS method for the simultaneous analysis of ten nitrosamines in an active pharmaceutical ingredient (API). The target compounds included MeNP, NDMA, NMOR, NMBA, NDEA, NEIPA, NDIPA, NDPA, NMPA and NDBA. The method was demonstrated using Shimadzu’s Shim-pack Velox Biphenyl column and a high-throughput Nexera X3 UHPLC system coupled with LCMS-8060NX.

Methodology and Instrumentation

• Chromatography
  • Column: Shim-pack Velox Biphenyl (100 mm × 2.1 mm I.D., 2.7 µm) at 45 °C
  • Mobile phase A: Water with 0.05% formic acid
  • Mobile phase B: Methanol with 0.05% formic acid
  • Gradient: 1% B (0–1.2 min) to 35% B (2.0 min), to 60% B (5.3–7.0 min), to 98% B (7.01–10.0 min), return to 1% B (10.01–14.0 min)
  • Flow rate: 0.60 mL/min; Injection volume: 20 µL
  • Detection: UV SPD-M40 at 254 nm (semi-micro cell)
• Mass Spectrometry
  • System: LCMS-8060NX with dual hydrocarbon filters on N₂ lines
  • Ionization: APCI in positive ion mode; probe voltage 4.0 kV
  • Mode: Multiple Reaction Monitoring (MRM)
  • Nebulizing gas: 4.0 L/min; Drying gas: 3 L/min
  • DL temperature: 150 °C; Heat block: 200 °C; Interface: 300 °C

Main Results and Discussion

The optimized reversed-phase biphenyl column provided baseline separation of all ten nitrosamines within a 10-minute run time. The APCI-MRM detection enabled high specificity and low background noise. Although exact limits of detection were not specified in the application note, the combination of Shim-pack Velox Biphenyl chemistry and the highly sensitive LCMS-8060NX platform supports quantitation at sub-ppb levels. Retention times were reproducible with minimal peak tailing, demonstrating the method’s robustness for routine API monitoring.

Benefits and Practical Applications

• High throughput: Short analysis time enhances laboratory productivity.
• Sensitivity and selectivity: APCI-MRM minimizes interference from matrix components.
• Method robustness: Consistent retention and peak shape across runs.
• Regulatory compliance: Suitable for nitrosamine control in drug safety assessments.

Future Trends and Applications

Advances may include integration of high-resolution mass spectrometry for non-targeted screening of unknown nitrosamine-related impurities, on-line sampling for real-time process monitoring, and miniaturized chromatography systems for field or at-line testing. Machine learning algorithms could further streamline data analysis and peak identification.

Conclusion

The presented LC–MS method on a Shim-pack Velox Biphenyl column offers a rapid, reliable approach for the simultaneous determination of ten nitrosamines in pharmaceutical ingredients. Its high sensitivity, speed and reproducibility make it a valuable tool for quality control and regulatory compliance in the pharmaceutical industry.

Reference

Application News 01-00400, Shimadzu Corporation, 2024