Routine, Ultra-Trace Analysis of Nitrosamines in Drugs using Gas-Chromatography – Orbitrap Mass Spectrometry

Importance of the Topic

The presence of nitrosamine impurities in pharmaceutical products poses a significant health risk due to their mutagenic and carcinogenic potential. Following the discovery of N-Nitrosodimethylamine in Valsartan in 2018, regulatory agencies worldwide have tightened limits and reporting requirements. Achieving reliable ultra-trace quantitation and broad-spectrum screening of nitrosamines is therefore critical for drug safety, regulatory compliance, and protecting public health.

Study Objectives and Overview

This study evaluates a combined static headspace gas chromatography coupled to an Orbitrap high-resolution mass spectrometer for routine analysis of nitrosamine impurities in Valsartan and Metformin. It aims to demonstrate compliance with current FDA guidelines for NDMA, NDEA, and NEIPA quantitation, while also providing an untargeted workflow to screen for other volatile organic contaminants.

Methodology and Instrumentation

Sample Preparation and Calibration:

• Standards for NDMA, NDEA, NEIPA and a deuterated internal standard were prepared in DMSO and headspace-grade DMSO following FDA and USP <467> guidelines.
• Valsartan and Metformin matrices were prepared, blank-checked, and spiked at multiple levels (below LOQ, at LOQ, and above LOQ).

Chromatographic Conditions:

• Static headspace sampling with incubation at 120 °C to prevent artifact formation.
• Separation on a 30 m × 0.25 mm × 0.5 μm TG-WAXMS B capillary column.

Mass Spectrometric Acquisition:

• Orbitrap high-resolution full-scan (60,000 resolution) and single ion monitoring (SIM) modes.
• Quantifier and confirmatory ions selected within ±2 ppm mass accuracy windows.
• Data systems: Xcalibur for untargeted screening and Chromeleon for quantitative analysis.

Instrumentation Used

• Thermo Scientific Exactive GC Orbitrap GC-MS
• Instant Connect Split/Splitless Injector
• TriPlus 500 HS valve and loop static headspace autosampler
• TraceGOLD TG-WAXMS B capillary column (30 m × 0.25 mm × 0.5 μm)
• Thermo Scientific Xcalibur, Freestyle, and Chromeleon CDS software

Key Results and Discussion

Linearity and Sensitivity:

• Calibration curves from 0.05 to 20 μg showed R2 ≥ 0.9998 and residual values (% RSD) below 10.5%.
• Method detection limits (0.01 ppm) and limits of quantitation (0.03 ppm) meet and exceed FDA requirements.

Quantitative Performance in Real Samples:

• Unspiked Valsartan and Metformin blanks showed NDMA and NEIPA traces below reporting limits.
• Spiked recovery ranged from 85% to 112%, with mass accuracy consistently below 1 ppm.

Untargeted Screening:

• Full-scan data searched against NIST 17 library enabled putative identification of volatile impurities (e.g., toluene) based on isotopic pattern and high mass accuracy.

Benefits and Practical Applications

• Combined SIM and full-scan acquisition allows simultaneous targeted quantitation and untargeted contaminant screening in a single injection.
• High resolution Orbitrap detection ensures selectivity and sensitivity at ultra-trace levels.
• Static headspace sampling simplifies preparation and reduces matrix effects for volatile analytes.
• Robust linearity and precision support regulatory submission and routine quality control workflows.

Future Trends and Potential Applications

Expansion of this approach to additional drug classes and broader volatile impurity profiles could further enhance pharmaceutical safety screening. Integration with advanced data-mining and machine learning algorithms may improve untargeted contaminant identification and trend monitoring. Miniaturized sampling techniques and automated workflows will continue to drive throughput and reproducibility in high-volume testing laboratories.

Conclusion

The combination of static headspace GC with Orbitrap high-resolution mass spectrometry provides a powerful solution for ultra-trace determination of NDMA, NDEA, and NEIPA in pharmaceutical products. This workflow meets stringent regulatory criteria while enabling comprehensive screening for other volatile impurities, supporting both compliance and patient safety.

Reference

1. FDA Combined Headspace N-Nitrosodimethylamine NDMA, N-Nitrosodiethylamine NDEA, NEIPA Impurity Assay by GC-MS/MS, 29/04/2019
2. USP <467> Organic Volatile Impurities, United States Pharmacopeia, December 2020
3. EMA Sartan medicines Review to Avoid Nitrosamine Impurities, EMA/44960/2019