Determination of Nitrosamines in Active Pharmaceutical Ingredients

Importance of the Topic

Genotoxic nitrosamine impurities have been detected in several pharmaceutical products, raising concerns about long-term exposure and carcinogenic risk. Regulatory agencies such as the FDA and EMA enforce strict limits on nitrosamine intake to protect patient safety. Sensitive analytical methods are essential to monitor trace levels of these genotoxic impurities in active pharmaceutical ingredients (APIs) and finished drug products.

Objectives and Study Overview

This application note demonstrates a robust, high-sensitivity LC/MS workflow for simultaneous quantification of multiple nitrosamines in APIs. Using the Agilent 1260 Infinity II Prime LC paired with the Agilent Ultivo LC/TQ, the study establishes limits of detection (LOD), limits of quantification (LOQ), linearity, accuracy, and precision at low ng/mL concentrations, exemplified by analysis of nitrosamines spiked into valsartan.

Methodology and Instrumentation

The analytical platform comprised:

• Agilent 1260 Infinity II Flexible Pump (G7104C)
• Agilent 1260 Infinity II Multisampler (G7167A)
• Agilent 1260 Infinity II Multicolumn Thermostat (G7116A)
• Agilent Ultivo LC/TQ Triple Quadrupole Mass Spectrometer (G6465B)
• Agilent MassHunter software suite for data acquisition and optimization
• ZORBAX Eclipse Plus C18 RRHD column, 2.1×100 mm, 1.8 µm

Chromatographic separation used a ternary gradient of water, methanol, and acetonitrile each with 0.1% formic acid. Flow rate was 0.35 mL/min at ambient column temperature. Sample preparation involved dissolving 80 mg valsartan in 20 mL methanol and spiking with nitrosamine standards to 50 ng/mL. APCI source conditions and dynamic MRM transitions were optimized for quantifier and qualifier ions of nine nitrosamines.

Main Results and Discussion

Calibration curves over 1–200 ng/mL exhibited excellent linearity (R² > 0.999). LOQs ranged from 0.25 to 5 ng/mL, and LODs were below 0.7 ng/mL. Retention time precision was ≤ 0.15% RSD and area precision better than 3% RSD at 10 ng/mL. In spiked valsartan samples, recoveries were 93–100% for most nitrosamines; N-nitroso-n-dibutylamine showed lower recovery (~82%), likely due to ion suppression by coeluting API. A post-run organic flush or quaternary solvent switching can remove residual matrix components.

Benefits and Practical Applications

• High sensitivity enables reliable quantification of nitrosamines at single-digit ng/mL levels, meeting regulatory thresholds.
• Sub-2 µm column compatibility delivers rapid separations with high resolution.
• Quaternary pump flexibility allows gradient modifications and post-run clean-up without hardware changes.
• Low cost of ownership and streamlined workflow support routine QA/QC in pharmaceutical laboratories.

Future Trends and Possibilities

Emerging directions include integration of on-line sample preparation to automate extraction and cleanup, adoption of high-resolution mass analyzers for enhanced specificity, expansion to additional drug classes and matrices, and development of ultra-fast gradients for high-throughput screening. Implementation of artificial-intelligence-driven data processing may further improve detection of unknown nitrosamine species.

Conclusion

The combination of the Agilent 1260 Infinity II Prime LC and Ultivo LC/TQ provides a sensitive, precise, and flexible solution for monitoring nitrosamine impurities in pharmaceutical APIs. The method achieves low ng/mL detection limits, robust quantification, and compliance with stringent regulatory limits while maintaining operational efficiency and cost-effectiveness.

References

1. European Medicines Agency. Guideline M7(R1) on Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. August 2015. EMA/CHMP/ICH/83812/2013.
2. Masada S, et al. Rapid and Efficient High-Performance Liquid Chromatography Analysis of N-Nitrosodimethylamine Impurity in Valsartan Drug Substance and Its Products. Scientific Reports. 2019;9:11852.
3. U.S. Food and Drug Administration. Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) Method for the Determination of Six Nitrosamine Impurities in ARB Drugs. May 21, 2019.