Improved LC/MS Performance to Determine Polar Nitrosamines Using the Agilent 1260 Infinity II Hybrid Multisampler

Significance of the Topic

This study addresses the critical need to monitor trace levels of polar nitrosamine impurities in pharmaceutical products. Nitrosamines are genotoxic compounds with carcinogenic potential, subject to strict regulatory limits. Reliable detection and quantification methods are essential to ensure patient safety and compliance with guidelines issued by agencies such as the FDA and EMA.

Objectives and Study Overview

The main goal was to compare an optimized feed injection workflow using the Agilent 1260 Infinity II Hybrid Multisampler against a conventional flow-through injection approach for LC/MS analysis of polar nitrosamines. The application note evaluates accuracy, precision, linearity, and sensitivity across a range of nitrosamine analytes in a high-organic solvent matrix.

Methodology and Instrumentation

The chromatographic separation employed an Agilent ZORBAX Eclipse Plus C18 RRHD column (2.1×100 mm, 1.8 µm) at 45 °C. Mobile phases were water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B), with a gradient from 2% to 95% B at 0.35 mL/min over 6 min.

Mass spectrometry was performed on an Agilent Ultivo LC/TQ with APCI source. MRM transitions were optimized for eight nitrosamines, with gas temperature 220 °C, vaporizer temperature 325 °C, nebulizer pressure 35 psi, and capillary voltage +2,500 V.

Sample preparation involved dissolving valsartan in methanol to 50 ng/mL per nitrosamine, simulating a 1 µg/day exposure. Calibration solutions ranged from 0.2 to 100 ng/mL. Repeated injections (N=10) were used to assess precision and linearity.

Main Results and Discussion

Feed injection markedly improved early-eluting peak shapes, preventing breakthrough seen with flow-through injections. At 10 µL injection volume, feed injection achieved peak area RSDs below 3% for most nitrosamines (versus 8–14% for flow-through). Linearity (R2) exceeded 0.999 with feed injection compared to 0.990–0.998 for flow-through. Limits of quantification for NDMA improved from 10 ppb (flow-through) to below 2 ppb (feed). Recoveries for a 50 ppb spike remained within 70–120% acceptance criteria using feed injection, while flow-through showed compromised recoveries for early-eluting analytes due to broadening and breakthrough.

Benefits and Practical Applications

• Enhanced peak shapes and reproducibility for polar nitrosamines in high-organic solvents
• Lower limits of quantification, enabling detection at sub-nanogram-per-milliliter levels
• Improved linearity and precision support robust quantification for quality control and regulatory compliance
• Applicable to routine screening of genotoxic impurities in drug substance development and release testing

Future Trends and Potential Applications

• Extension of feed injection strategies to other challenging polar analyte classes
• Integration with high-resolution MS workflows for broader impurity profiling
• Automation and high-throughput adaptation in regulated laboratories
• Further optimization of sampler parameters to enhance trapping and desolvation efficiency

Conclusion

Implementing the Agilent 1260 Infinity II Hybrid Multisampler in feed injection mode substantially improves LC/MS performance for polar nitrosamines. The approach delivers superior precision, sensitivity, and linearity compared to conventional flow-through injection, offering a robust solution for trace-level analysis of genotoxic impurities in pharmaceutical matrices.

Reference

1. European Medicines Agency. CH Guideline M7(R1) on Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. EMA/CHMP/ICH/83812/2013, 2015.
2. Masada S. et al. Rapid and Efficient HPLC Analysis of N-Nitrosodimethylamine Impurity in Valsartan. Sci. Rep. 2019, 9(1):11852.
3. U.S. Food and Drug Administration. LC-HRMS Method for Determination of Six Nitrosamine Impurities in ARB Drugs, 2019.