High-Sensitivity Quantitative Analysis of Nitrosamines Using Triple Quadrupole LC/MS/MS

Importance of the Topic

Nitrosamine impurities such as NDMA and NDEA are classified as probable human carcinogens by IARC and regulated under ICH M7 guidelines. Achieving trace-level detection and quantification is essential for pharmaceutical safety and regulatory compliance.

Objectives and Study Overview

This application news describes a high-sensitivity quantitative LC-MS/MS method for six nitrosamines performed on a Shimadzu LCMS-8060NX triple quadrupole system. The study aims to establish calibration ranges, assess linearity, and determine repeatability and limits of quantification.

Methodology

The analytical workflow combines UHPLC separation on a Shim-pack Scepter C18 (100 × 2.1 mm I.D., 1.9 µm) column with APCI-MRM detection. Mobile phases are 0.05% formic acid in water (A) and in methanol (B). A gradient from 1% B to 80% B over 8.5 min at 0.4 mL/min and 45 °C is employed. Injection volume is 10 µL. Mass spectrometric conditions include positive-mode APCI with a probe voltage of 4.0 kV, nebulizing gas at 4 L/min, drying gas at 3 L/min, DL temperature 150 °C, heat-block at 200 °C and interface at 300 °C.

Instrumentation Used

• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer
• APCI ionization probe
• Nexera X3 UHPLC system
• Shim-pack Scepter C18 column

Main Results and Discussion

Calibration curves were linear over 0.05–10 ng/mL for NDMA, NDEA, NDIPA, NMBA and 0.025–10 ng/mL for NEIPA and NDBA, with coefficients of determination (R2) ≥ 0.998. The lower limit of quantification was ≤ 0.05 ng/mL for all six compounds using a 10 µL injection. Repeatability (n=6) showed RSD ≤ 6.5% and accuracy ranged from 102.0% to 114.5% at the lowest calibration levels. Chromatograms displayed sharp, well-resolved peaks near LLOQ and consistent response across the calibration range.

Benefits and Practical Applications

• Enables reliable quantification of nitrosamine impurities at regulatory thresholds
• Supports risk assessment and quality control in pharmaceutical development and production
• Offers a broad dynamic range and excellent linearity for diverse nitrosamine concentrations

Future Trends and Applications

Advances may include integration of automated sample preparation, higher throughput workflows, extension to additional trace‐level organic contaminants and coupling with high-resolution mass spectrometry for untargeted screening of genotoxic impurities.

Conclusion

The described LC-MS/MS method on the LCMS-8060NX provides ultra-sensitive, precise and accurate quantification of six nitrosamines, meeting stringent regulatory requirements and supporting robust impurity control in pharmaceutical analysis.

Reference

1) ICH M7 (R1) Addendum: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk