Simultaneous Quantitation of N-nitrosamines and NDSRI in API and Formulation by Using a DUISTM Ionization Source in LC-MS/MS

Significance of the Topic

Monitoring N-nitrosamines and N-nitrosamine drug substance related impurities (NDSRIs) in pharmaceuticals has become critical due to stringent regulatory limits and potential carcinogenic risks. Traditional approaches require separate LC-MS/MS methods with APCI for N-nitrosamines and ESI for NDSRIs, increasing solvent use, analysis time and operational complexity. A unified workflow enabling simultaneous detection of both classes in active pharmaceutical ingredients and finished formulations addresses these challenges and enhances laboratory efficiency.

Objectives and Study Overview

The primary goal of this study was to develop and partially validate a single LC-MS/MS method for quantifying nine common N-nitrosamine impurities and one N-nitroso-Afatinib impurity (N-AFA) in Afatinib API and its formulation. Using Shimadzu’s Nexera X3 UHPLC coupled to an LCMS-8060NX triple quadrupole equipped with a dual ionization source (DUIS), the method aimed to deliver high sensitivity, low limits of quantitation and robust performance without complex sample pretreatment.

Methodology and Instrumentation

Standards for NDMA, NMEA, NPYR, NDEA, NPIP, NEIPA, NDIPA, NDPA, NDBA and N-AFA were prepared in diluent. MRM transitions and collision energies were optimized automatically in LabSolutions auto MRM mode. Chromatographic separation was achieved on a Shim-pack Scepter PFPP (metal free) column (2.1 × 100 mm, 3 µm) at 45 °C using 10 mM ammonium formate in water (A) and methanol:acetonitrile 9:1 (B) at 0.45 mL/min with a gradient from 2 % to 100 % B in 20 min. Injection volume was 40 µL. The LCMS-8060NX DUIS source combined ESI and APCI-like conditions with IonFocus™ and UF-Qarray™ II lens technology, operating at 400 °C interface and 250 °C desolvation line temperatures.

Instrumentation Used

• UHPLC: Nexera X3
• Column: Shim-pack Scepter PFPP, 2.1 × 100 mm, 3 µm
• MS: LCMS-8060NX triple quadrupole
• Ionization: DUIS with IonFocus™ and UF-Qarray™ II
• Software: LabSolutions™ for MRM optimization and data analysis

Main Results and Discussion

Chromatographic peaks for all analytes and Afatinib API were resolved with clear baseline separation. Limits of quantitation were established at 1.0 ppb for NDMA and 0.5 ppb for the other nine compounds, each achieving signal-to-noise ratios above 30 and repeatability (RSD) below 15 %. Calibration curves demonstrated excellent linearity (r2 ≥ 0.992). Real API and formulation samples showed no detectable levels (

Benefits and Practical Applications

Implementing this unified LC-MS/MS approach offers:

• Reduced solvent consumption and faster turnaround by combining ESI and APCI in one run
• High sensitivity for structurally diverse nitrosamines
• Simplified sample preparation with direct dilution and filtration
• Regulatory compliance for impurity control in drug substances and products

Future Trends and Potential Applications

Expanding this methodology to other drug classes and more complex matrices could further elevate impurity profiling. Integration with automated sample handling and high-throughput platforms may enhance laboratory productivity. Emerging ionization technologies and advanced data processing algorithms are expected to push sensitivity and selectivity boundaries, supporting next-generation impurity screening.

Conclusion

A single LC-MS/MS method using DUIS on the LCMS-8060NX enables simultaneous, sensitive quantitation of nine N-nitrosamines and one NDSRI in Afatinib API and formulation. The approach delivers low LOQs, robust repeatability, accurate recoveries and streamlined workflow, fulfilling regulatory requirements and improving operational efficiency.

References

No external literature references were cited in the original text.