Simultaneous quantitation of N-nitrosamines and NDSRI in API and formulation by using a DUIS ionization source in LC-MS/MS

Simultaneous Quantitation of N-nitrosamines and NDSRI in Afatinib API and Formulation by LC-MS/MS with DUIS Source

Significance of the Topic
N-nitrosamines (R2N–N=O) are class 1 mutagens with strict regulatory limits in pharmaceuticals. Their detection—and that of structurally related nitroso drug substance related impurities (NDSRIs)—is critical for ensuring patient safety and regulatory compliance.

Objectives and Study Overview
This work describes a partially validated UHPLC-MS/MS approach for simultaneous quantification of nine N-nitrosamines and one nitroso-Afatinib impurity (N-AFA) in Afatinib active pharmaceutical ingredient (API) and its oral formulation. The method aims to achieve sub-ppb sensitivity, high throughput, and alignment with ICH M7 and FDA requirements.

Methodology and Instrumentation
Used Instrumentation

• UHPLC System: Shimadzu Nexera X3
• Column: Shim-pack Scepter PFPP-120 (2.1 × 150 mm, 3 µm)
• Mass Spectrometer: LCMS-8060NX triple quadrupole with DUIS dual ionization source

Chromatographic Conditions

• Mobile Phase A: 10 mM ammonium formate in water; B: methanol:acetonitrile (9:1)
• Gradient: 2%–40% B (0–8 min), 40%–70% B (8–20 min), hold then re-equilibrate
• Flow Rate: 0.45 mL/min; Column Temp.: 45 °C; Injection Volume: 40 µL

MS Parameters and Sample Preparation

• MRM transitions optimized via auto MRM (LabSolutions); collision energies fine-tuned for each analyte
• Limits of Quantitation: 1.0 ppb for NDMA, 0.5 ppb for other nitrosamines; S/N ratios >30 at LOQ
• Sample Prep: 10 mg of API or equivalent formulation dissolved in diluent, sonication, 0.22 µm filtration
• Recovery Studies: Spiking at 500 ppb (NDMA) and 250 ppb (other analytes)

Main Results and Discussion

• Linearity: Calibration curves showed r² ≥ 0.994 across 0.5–20 ppb ranges
• Precision: Repeatability at LOQ under 15% RSD
• Recovery: 70–130% for all target compounds in both API and formulation matrices
• Real Sample Analysis: All nitrosamines and N-AFA were below LOQ in Afatinib API and tablet formulation

Benefits and Practical Applications

• Simultaneous APCI- and ESI-like ionization via DUIS reduces analysis time and reagent consumption
• Sub-ppb sensitivity supports stringent regulatory monitoring of mutagenic impurities
• Streamlined workflow suitable for QC laboratories in pharmaceutical manufacturing

Future Trends and Opportunities

• Expansion of DUIS-based LC-MS/MS methods to other drug substances and broader nitrosamine classes
• Full method validation per ICH and FDA guidelines to facilitate inter-laboratory transfer
• Automation of sample preparation and data analysis to boost throughput
• Integration with high-resolution mass spectrometry for non-targeted impurity profiling

Conclusion
The developed UHPLC-DUIS-MS/MS protocol offers a robust, sensitive, and efficient strategy for simultaneous quantitation of N-nitrosamines and NDSRI in Afatinib API and formulation. It provides regulatory compliance, improved throughput, and practical utility for pharmaceutical quality control.

Reference
[1] WHO Information Note; Update on Nitrosamine Impurities, Nov. 2019
[2] Cioc R. C. et al.; Formation of N-Nitrosamine Drug Substance Related Impurities in Medicines: A Regulatory Perspective on Risk Factors and Mitigation Strategies, ACS Publications, 2023