LC-MS/MS Method for Identification & Quantification of Varenicline Nitroso Impurities (NDSRi) by Utilizing QTRAP 4500 Mass Detector from Formulation Dosage Form

Background: Nitrosamines are known carcinogens. Regulatory bodies like the FDA and EMA have set strict limits for their presence in pharmaceuticals. Monitoring and controlling nitrosamine level ensures product quality and purity. Therefore, suitable analytical tools are essential for estimating nitrosamine impurities, ensuring safety, quality, and regulatory compliance. Purpose of study: The research aimed to develop and validate an LC-MS/MS method for the estimation of three potential nitrosamine impurities related to Varenicline, namely nitroso-Varenicline impurity, nitrosamine impurity A, and nitrosamine impurity B, and its application to pharmaceutical dosage forms.  Method: The optimized method includes LC-MS/MS coupled with a Sciex QTRAP 4500 mass detector. Analytical separation was achieved with a biphenyl column (150 x 4.6 mm, 2.6 µm), employing a gradient system with a flow rate of 0.4 mL/min and an injection volume of 20 µL. The auto-sampler and column oven temperatures were maintained at 15°C and 30°C, respectively. Samples were introduced to the mass spectrometer via electrospray ionization using the following conditions: Scan type: MRM; polarity: positive; ion source: ESI; curtain gas: 40 psi; probe temperature: 500°C; GS: 30 psi; GS2: 30 psi; ion spray voltage: 5500 V. Results: The developed method was validated and observed to be selective, specific, precise, linear, accurate, and highly sensitive, as the observed LOQ level for Nitroso-Varenicline impurity and Nitrosamine impurity B is 0.5 ppm, whereas for Nitrosamine impurity A, it is 0.4 ppm. The method was applied to multiple batches of Varenicline tablets. Conclusion: The newly developed and validated LC-MS/MS method is an essential tool for analyzing nitrosamine impurities in pharmaceuticals, meeting regulatory requirements and industry standards. It is suitable for its intended purpose and serves as a robust tool for estimating carcinogenic impurities in pharmaceutical drug substances and products during the drug development process and quality control analysis.