race Level Quantitation of 8 Nitrosamines Including Varenicline Nitroso-Drug Substance Related Impurity (VNDSRI) in Varenicline Tablets Using LCMS-8045

Importance of the topic

Varenicline is a frontline prescription therapy for smoking cessation, acting as a partial agonist at the α4β2 nicotinic acetylcholine receptor. Regulatory bodies such as the USFDA set stringent limits on nitrosamine impurities, including Varenicline Nitroso‐Drug Substance Related Impurity (VNDSRI), due to their potential carcinogenic risk at trace levels. Ensuring accurate, sensitive quantification of these compounds in pharmaceutical products is critical for patient safety and regulatory compliance.

Objectives and study overview

This study aimed to develop and validate a robust LC‐MS/MS method for simultaneous trace‐level quantitation of eight nitrosamines (VNDSRI, NDMA, NMBA, NDEA, NEIPA, NDIPA, NDPA, NDBA) in varenicline tablets. The goal was to achieve lower limits of quantitation (LOQs) than those specified in existing USFDA methods and to demonstrate method performance in real samples and spiked placebos.

Instrument used

• UHPLC system: Nexera X3
• Analytical column: Shim-pack GIST C18 (150 × 4.6 mm, 5 µm)
• Mass spectrometer: LCMS-8045 triple quadrupole with APCI source
• Data acquisition: Multiple reaction monitoring (MRM) in positive mode

Methodology and instrumentation

Chromatographic separation employed a gradient elution with 0.1% formic acid in water (A) and methanol (B) at 0.7 mL/min and 40 °C. A divert valve removed the high‐concentration varenicline peak to waste to protect the MS. MRM transitions and optimized collision energies were established for each analyte and two isotopically labeled internal standards. Sample preparation involved simple dilution of crushed tablet or placebo in water containing internal standards, followed by vortexing, sonication, centrifugation, and filtration.

Main results and discussion

Calibration curves for all eight nitrosamines displayed excellent linearity (r² ≥ 0.998) across 0.1–100 ppb ranges, with LOQs of 0.1 ppb for VNDSRI, NDEA, NEIPA and 0.2–0.5 ppb for the remaining compounds. Signal‐to‐noise ratios at LOQ ranged from 13 to 62. Analysis of commercial varenicline tablets (0.5 mg/mL) detected VNDSRI at 16.83 ppm, well below the 18.5 ppm limit, while other nitrosamines were below LOQ. Spiked placebo recovery at 0.2, 0.5 and 1.0 ppb levels yielded 70–130% recoveries, meeting acceptance criteria.

Benefits and practical applications

• The method unifies quantitation of eight regulated nitrosamines in a single run.
• Ultra‐fast scanning and polarity switching of LCMS-8045 enhance throughput and sensitivity.
• Simplified sample prep suits high‐throughput quality control workflows.
• LOQs exceed regulatory requirements, supporting routine compliance testing.

Future trends and possibilities

Advances in triple quadrupole and high‐resolution MS will further lower detection limits and expand target lists. Automated sample handling and data processing pipelines may accelerate nitrosamine screening across various drug products. Integration of orthogonal separation techniques and spectral libraries could improve confirmation of novel or unknown nitrosamines in complex matrices.

Conclusion

A single, highly sensitive LC‐MS/MS method was successfully developed on Shimadzu’s Nexera X3‐LCMS-8045 platform to quantify eight nitrosamines in varenicline tablets. The validated approach meets stringent regulatory criteria, demonstrates excellent linearity and recovery, and confirms product safety by detecting VNDSRI well below acceptable intake limits.

Reference

Shimadzu Analytical (India) Pvt. Ltd., Application News, Liquid Chromatograph Mass Spectrometer LCMS-8045, July 2022.