Quantitation of 5 NSA in Metformin API as per proposed USP General Chapter <1469> Procedure-4 by GC-MS/MS

Significance of the Topic

Recent findings of nitrosamine impurities (NSA) in pharmaceutical substances have raised significant safety concerns due to their mutagenic and carcinogenic potential. Regulatory bodies such as the United States Pharmacopeia (USP) introduced General Chapter <1469> to enforce strict controls on NSA levels in both active pharmaceutical ingredients (API) and finished dosage forms (FDF). Metformin, a widely prescribed drug for type 2 diabetes, is prone to NSA contamination, necessitating sensitive and reliable analytical methods to ensure patient safety and regulatory compliance.

Objectives and Study Overview

The primary goal of this study was to implement and validate USP <1469> Procedure 4 for the quantitation of five key nitrosamines—NDMA, NDEA, NEIPA, NDIPA and NDBA—in Metformin API. The work demonstrates the application of the Shimadzu GCMS-TQ 8050 NX system coupled with the AOC-20i Plus autosampler, evaluating method performance in terms of sensitivity, precision, accuracy and regulatory suitability.

Methodology and Instrumentation

Sample Preparation:

• Weighed 500 mg Metformin API and added 5 mL dichloromethane (DCM) or LOQ-level spiking solution for recovery studies.
• Vortexed for 1 minute and centrifuged at 4 000 rpm for 2.5 minutes.
• Filtered the supernatant through a 0.45 μm nylon filter into a GC vial; injected 2 μL splitless.

Quantitative Method Development:

• Analytes were identified by full‐scan MS; multiple reaction monitoring (MRM) transitions were optimized at various collision energies to achieve maximum sensitivity.
• Eight‐point calibration curves (0.25–15 ppb) employed NDMA C13 d6 as internal standard (5.0 ppb).
• LOD and LOQ for all five NSA were determined as 0.1 ppb and 0.25 ppb, respectively.

Instrumentation Used:

• GC-MS/MS: Shimadzu GCMS-TQ 8050 NX with AOC-20i Plus autosampler.
• Column: Wax MS, 30 m × 0.25 mm I.D., 1.0 μm film thickness.
• Carrier gas: Helium, column flow 1.0 mL/min.
• Injector: Splitless mode, 250 °C; Interface 230 °C; Ion source 230 °C.
• Ionization: Electron Ionization (EI); Collision-induced dissociation with argon at 200 kPa.

Main Results and Discussion

The method achieved excellent linearity (r2 > 0.995) for all analytes. Repeatability at the LOQ level (%RSD, n=6) remained below 10 %. Spiked recovery studies at LOQ (2.5 ppb) yielded 70–110 % recovery across the five nitrosamines. Compared to USP <1469> default LOQs (5.0 ppb), the Shimadzu application lowered LOQs to 2.5 ppb for NEIPA and equivalent or better limits for other compounds, demonstrating superior sensitivity.

Benefits and Practical Applications of the Method

The presented GC-MS/MS approach offers:

• High sensitivity allowing trace-level detection well below regulatory thresholds.
• Robust precision and accuracy suitable for routine QC environments.
• Streamlined sample preparation with minimal solvent consumption.
• Compatibility with USP <1469> guidelines, facilitating regulatory compliance.

Future Trends and Opportunities

Developments expected to enhance nitrosamine analysis include:

• Integration of high-resolution mass spectrometry (HRMS) for advanced screening and non‐targeted impurity profiling.
• Automated sample cleanup techniques (e.g., online SPE) to improve throughput and reduce manual handling.
• Expansion of validated methods to other pharmaceutical matrices beyond Metformin.
• Application of chemometric and machine learning tools for data processing and anomaly detection in impurity profiles.

Conclusion

The Shimadzu GCMS-TQ 8050 NX system with AOC-20i Plus demonstrates a reliable, sensitive and precise GC-MS/MS method for quantifying five critical nitrosamines in Metformin API. The workflow meets and exceeds USP <1469> requirements, ensuring robust control of mutagenic impurities to maintain patient safety and regulatory adherence.

Reference

Suryawanshi N, Handique D, Hase P, Chiplunkar S, Sawant D, Wagle A, Dwivedi R, Kelkar J, Rasam P. Quantitation of Five Nitrosamines in Metformin API by GC-MS/MS (USP <1469> Procedure 4). Shimadzu Analytical (India) Pvt. Ltd.; First Edition September 2021.