Quantitation of 6 N-Nitrosamines in Metformin and 5 Sartan APIs as per the EDQM method Procedure C

Significance of the Topic

Trace levels of N-nitrosamines in active pharmaceutical ingredients pose a significant carcinogenic risk. Regulatory bodies such as the EDQM and US FDA require stringent control of these impurities. Developing highly sensitive and reliable methods to quantify nitrosamines down to low parts-per-billion levels is essential for ensuring drug safety and maintaining compliance during drug development and production.

Objectives and Study Overview

This study aimed to demonstrate the applicability of the EDQM Procedure C for quantitative determination of six N-nitrosamine impurities in Metformin and five Sartan APIs using Shimadzu’s GCMS-TQ8050 NX coupled with the AOC-20i+s Plus autosampler. Key objectives included:

• Extending EDQM’s validated scope—originally limited to Sartans—to include Metformin API.
• Achieving a lower limit of quantitation (LOQ) than the 30 ppb specified by EDQM, targeting 15 ppb while meeting signal-to-noise and recovery criteria.
• Validating method performance in terms of linearity, repeatability, sensitivity, and accuracy.

Methodology and Instrumentation

Sample preparation involved alkaline extraction of 250 mg API with a sodium hydroxide–acetonitrile–water mixture, followed by dichloromethane partitioning. Linearity standards were prepared at 7.5, 15, and 30 ppb by spiking certified reference nitrosamine solutions and using N-nitroso-ethylmethylamine (NEMA) as an internal standard. Shimadzu GCMS-TQ8050 NX with AOC-20i+s Plus autosampler was configured in splitless injection mode and equipped with a SH-I-624Sil MS column (30 m×0.25 mm I.D., 1.4 µm film). Electron ionization and optimized multiple reaction monitoring (MRM) transitions for NDMA, NDEA, NDBA, NMBA, NEIPA, NDIPA, and NDPA were employed. Collision energies were tuned for each transition to maximize sensitivity at trace levels.

Results and Discussion

Calibration curves for all six nitrosamines exhibited excellent linearity (r2 ≥ 0.999) across the tested range. The method achieved an LOQ of 15 ppb, half the EDQM requirement, while maintaining signal-to-noise ratios well above the minimum criteria (principal ≥ 10, qualifier ≥ 3). Repeatability experiments (n = 6) at both 15 ppb and 30 ppb showed relative standard deviations below 7 % for all analytes. Recovery studies at 15 ppb and 30 ppb produced values between 70 % and 130 %, meeting EDQM acceptance limits. Analysis of blank API samples confirmed nitrosamine concentrations below the 15 ppb LOQ. A chromatographic overlay of spiked Sartans and Metformin demonstrated consistent retention and peak resolution across all seven compounds.

Benefits and Practical Applications

• The method extends EDQM Procedure C to Metformin, providing a unified GC-MS/MS workflow for multiple API classes.
• Lower LOQ enhances detection capability for critical impurities, supporting tighter quality control.
• Robust repeatability and recovery ensure reliable routine monitoring in QC laboratories.
• Automated sampling with AOC-20i+s Plus increases throughput and reproducibility.

Future Trends and Applications

Advances may include automated online extraction techniques, expansion to a broader spectrum of nitrosamine analogues, and cross-platform transfer to other GC-MS/MS instruments. Integration of high-resolution MS could further improve specificity. Applicability to complex drug matrices and biologics is a promising area for method adaptation and regulatory harmonization.

Conclusion

The Shimadzu GCMS-TQ8050 NX with AOC-20i+s Plus autosampler successfully quantifies six nitrosamines in Sartans and Metformin at 15 ppb LOQ, surpassing EDQM requirements. The method demonstrates excellent linearity, sensitivity, repeatability, and accuracy, making it a powerful tool for pharmaceutical impurity control.

Instrumentation Used

• Shimadzu GCMS-TQ8050 NX triple quadrupole mass spectrometer
• AOC-20i+s Plus autosampler
• SH-I-624Sil MS capillary column (30 m×0.25 mm I.D., 1.4 µm)