Quantitation of 5 Nitrosamines in Metformin API and Formulation by HS-GCMS/MS

Significance of the Topic

Many pharmaceuticals have been found to contain trace levels of nitrosamines, potent genotoxic impurities, raising concerns about patient safety. The USFDA has set stringent intake limits for nitrosamines due to their carcinogenic potential. Metformin, a widely prescribed antidiabetic agent, must be monitored for nitrosamine content to assure safe therapy. Developing a robust, sensitive analytical method to quantify nitrosamines below regulatory thresholds in both active pharmaceutical ingredient (API) and finished dosage forms (FDF) is therefore critical.

Objectives and Overview of the Study

This application note describes a headspace GC–MS/MS method for simultaneous quantitation of five nitrosamines in metformin API and formulation. The target analytes are N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosoethylisopropylamine, N-nitrosodiisopropylamine, and N-nitrosodibutylamine. The method aims to achieve a combined limit of quantitation (LOQ) of 16.5 ppb, meeting USFDA guidance for maximum daily exposure.

Methodology and Instrumentation Used

A five-point calibration (1–20 ppb) was prepared in dimethyl sulfoxide (DMSO). Instrumental analysis was performed on a Shimadzu GCMS-TQ8040 NX triple quadrupole mass spectrometer coupled to an HS-20 headspace autosampler. Chromatographic separation employed an SH-Stabilwax DA capillary column (60 m × 0.25 mm I.D., 0.25 μm df). Electron ionization at 70 eV and multiple reaction monitoring (MRM) enabled selective detection. Calibration and MRM conditions, including precursor/product transitions and collision energies, were optimized in segmented acquisition. The headspace parameters (oven 135 °C, sample line 150 °C, transfer line 180 °C) ensured efficient transfer of volatile nitrosamines.

Main Results and Discussion

Calibration curves showed excellent linearity (r² > 0.996) over the 1–20 ppb range for all five nitrosamines. Limits of detection and quantitation were 0.3 ppb and 1.0 ppb, respectively. Signal-to-noise ratios at LOQ ranged from 51 to 366. Repeatability at LOQ (n=6) yielded relative standard deviations below 11%. Spiked recovery studies at 3.3 ppb demonstrated accuracy between 90% and 115% in both metformin API and FDF matrices, confirming method reliability for trace‐level quantitation.

Benefits and Practical Applications of the Method

• High sensitivity and selectivity enable reliable detection of nitrosamines below regulatory limits in pharmaceutical matrices.
• Headspace sampling minimizes matrix effects and reduces sample preparation complexity.
• Shimadzu GCMS-TQ8040 NX provides long-term stability and high precision, supporting routine quality control.
• Rapid analysis cycle (30 min) enhances laboratory throughput.

Instrumentation

• Shimadzu GCMS-TQ8040 NX triple quadrupole mass spectrometer with high-sensitivity ion source.
• Shimadzu HS-20 headspace autosampler.
• SH-Stabilwax DA capillary column (60 m × 0.25 mm I.D., 0.25 μm df).

Future Trends and Potential Applications

Ongoing trends in pharmaceutical analysis emphasize automation, improved sensitivity, and reduced environmental impact. Future work may explore:

• Integration with microextraction techniques for lower sample volumes and enhanced throughput.
• High-resolution mass spectrometry to expand impurity profiling capabilities.
• Advanced data processing algorithms for real-time compliance monitoring and reporting.
• Application of the validated method to other drug substances susceptible to nitrosamine contamination.

Conclusion

The HS-GC–MS/MS method on Shimadzu GCMS-TQ8040 NX with HS-20 headspace autosampler offers a robust, sensitive, and reproducible approach for quantifying five nitrosamines in metformin API and FDF. The protocol meets USFDA requirements, providing high confidence in ensuring pharmaceutical safety.