Simultaneous Estimation of Eleven Nitrosamine Impurities in Metformin Drug Product Using an Agilent 6495D LC/TQ

Importance of the Topic

The presence of nitrosamine impurities in pharmaceuticals poses significant carcinogenic risks even at trace levels. Regulatory bodies such as the FDA and EMA have established stringent acceptable intake limits, driving the need for highly sensitive and reliable analytical methods. Metformin, a widely used antidiabetic drug, must be monitored to ensure patient safety and compliance with updated nitrosamine guidelines.

Objectives and Overview of the Study

This work aimed to develop and validate a robust LC/MS/MS method for the simultaneous estimation of eleven nitrosamine impurities in metformin drug product. Using an Agilent 1290 Infinity II LC system coupled to an Agilent 6495D triple quadrupole mass spectrometer, the study optimized sample preparation, chromatographic separation, and MRM detection to achieve trace-level sensitivity and high reproducibility.

Instrumentation

• Agilent 1290 Infinity II High Speed Pump, Multisampler, Column Thermostat, Variable Wavelength Detector
• Agilent 6495D Triple Quadrupole MS with APCI source
• Agilent InfinityLab Poroshell Phenylhexyl column (150 × 3.0 mm, 2.7 µm)
• Diverter valve for metformin diversion to waste

Methodology

Sample Preparation:
Metformin IR tablets were crushed, weighed to 100 mg API, extracted in methanol/water, sonicated, shaken, centrifuged, and filtered through 0.22 µm PVDF. Working standards (10–1,000 pg/mL) were prepared in the same diluent.
Chromatography:
Mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in methanol) at 0.4 mL/min; gradient from 100% A to 90% B over a 22 min run; column at 40 °C; diverter valve timed to exclude metformin from MS.
Mass Spectrometry:
APCI positive mode; optimized source parameters (250 °C gas temp, 11 L/min flow, 25 psi nebulizer, 2,000 V capillary, 8 µA corona, 350 °C vaporizer); dynamic MRM transitions for each nitrosamine with specific precursor/product ions, collision energies, and retention time windows.

Main Results and Discussion

• Linearity: 10–1,000 pg/mL with R² > 0.999 for all eleven nitrosamines.
• Sensitivity: LOD of 5 pg/mL (S/N ≥ 10) and LOQ of 10 pg/mL (S/N ≥ 20).
• Precision: RSD ≤ 5% at LOD, LOQ, and 100 pg/mL levels (six replicates).
• Accuracy: Recovery between 80% and 120% in spiked metformin samples at 10 and 100 pg/mL.
• Selectivity: Qualifier/quantifier ion ratios within ±20%, with clear chromatographic separation from metformin and among impurities.

Benefits and Practical Applications

The method provides high-throughput, automated screening of nitrosamines at ppb levels in metformin and can be extended to other APIs. Its robustness and compliance with regulatory thresholds support routine quality control and risk assessment in pharmaceutical manufacturing.

Future Trends and Applications

Advances may include coupling with high-resolution MS for non-targeted screening, integration of UHPLC for faster runs, expansion to broader impurity panels, and use of AI-driven optimization for method development and data processing.

Conclusion

An Agilent 6495D LC/TQ–based MRM method was established for eleven nitrosamine impurities in metformin, demonstrating exceptional sensitivity, linearity, precision, and accuracy. It fulfills stringent regulatory requirements and offers a versatile platform for ongoing pharmaceutical safety monitoring.

References

1. Kruhlak N. et al. Regul. Toxicol. Pharmacol. 2024;150:105640.
2. FDA. Recommended Acceptable Intake Limits for Nitrosamine Impurities. 2023.
3. EMA. Q&A on Nitrosamine Impurities in Human Medicinal Products. 2020.
4. FDA. Control of Nitrosamine Impurities in Human Drugs Guidance. 2024.
5. USP. Quantitative Analysis of Nitrosamine Impurities by LC-MS Methods. 2024.
6. ICH Q2(R2). Validation of Analytical Procedures. 2023.
7. FDA. RapidFire-MS/MS Method for Nitrosamine Impurities in ARB Drugs. 2019.
8. FDA. LC-HRMS NDMA in Ranitidine Method. 2019.
9. FDA. LC-HRMS Method for Six Nitrosamines in ARB Drugs. 2019.