A validated method for the rapid determination of fifteen nitrosamines in metformin drug substance

Significance of the topic

The presence of nitrosamine impurities in pharmaceutical drugs poses serious mutagenic and carcinogenic risks. Regulatory agencies worldwide demand ultra-trace detection of these compounds to ensure patient safety and product compliance.

Objectives and study overview

This work aimed to develop and validate a rapid, high-resolution GC-MS method for quantifying fifteen nitrosamines in metformin drug substance. The study evaluated chromatographic performance, sensitivity, selectivity, accuracy, precision, and long-term robustness using the Thermo Scientific™ Orbitrap Exploris™ GC mass spectrometer.

Methodology and instrumentation

• Sample preparation: 100 mg metformin was extracted with water and dichloromethane containing isotopically labeled standards. The workflow (vortexing, centrifugation, transfer) required 6 minutes per sample.
• Chromatography: separation on a TraceGOLD TG-1701MS column (30 m × 0.25 mm, 0.50 µm) with splitless injection and a 13.6 min temperature program.
• Mass spectrometry: full scan (m/z 40–440) with timed selected ion monitoring (tSIM) at 30,000 FWHM, using an ExtractaBrite™ EI source for high sensitivity and minimal maintenance.
• Data acquisition and processing: Thermo Scientific Chromeleon™ 7.3 CDS enabled automated calibration, integration, and retrospective data review.

Results and discussion

• Chromatographic resolution: all fifteen analytes were baseline-separated in under 12 minutes with Gaussian peak shapes and EP asymmetry <1.5.
• Sensitivity: instrument detection limits ranged from 42 to 388 fg on-column (0.1–1.3 ng/g). Method detection limits were 51–550 fg OC (0.2–1.8 ng/g), and method LOQs of 0.6–3.0 ng/g easily met FDA limits of 30 ng/g total daily intake.
• Linearity: demonstrated from 0.2 to 2,000 ng/mL (0.6–6,000 ng/g) with R² ≥ 0.995 across four orders of magnitude.
• Accuracy and precision: recoveries ranged from 88% to 105% (mean 96%) with RSD < 20% (mean 8.3%) at spiking levels of 1–20 ng/g.
• Selectivity and specificity: no carryover in solvent blanks, no matrix interferences in unspiked extracts, and mass errors < 1 ppm for all compounds.
• Robustness: over 100 metformin extracts injected across two weeks showed internal standard peak area RSD of 6.9% and mass accuracy within ±3 ppm with only daily calibration and minimal maintenance.

Benefits and practical applications

This validated method delivers high throughput analysis for pharmaceutical QC labs, enabling confident quantification of multiple nitrosamine impurities in a single run. The use of high-resolution MS reduces false positives from matrix interferences and allows retrospective data interrogation.

Future trends and potential applications

Advances may include expanding the panel to emerging nitrosamines, integrating automated sample preparation, and applying AI-driven data analysis. High-resolution GC-MS methods can be adapted for other drug substances and drug-product assays, supporting ongoing regulatory requirements and data integrity initiatives.

Conclusion

The Orbitrap Exploris GC method provides rapid, sensitive, and selective quantification of fifteen nitrosamines in metformin. It meets stringent regulatory criteria for detection limits, accuracy, precision, and robustness, making it a powerful tool for pharmaceutical impurity monitoring.

Reference

• ICH M7(R1) Guideline, Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals, 2017.
• IARC Monographs Volume 89, Smokeless Tobacco and Some Tobacco-specific N-Nitrosamines, 2007.
• WHO Information Note on Nitrosamine Impurities, 2019.
• EMA/351053/2019 rev 1: Temporary interim limits for NMBA, DIPNA and EIPNA in sartan medicines, 2020.