LC-MS/MS method for the quantification of 10 nitrosamine impurities in metformin

Importance of the topic

Metformin is a widely used first-line treatment for type 2 diabetes, and the presence of trace nitrosamine impurities in pharmaceuticals has raised significant safety concerns. Regulatory agencies have issued recalls of batches containing nitrosamines above acceptable daily intake limits. A robust analytical method capable of detecting and quantifying multiple nitrosamines in metformin is essential to ensure product safety, regulatory compliance, and patient health.

Objectives and study overview

This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of ten nitrosamine impurities in metformin drug product. The target analytes include NDMA, NDEA, NEIPA, NDIPA, NDPA, NMPA, NDBA, NMBA, NMOR and NPIP. The method follows ICH Q2(R1) guidelines, achieving sensitivity below regulatory limits.

Used instrumentation

• Thermo Scientific Vanquish Flex Binary UHPLC with temperature-controlled autosampler and column compartment
• Thermo Scientific TSQ Quantis triple-stage quadrupole mass spectrometer with APCI source
• Acclaim 120 C18 column, 4.6 × 150 mm, 3 µm
• Optima grade methanol, water and formic acid

Methodology

Sample preparation involves pulverizing tablets to achieve 100 mg/mL active ingredient in a methanol–water (80:20) diluent, followed by vortex mixing, sonication, centrifugation and PVDF filtration. Chromatographic separation uses a reversed-phase C18 column at 40 °C with a gradient from 14% to 80% methanol containing 0.1% formic acid over 32 minutes. Detection employs selected reaction monitoring (SRM) in positive APCI mode. Calibration standards span 0.5 to 40 ng/mL, defining LODs at 0.2–0.4 ppb and LOQs at 0.5–1 ppb.

Main results and discussion

System suitability tests showed peak area RSDs below 10% and cumulative RSDs below 6% over a 35-hour sequence, demonstrating robust long-term performance. Linearity for all analytes yielded R2 values greater than 0.99. Signal-to-noise ratios at LOQ exceeded 68 for all compounds, with NMBA and NDPA achieving the highest values. Recovery studies at low, mid and high levels produced results within 80–120%, confirming accuracy. The method resolves syn/anti conformers of NMBA and NEIPA, integrating combined peak areas for quantitation.

Benefits and practical applications

• Simultaneous quantification of ten nitrosamines in a single analysis
• Meets or exceeds regulatory sensitivity requirements for metformin impurities
• High reproducibility and recovery supporting routine QA/QC workflows
• Long-term robustness enabling extended analytical sequences without performance loss
• Adaptable to other drug matrices with minor adjustments

Future trends and possibilities

• Integration of automated sample preparation and online extraction for higher throughput
• Use of high-resolution mass spectrometry to expand analyte scope beyond ten nitrosamines
• Green chromatography approaches to reduce solvent consumption and environmental impact
• Implementation of chemometric tools for trend analysis in impurity profiling
• Application to multi-product platforms for broader pharmaceutical quality control

Conclusion

An LC-MS/MS method for ten nitrosamine impurities in metformin was successfully developed and validated, demonstrating excellent sensitivity, precision, linearity and recovery within regulatory expectations. The use of APCI-SRM on a triple-quadrupole platform ensures robust performance, supporting routine monitoring of nitrosamine levels in pharmaceutical quality control.

References

• US FDA. LC-HRMS Method for the Determination of Six Nitrosamine Impurities in ARB Drugs. 2019.
• US FDA. LC-HRMS Method for the Determination of NDMA in Ranitidine Drug Substance and Drug Product. 2019.
• US FDA. LC-HRMS Method for the Determination of NDMA in Metformin Drug Substance and Drug Product. 2020.
• US FDA. Rigorous Detection of Nitrosamine Contaminants in Metformin Products. 2020.
• US FDA. LC-ESI-HRMS Method for the Determination of Nitrosamine Impurities in Metformin Drug Substance and Drug Product. 2020.