Highly sensitive and robust LC-MS/MS solution for quantitation of nitrosamine impurities in metformin drug products

Significance of the Topic

Nitrosamine impurities have emerged as critical genotoxic contaminants in pharmaceuticals, prompting global regulatory bodies to enforce stringent limits on allowable daily intake. These compounds pose serious health risks even at trace levels, making highly sensitive and reliable analytical methods essential for safeguarding drug quality and patient safety.

Objectives and Study Overview

This study presents the development and validation of a targeted LC-SRM-MS workflow for simultaneous quantitation of ten nitrosamines in metformin extended-release tablets. The goals were to achieve detection limits below regulatory thresholds, demonstrate method robustness over large sample sets, and ensure compliance with EMA and FDA guidelines.

Methodology and Instrumentation

• Sample Preparation: Tablets were pulverized and extracted with methanol, followed by protein-cutoff filtration, cold centrifugation, and aqueous dilution to reduce polymer interference.
• Chromatography: A Thermo Scientific Vanquish Horizon UHPLC system fitted with a Hypersil GOLD phenyl column (100 × 4.6 mm, 3 µm) operated at 30 °C and 0.5 mL/min gradient (water/methanol with 0.1% formic acid).
• Mass Spectrometry: Thermo Scientific TSQ Quantis triple quadrupole in both heated electrospray ionization (HESI) and atmospheric pressure chemical ionization (APCI) modes, using optimized SRM transitions and a diverter valve to protect the source from high-load API.
• Data System: Chromeleon CDS v7.2.10 provided compliant data acquisition and processing with full audit trails and 21 CFR Part 11 capability.

Main Results and Discussion

• Limits of Quantitation: Achieved LOQs ≤ 10 ppb by HESI and ≤ 5 ppb by APCI, meeting both EMA and FDA acceptable intake levels.
• Linearity and Precision: Calibration curves over 1–200 ppb showed R² > 0.995 with 1/x weighting. Accuracy and precision at low ppb levels remained within ±15%.
• Robustness: Over 1,000 consecutive injections of spiked samples, peak area RSDs remained below 20% at 5 ppb and below 10% at 20 ppb. Retention times and peak shapes were stable, with theoretical plate counts varying by <5%.
• Matrix Interference: No significant endogenous interferences observed in blank metformin extracts, confirming specificity of SRM transitions.

Benefits and Practical Applications

• Routine screening and quality control of nitrosamine impurities in metformin and similar drug products.
• Regulatory Compliance: Fully compliant workflow for cGMP environments, addressing EMA and FDA guidance including 21 CFR Part 11.
• Flexibility: Applicable to a broad panel of nitrosamines, extendable to other active pharmaceutical ingredients.

Future Trends and Potential Uses

As regulatory scrutiny intensifies, future developments may focus on expanding the nitrosamine panel, integrating high-resolution mass spectrometry for confirmatory analysis, automating sample preparation for higher throughput, and applying similar workflows to other drug classes and complex matrices.

Conclusion

The presented LC-SRM-MS method delivers high sensitivity, specificity, and reproducibility for quantifying ten nitrosamine impurities in metformin extended-release tablets. Its robust performance over extensive injection series, coupled with compliant data management, makes it a valuable solution for routine pharmaceutical quality assurance and regulatory adherence.

Reference

• ICH M7(R1) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk.
• FDA Guidance for Industry: Control of Nitrosamine Impurities in Human Drugs (2020).
• EMA Assessment Report: Nitrosamine Impurities in Human Medicinal Products (2020).