Simultaneous quantitation of nine nitrosamines using a highly sensitive and LC-HRAM mass spectrometry method in multiple drug products

Significance of the Topic

Exposure to nitrosamine impurities in pharmaceuticals has become a major safety concern due to their potential genotoxic and carcinogenic properties. Regulatory agencies worldwide have issued guidelines to limit nitrosamine levels in commonly prescribed drugs such as sartans and metformin. Reliable, sensitive, and efficient analytical methods are essential to ensure patient safety and support quality control in pharmaceutical manufacturing.

Objectives and Study Overview

The study aimed to develop and validate a single, high-resolution LC-HRAM Orbitrap MS method for simultaneous quantitation of nine nitrosamines in sartan APIs/tablets and metformin products. The method follows U.S. FDA recommendations with minor optimizations to improve sensitivity, selectivity, and throughput in routine pharmaceutical testing laboratories.

Methodology

Sample Preparation:

• Stock solutions of each nitrosamine were prepared in methanol and serially diluted to cover 0.1–20 ng/mL.
• Valsartan and metformin tablets were powdered, extracted with 100% methanol, shaken, centrifuged, and filtered.

Chromatographic Conditions:

• 2.6 μm biphenyl column (150 × 3.0 mm) with a three-solvent gradient (0.1% formic acid in water, methanol/acetonitrile 80:20, and acetonitrile).
• Flow rate 0.4 mL/min, column 40 °C, injection volume 3 μL, run time 28 min.

Mass Spectrometry:

• Q Exactive Plus Orbitrap with HESI-II source, polarity switching for targeted PRM and t-SIM scans.
• Resolution 35,000–70,000 (full width at half maximum), mass isolation width 1.5 m/z, AGC target and injection times optimized for each analyte.

Used Instrumentation

• Thermo Scientific Vanquish Flex Quaternary UHPLC system with diode array detector and split sampler.
• Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer.
• Optima LC/MS solvents (water, methanol, acetonitrile, formic acid).
• Clean Chem nitrosamine impurity standards.
• Laboratory equipment: analytical balance, vortex mixer, centrifuge, syringe filters.

Key Results and Discussion

• All nine nitrosamines achieved limits of detection down to 0.1–0.5 ng/mL and LOQs of 0.5–2.0 ng/mL with signal-to-noise ratios >30.
• Linearity over 0.5–20 ng/mL yielded correlation coefficients (R2) ≥0.997.
• Reproducibility at LOQ and standard levels showed %RSD ≤7% across analytes.
• Recovery studies in sartan and metformin matrices (1, 3, 20 ng/mL spikes) returned 83–114% across all compounds.
• High mass resolution (≥120,000) effectively resolved near-isobaric interferences (e.g., NDMA vs. DMF isotope) without compromising sensitivity.

Benefits and Practical Applications

The validated method enables rapid, sensitive, and accurate monitoring of multiple nitrosamine impurities in diverse pharmaceutical matrices. Its single-run capability and small injection volume support high throughput and can be easily integrated into quality control workflows to meet stringent regulatory standards.

Future Trends and Potential Applications

Future developments may include lower detection limits through larger injection volumes or enhanced sample concentration techniques, application to additional drug classes, integration with automated sample handling systems, and real-time data evaluation using advanced software or artificial intelligence to further streamline impurity monitoring.

Conclusion

The presented LC-HRAM Orbitrap MS method demonstrates a robust, sensitive, and multiplexed approach for quantifying nine nitrosamine impurities at sub-ppb levels in sartan and metformin products. High resolution and tailored acquisition parameters ensure reliable differentiation of target analytes from interferences, supporting compliance with regulatory requirements and enhancing pharmaceutical quality assurance.

References

1. U.S. FDA. Control of Nitrosamine Impurities in Human Drugs – Guidance for Industry.
2. Rostkowska K., et al. Formation and metabolism of N-Nitrosamines. Pol. J. Environ. Stud. 1998;7:321–325.
3. U.S. FDA. LC-ESI-HRMS Method for Determination of Nitrosamines in Metformin.
4. U.S. FDA. LC-HRMS Method for Determination of NDMA in Metformin.
5. U.S. FDA. LC-HRMS Method for Six Nitrosamine Impurities in ARB Drugs.
6. Thermo Scientific Application Note 21922. GC-MS Determination of Genotoxic Nitrosamines in Valsartan.
7. Thermo Scientific. HRAM LC-MS Method for Determination of Nitrosamines in Drugs.