Determination of Nitrosamine Impurities Using the Agilent 6495D Triple Quadrupole LC/MS System

Significance of the topic

Nitrosamine impurities are highly potent genotoxic and probable carcinogenic byproducts that can form during pharmaceutical synthesis. Regulatory bodies have recalled several ARB drugs due to nitrosamine contamination, driving the need for ultra-sensitive and robust analytical methods for routine quality control and regulatory compliance. The ability to detect and quantify nitrosamines at low ppt levels is critical to ensure drug safety and protect public health.

Objectives and overview of the study

The study aimed to evaluate the quantification performance of eight representative nitrosamine compounds using the Agilent 6495D triple quadrupole LC/MS system coupled with an Agilent 1290 Infinity II bio LC and APCI source. By establishing calibration curves, assessing limits of quantitation, precision, and accuracy, the work demonstrates method suitability for trace-level analysis in both solvent and pharmaceutical matrices.

Methodology and instrumentation

Sample preparation involved spiking nitrosamine standards into solvent blanks (methanol:water 10:90) across nine concentration levels (0.0125–10 ng/mL). A losartan potassium drug product extract was prepared (100 mg dissolved in methanol:water 50:50, sonicated, centrifuged, diluted) and spiked at 0.005–20 ng/mL.

• Liquid chromatography: Agilent 1290 Infinity II bio LC with Poroshell 120 EC-C18 column (3.0×150 mm, 2.7 µm), 0.5 mL/min flow, 0.1% formic acid in water and methanol gradient, column at 40 °C, injection volume 20 µL.
• Mass spectrometry: Agilent 6495D triple quadrupole in positive APCI mode, gas temperature 300 °C, drying gas 7 L/min, vaporizer temperature 350 °C, corona current 4 µA, MRM with iFunnel fragile mode, data acquired using MassHunter 12.1.
• Quantitative settings: two MRM transitions per analyte, 1/x weighting, linear regression with origin ignored.

Main results and discussion

The method achieved excellent chromatographic separation and peak shape for all eight nitrosamines, with retention times ranging approximately 2.5–13.5 minutes. Calibration curves exhibited R² values above 0.997 across 0.005–20 ng/mL. Limits of quantitation were at or below 0.01 ng/mL (10 ppt) for all compounds, meeting regulatory sensitivity requirements.
Precision (%RSD) was below 5% and accuracy within 80–120% at the LLOQ and higher levels. In losartan matrix, NDMA was quantified at 0.03 ng/mL with S/N > 22 and excellent reproducibility, demonstrating negligible matrix interference and robust performance in a real drug extract.

Benefits and practical application

• High sensitivity and specificity enable confident trace analysis of genotoxic impurities.
• Compatibility with routine bio-LC platforms and triple quadrupole detection supports seamless integration into existing QC workflows.
• Fast analysis (<20 min) and reliable MRM transitions facilitate high-throughput screening of multiple nitrosamines across various drug matrices.
• Compliance with regulatory guidelines for nitrosamine limits in final pharmaceutical products.

Future trends and applications

• Extension to additional nitrosamine analogues and metabolites as regulatory scopes evolve.
• Integration with automated sample preparation and high-throughput platforms to streamline routine monitoring.
• Adoption of high-resolution mass spectrometry for non-targeted screening of unknown nitrosamine impurities.
• Implementation of machine learning algorithms for enhanced peak detection and data quality assessment in complex matrices.

Conclusion

The Agilent 6495D triple quadrupole LC/MS method provides a robust, sensitive, and accurate approach to quantify eight key nitrosamine impurities down to 10 ppt levels. Its demonstrated performance in both solvent and losartan matrix highlights its suitability for regulatory-driven pharmaceutical quality control and ensures patient safety through reliable detection of genotoxic contaminants.

References

• FDA Updates and Press Announcements: Angiotensin II receptor blocker (ARB) recalls (2023).
• Control of Nitrosamine Impurities in Human Drugs: Guidance for Industry. U.S. FDA (2021).
• Nitrosamines Impurities in Human Medicinal Products: EMA Assessment Report (2020).