Highly sensitive and robust LC-HRAM-MS method for simultaneous quantitation of sixteen nitrosamines in multiple drug products

Significance of the Topic

Detection and quantitation of nitrosamine impurities in pharmaceuticals are critical due to their classification as probable carcinogens. Contamination events in sartans and metformin have led to global recalls and regulatory limits at low sub-ppm levels. A single, robust analytical method capable of monitoring multiple nitrosamines supports drug safety and regulatory compliance.

Objectives and Study Overview

This study aimed to develop and validate a highly sensitive LC-HRAM-MS method for simultaneous quantitation of 16 genotoxic nitrosamines in sartan and metformin drug products. Using a Thermo Scientific Vanquish Flex UHPLC system coupled to a Q Exactive Plus Orbitrap mass spectrometer, the method covers impurities ranging from NDMA and NDEA to NDPhA.

Instrumentation

A Vanquish Flex Quaternary UHPLC system with Hypersil GOLD and Hypercarb columns was employed along with a Q Exactive Plus Hybrid Orbitrap mass spectrometer. The HESI source and PRM/t-SIM acquisition modes enabled high resolution and accurate mass measurements.

Methodology and Sample Preparation

Certified nitrosamine standards were prepared in methanol to yield calibration levels from 0.3 to 50 ng/mL. Tablet samples were homogenized, extracted in methanol, centrifuged, and filtered before injection. Chromatography used a formic acid gradient at 0.4 mL/min with 10 µL injections and column temperature at 40 °C. MS parameters included positive and negative ion modes, isolation widths of 1.5 m/z, resolutions up to 70,000, and 15 ppm extraction tolerance.

Main Results and Discussion

The method achieved limits of detection of 0.08 ng/mL (0.008 ppm) and limits of quantitation at 0.3 ng/mL (0.003 ppm) with signal-to-noise ratios exceeding 19 for all analytes. Calibration curves were linear (R2 > 0.999) over the full range with 1/x weighting. Retention times separated metformin (< 6.8 min) and valsartan (~24.5 min) matrices. Intra-day reproducibility at LOQ and standard levels showed RSDs below 10%. Recovery studies in both matrices yielded 80–120% across low, medium, and high spikes.

Benefits and Practical Application

By consolidating the quantitation of 16 nitrosamines into a single LC-HRAM-MS run, this method streamlines laboratory workflows, reduces solvent and sample consumption, and ensures stringent quality control in API and drug product analysis. It directly addresses regulatory guidelines for nitrosamine limits and supports rapid screening in manufacturing environments.

Future Trends and Opportunities

Advances in high-resolution mass spectrometry and data processing are expected to expand multi-analyte monitoring capabilities, lower detection thresholds, and incorporate automated data review. Integration with artificial intelligence and larger impurity panels can further enhance early detection of genotoxic contaminants across diverse drug classes.

Conclusion

The validated LC-HRAM-MS method demonstrates the capability to detect and quantify 16 nitrosamine impurities with high sensitivity, precision, and accuracy in sartan and metformin products. Its robustness and compliance with regulatory requirements make it a valuable tool for pharmaceutical quality assurance.

References

• U.S. FDA. LC-HRMS Method for Six Nitrosamine Impurities in ARB Drugs.
• U.S. FDA. LC-ESI-HRMS Method for Nitrosamines in Metformin.
• U.S. FDA. LC-HRMS Method for NDMA in Metformin.
• U.S. FDA. Control of Nitrosamine Impurities in Human Drugs Guidance.
• Thermo Fisher Scientific Application Note 21922: GC-MS Determination of Nitrosamines.
• Thermo Fisher Scientific. HRAM LC-MS Determination of Nitrosamine Impurities.