Reliable quantitation of 11 nitrosamine impurities in metformin drug products using Orbitrap Exploris 120 mass spectrometry

Significance of the Topic

The presence of nitrosamine impurities in metformin poses a serious genotoxic and carcinogenic risk, prompting regulatory agencies such as the U.S. FDA and EMA to enforce stringent limits. Reliable detection and quantitation of these compounds at trace levels are critical for ensuring patient safety and compliance with updated guidelines.

Objectives and Study Overview

This study aimed to develop and validate a high-resolution accurate-mass LC-MS method to quantify eleven nitrosamines in extended-release metformin tablets. The approach targets clear chromatographic separation and meets the U.S. FDA daily acceptable intake threshold of 26.5 ng/day per analyte panel.

Methodology and Sample Preparation

Reagents and consumables included UHPLC-MS-grade solvents, formic acid, deuterated internal standards, and metformin tablets. Key steps:

• Extraction: Ground tablets were extracted with methanol containing internal standards.
• Filtration: Supernatant was cleared by cold centrifugation and passage through a 100 kDa cutoff filter.
• Calibration: Neat standards ranged from 0.2 to 100 ng/mL; spiked samples covered 0.2–100 ng/mL.
• Analysis: Targeted selected ion monitoring (tSIM) and data-dependent MS2 (tMS2) modes for optimal sensitivity.

Used Instrumentation

The analytical setup comprised:

• Thermo Scientific Vanquish Horizon UHPLC system with Hypersil GOLD C18 column (150 × 4.6 mm, 3 µm).
• Orbitrap Exploris 120 mass spectrometer operated in HESI or APCI ionization modes.
• Chromeleon 7.2.10 CDS for compliant data acquisition and processing.

Main Results and Discussion

The method achieved baseline separation of metformin and eleven nitrosamines, including NDMA, NMBA, and NMPA. Chromatographic diversion prevented source contamination by high-concentration metformin. Instrumental LODs ranged from 0.2 to 1 ng/mL and LOQs from 0.5 to 2 ng/mL, corresponding to sub-20 ppb in drug product. Accuracy was within 87–99% and precision below 9% RSD. APCI enhanced detection of NDEA, NDIPA, and NDPA, while HESI was preferable for labile NMBA.

Benefits and Practical Applications

The validated method offers high selectivity and sensitivity for routine screening of nitrosamine impurities in pharmaceutical quality control. Compliance-ready workflows support U.S. FDA 21 CFR Part 11 and EU Annex 11 regulations, ensuring data integrity and audit readiness.

Future Trends and Potential Applications

Emerging directions include expanding panels to additional nitrosamines, adapting methods for biologics and complex formulations, integrating automated sample handling, and leveraging higher-resolution Orbitrap platforms to improve throughput and detection capabilities.

Conclusion

This fit-for-purpose LC-HRAM-MS approach on the Orbitrap Exploris 120 provides robust, trace-level quantitation of eleven nitrosamines in metformin, meeting global regulatory requirements and facilitating enhanced patient safety.

Reference

1. US FDA (2020) Control of Nitrosamine Impurities in Human Drugs. Guidance for Industry.
2. EMA (2020) Nitrosamine Impurities in Human Medicinal Products. EMA/369136/2020.
3. Thermo Fisher Scientific (2021) Application Note 73814: HRAM LC-MS Method for Nitrosamine Impurities.
4. Yang H, Bardsley J, Du M (2020) A cautionary tale: Quantitative LC-HRMS procedures for N-nitrosodimethylamine in metformin. AAPS Journal 22:89.
5. Thermo Fisher Scientific (2021) Application Note 74059: LC-MS/MS Quantitation of Nitrosamines in Metformin.