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N-nitrosamine analysis has raised significant concerns in the pharmaceutical industry since 2018, when these potential carcinogens were found in several angiotensin II receptor blockers (sartans). Subsequent discoveries in ranitidine and some slow-release metformin medications prompted widespread product recalls. The industry has implemented stricter manufacturing requirements and intensified efforts to evaluate and control N-nitrosamine contamination. To meet stringent regulatory requirements, it is essential to develop effective chromatography and mass spectrometry (MS) methods for N-nitrosamine analysis. This blog addresses the challenges associated with detecting N-nitrosodimethylamine (NDMA) in metformin drug products.

Analytical requirements for N-nitrosamine assays

A comprehensive N-nitrosamine assay involves 3 main components: sample preparation, chromatographic separation and MS measurement. Sample preparation aims to solubilize the target analyte, ensuring suitability for high-performance liquid chromatography (HPLC) analysis. However, the volatile and small molecular size of nitrosamines, particularly NDMA, require careful handling to prevent recovery issues. Chromatography plays a crucial role in separating the analyte from the active pharmaceutical ingredient (API) and achieving reliable peak shapes for accurate quantitation. MS is used for detection and quantitation, requiring sensitivity and selectivity to analyze low levels of nitrosamines in complex matrices.

Challenges in chromatography and MS techniques

Analyzing NDMA in metformin poses specific challenges due to the small size and high polarity of both the drug and the contaminant. Additionally, extended-release formulations present difficulties during sample preparation, often resulting in unsuitable sample matrices for analysis that uses liquid chromatography combined with mass spectrometry (LC-MS). A study involving 9 metformin lots aimed to overcome these challenges.

Addressing the challenges: analysis of NDMA in metformin

This study followed a published US Food and Drug Administration (FDA) method for metformin sample preparation that involves crushing tablets to a powder and dissolving them in pure methanol. Vortex mixing and sonication enable complete exposure of the sample to the solvent. The samples were clarified through centrifugation and the supernatant was extracted to ensure suitability for analysis.

To select the appropriate chromatography column for NDMA analysis, various stationary phases were compared using NDMA diluted in metformin. The chosen column for subsequent analysis was the Luna Omega PS C18 column from Phenomenex, which demonstrated clear separation and acceptable peak shape for quantitation. Incorporating a divert valve enabled selective collection of analyte peaks for MS analysis, preventing contamination of the mass spectrometer with the API and other formulation materials.

The study utilized the QTRAP 6500+ system from SCIEX, including a SelexION device for performing differential mobility separation (DMS). The addition of the SelexION device enhanced selectivity and specificity by separating ions orthogonally before tandem MS analysis. An atmospheric pressure chemical ionization (APCI) step was also used for precise quantitation and identification. The calibration using methanol samples and spiked samples allowed accurate quantitation of NDMA in metformin. The method achieved good reproducibility, linearity and sensitivity, with an improved signal-to-noise ratio and a lower limit of quantitation (LLOQ) of 2.

Analyzing similar contamination issues in the future

By combining these strategies and continually refining analytical methodologies, researchers are working towards a reliable and robust method for future similar drugs and contamination issues. To learn more, visit our website

Resources

https://link.springer.com/article/10.1208/s12248-020-00473-w

https://www.phenomenex.com/ViewDocument/?id=lc-mrm-ms%20method%20for%20the%20detection%20and%20quantification%20of%20six%20nitrosamine%20impurities%20in%20sartan%20%28arbs%29%20drugs