Objective: This research foucuses on the development of a liquid chromatographic method for the rapid and reliable separation and identification of major nitrosamine impurities, ensuring both short analysis time and adequate resolution. Given the toxicological relevance of nitrosamines, their occurrence in pharmaceutical formulations has raised substantial concerns, leading to the reassessment of multiple drug products. In response, reverse-phase HPLC with UV detection and LC-MS techniques have been widely applied as powerful analytical tools for their detection and control.
Methods: The following impurities of the N-nitrosamine class are separated and identified by the LC-MS technique: NDMA (N-nitrosodimethylamine), NDEA (N-nitrosodiethylamine), NMEA (N-nitrosomethylethylamine) NDIPA (N-nitrosodiisopropylamine), NDBA (N-nitrosodibutylamine) NPIP (N-nitrosopiperidine). A standard solution of nitrosamines mix was prepared and subsequently diluted in methanol to achieve a final concentration of 20 µg/mL for each compound. The analysis was performed using a UHPLC chromatography system Flexar FX10 (Perkin Elmer) with MS QTOF (AB Sciex TripleTOF4600), Phenomenex Luna Omega 3 C18 (150×4.6mm, 3μm) column, column temperature 450C, mobile phase methanol and formic acid 0.1% in ultrapure water, gradient elution, flow 0.45 mL/min., injected volume 5 µl. The proposed LC-MS conditions are significantly improved compared to the European Pharmacopoeia recommendations for N-Nitrosamines impurities in active substances analysis.
Results: Based on the mass fragmentation profiles of the six investigated nitrosamines, chromatographic separation was successfully accomplished in less than 25 minutes, with the elution sequence established as follows: NDMA, NMEA, NDEA, NPIP, NDIP, NDBA.
Conclusions: The development of optimal chromatographic conditions allows further separation and identification of nitrosamines impurities in pharmaceutical products.