Development of LC-MS method for nitrosamine impurities separation and quantification

Abstract

Objective: Development of a liquid chromatographic method for separation and identification of the most important nitrosamine impurities, in a relatively short time and with a good solution represented the main focus point of the article. The presence of nitrosamine impurities in various pharmaceutical forms is an element of concern, a number of drugs have been reassessed from the point of view of the possible presence of these impurities. Reverse phase HPLC-UV methods or LC-MS detection are intensively applied. The mentioned methods present some limitations related to the polar character of some nitrosamines, which leads to their column retention, in this respect, it is important to identify the optimal proportion of organic solvent in the mobile phase.

Methods: The following impurities of the N-nitrosamine class are quantified by the LC-MS technique: NDMA (N-nitrosodimethylamine), NDEA (N-nitrosodiethylamine), NMEA (N-nitrosomethylethylamine) NDIPA (N-nitrosodiisopropylamine), NDBA (N-nitrosodibutylamine) NPIP (N-nitrosopiperidine). Nitrosamines standard solution were diluted in methanol with a final concentration of 20µg/ml. UHPLC chromatography system Flexar FX10 (Perkin Elmer) with MS QTOF- triplu quadrupol 4600 (AB Sciex TripleTOF4600) detector, APCI- Atmospheric pressure chemical ionization, Phenomenex Luna Omega 3 C18 (150x4,6mm, 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.

Results: Following the fragmentation of the masses of the 6 nitrosamines studied, the separation was managed under 25 minutes and the elution order was: NDMA, NMEA, NDEA, NPIP, NDIP, NDBA.

Conclusions: the development of optimal chromatographic conditions allows further separation and identification of nitrosamines impurities in pharmaceutical products.

Keywords: nitrosamines, impurities, chromatography, development