On-column nitrosation of amines observed in liquid chromatography impurity separations employing ammonium hydroxide and acetonitrile as mobile phase

•We observed an on-column reaction with amines using HPLC at high pH.•We identified the products of the reaction as nitrosamines.•The reaction requires ammonium hydroxide and acetonitrile in the mobile phase.•The stainless steel column entrance frit is involved in providing a reactive metal.•Multiple compounds with amine groups formed the on-column impurities. The availability of high performance liquid chromatography (HPLC) columns capable of operation at pH values up to 12 has allowed a greater selectivity space to be explored for method development in pharmaceutical analysis. Ammonium hydroxide is of particular value in the mobile phase because it is compatible with direct interfacing to electrospray mass spectrometers. This paper reports an unexpected N-nitrosation reaction that occurs with analytes containing primary and secondary amines when ammonium hydroxide is used to achieve the high pH and acetonitrile is used as the organic modifier. The nitrosation reaction has generality. It has been observed on multiple columns from different vendors and with multiple amine-containing analytes. Ammonia was established to be the source of the nitroso nitrogen. The stainless steel column frit and metal ablated from the frit have been shown to be the sites of the reactions. The process is initiated by removal of the chromium oxide protective film from the stainless steel by acetonitrile. It is hypothesized that the highly active, freshly exposed metals catalyze room temperature oxidation of ammonia to NO but that the actual nitrosating agent is likely N2O3.