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.