A Diagnostic Nitrosamine Detection Approach for Pharmaceuticals by Using Tandem Mass Spectrometry Based on Diagnostic Gas-Phase Ion-Molecule Reactions.

<i>N</i>-Nitrosamines are strictly regulated in pharmaceutical products due to their carcinogenic nature. Therefore, the ability to rapidly and reliably identify the <i>N</i>-nitroso functionality is urgently needed. Unfortunately, not all ionized <i>N</i>-nitroso compounds produce diagnostic fragment ions and hence tandem mass spectrometry based on collision-activated dissociation (CAD) cannot be used to consistently identify the <i>N</i>-nitroso functionality. Therefore, a more reliable method was developed based on diagnostic functional-group selective ion-molecule reactions in a linear quadrupole ion trap mass spectrometer. 2-Methoxypropene (MOP) was identified as a reagent that reacts with protonated <i>N</i>-nitrosamines in a diagnostic manner by forming an adduct followed by the elimination of 2-propenol (CH₃C(OH)═CH_<b>2</b>]). From 18 protonated <i>N</i>-nitrosamine model compounds studied, 15 formed the diagnostic product ion. The lack of the diagnostic reaction for three of the <i>N</i>-nitrosamine model compounds was rationalized based on the presence of a pyridine ring that gets preferentially protonated instead of the <i>N</i>-nitroso functionality. These <i>N</i>-nitrosamines can be identified by subjecting a stable adduct formed upon ion-molecule reactions with MOP to CAD. Further, the ability to use ion-molecule reactions followed by CAD to differentiate protonated <i>O</i>-nitroso compounds with a pyridine ring from analogous <i>N</i>-nitrosamines was demonstrated This methodology is considered to be robust for the identification of the <i>N</i>-nitroso functionality in unknown analytes. Lastly, HPLC/MS² experiments were performed to determine the detection limit for five FDA regulated <i>N</i>-nitrosamines.