Data-dependent neutral gain MS3: toward automated identification of the N-oxide functional group in drug metabolites.

We report here an automated method for the identification of N-oxide functional groups in drug metabolites by using the combination of liquid chromatography/tandem mass spectrometry (LC/MS(n)) based on ion-molecule reactions and collision-activated dissociation (CAD). Data-dependent acquisition, which has been readily utilized for metabolite characterization using CAD-based methods, is adapted for use with ion-molecule reaction-based tandem mass spectrometry by careful choice of select experimental parameters. Two different experiments utilizing ion-molecule reactions are demonstrated, data-dependent neutral gain MS(3) and data-dependent neutral gain pseudo-MS(3), both of which generate functional group selective mass spectral data in a single experiment and facilitate increased throughput in structural elucidation of unknown mixture components. Initial results have been generated by using an LC/MS(n) method based on ion-molecule reactions developed earlier for the identification of the N-oxide functional group in pharmaceutical samples, a notoriously difficult functional group to identify via CAD alone. Since commercial software and straightforward, external instrument modification are used, these experiments are readily adaptable to the industrial pharmaceutical laboratory.