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. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.