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Factors Affecting the Limit of Detection For HPLC/Tandem Mass Spectrometry Experiments Based on Gas-phase Ion-Molecule Reactions.

Diagnostic and predictable gas-phase ion-molecule reactions have emerged as a potential alternative to collision-activated dissociation in tandem mass spectrometry (MS2) experiments performed to gain structural information for unknown organic compounds, such as drug metabolites, in complex mixtures. However, the applicability of this approach for analyzing metabolites at physiologically relevant concentrations has not been determined. In this study, HPLC/MS2 experiments based on gas-phase ion-molecule reactions of protonated model compounds were successfully conducted at nanomolar and picomolar analyte concentrations. As the analyte concentration decreased, the signal-to-noise ratio of the HPLC peaks decreased more than the signal-to-noise ratio of the mass spectrometer peaks. Therefore, the HPLC part of this analysis was the primary limiting factor for each analyte (rather than the ion-molecule reactions). The ion-molecule reaction limits of detection ranged from 50 pM to 250 nM, with the average being 50-100 nM. Since all compounds had ion-molecule reaction detection limits below 500 nM, the detection limits are within the physiologically relevant range for in vivo studies of drugs and drug metabolites. When considering only mass spectrometry, the number of ion isolation events (one in MS2 experiments involving ion-molecule reactions or two in MS3 experiments involving CAD of products formed upon ion-molecule reactions) and the subsequent CAD in the MS3 experiments were the most important limiting factors. Indeed, the limit of detection for the MS3 experiments was 250 nM, about three times higher than the average ion-molecule reaction detection limit of 75 nM but still within physiologically relevant concentrations.