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Analysis of a Verapamil Microsomal Incubation using Metabolite ID and Mass FrontierTM

using a FinniganTM LCQTM Advantage ion trap mass spectrometer equipped with a Finnigan Surveyor HPLC system. The mass spectrometer was operated in a data-dependent acquisition mode in which both MS and MS2 spectra were acquired without the need to specify parent masses. In this mode, the acquisition software probed the MS spectra in real-time on a scan-by-scan basis to select the two most intense parents ions for MS2 analysis. By analyzing both the first and the second most intense parent ions, information on co-eluting metabolites was obtained. Dynamic ExclusionTM was not employed in these analyses in order to avoid the possibility of missing modifications such as hydroxylations that can occur multiple times with closely spaced retention times. The ion source of the mass spectrometer was operated in the positive ion electrospray ionization mode. The HPLC separation was achieved using a 150 2.1 mm 5 μm BetaBasic 18 column (Thermo Electron) and the following HPLC gradient conditions:

Metabolite ID Analysis
Background subtraction is an optional step that was used for the verapamil example presented here. The background subtraction feature includes a scaling factor by which the control files intensity can be multiplied (to ensure complete background subtraction) and a time offset (to correct for any retention time drift between the source and the control file). Metabolite ID also provides a manual background subtraction feature that allows the user to specify a specific mass range and time range to be subtracted.

After the optional background subtraction, the appropriate data file is opened and the parent drugs of interest are specified as illustrated in Figure 1. Users can select from previously created drug lists or create new entries by pressing the new Item tab. The Modifications button allo
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