Using MMDF in combination with HCD and CID MS/MS (Figure 2), 13 Irinotecan metabolites were identified from the incubation samples with an accuracy of greater than three ppm. All 13 metabolites were found with peak areas less than 1% of that of the parent and are well buried in the original chromatogram (Figure 3A).
The most abundant metabolite peaks become visible after applying a single MDF (Figure 3B), however, at this stage peaks from background matrix ions that are unrelated to the metabolite are still prominent. This is due to the fact that in order to use only one MDF to capture all of the metabolites, a relatively wide mass defect range has to be used. As such, a portion of the background ions remains after a single filter.
Next, four different mass defect filters are applied to the original data using the Thermo Scientific MetWorks software package that enables up to six mass defect filters to be applied at once. The resulting chromatogram is much cleaner (Figure 3C).
Figure 4 further illustrates how the full MS spectrum changed after mass defect filtering. The peak at 603.2805 m/z is a hydroxylation metabolite (M3) that elutes at 8.45 minutes. The original full scan MS is dominated by background ions and the M3 peak has less than 15% relative abundance (Figure 3A). After a single MDF was applied, the M3 peak becomes the base peak; however, there are still a lot of background peaks remaining in the spectrum (Figure 3B). After MMDFs were applied, only the M3 peak and trace of the parent remain in the spectrum while almost all the background ions are gone (Figure 4C).
The combination of HCD and CID provides fragmentation information. MMDF offers superior background discrimination as compared to a single filter and enables metabolites with peak areas less than 1% of that of the parent drug to be identified.