( his one analysis. Additional structural...)In-Vitro Metabolism Studies Using Data-Dependent LC/MSn

his one analysis. Additional structural data were collected from subsequent LC/MSⁿ analyses designed to collect MSⁿ data for specific ions of interest. Although the Data Dependent analyses provide a tremendous amount of data from a single chromatographic analysis, a simple LC/MS analysis is still frequently valuable, especially when there are closely eluting analytes with the same molecular weight. The data from an LC/MS analysis will have more data points (since it is not interrupted with MSⁿ scans) to describe the chromatographic peaks better and reveal shoulders or minor peaks more clearly.

Results and Discussion

Glyburide was characterized using MS², MS³, and MS⁴ experiments in positive ion mode during an infusion of a 1 g/mL stock solution. A 7 isolation width was used to collect both 35Cl- and 37Cl-containing ions. Thus, the product ions included the Cl isotope pattern. These data are summarized in Scheme 1. These spectra were used as references to aid in interpretation of the spectra of metabolites. Shifts in masses observed in spectra for metabolites relative to spectra for glyburide, as well as differing fragmentation patterns facilitated characterization of the metabolite structures.

An LC/MSⁿ experiment was performed on a microsomal sample. The Data-Dependent analysis afforded MS¹, MS² and MS³ data providing retention time, molecular weight and structural information. The reconstructed ion chromatogram (RIC) reproduced in Figure 1 indicated that there were seven metabolites that resulted from the incorporation of a single oxygen molecule (at 8.72, 9.92, 10.65, 11.89, 13.01, 16.79 and 23.87 min.). The MS¹ spectra of all these metabolites were
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