( olite characterization is to 1) obtain ...)In-Vitro Metabolism Studies Using Data-Dependent LC/MSn

olite characterization is to 1) obtain MSⁿ data on the unmetabolized drug, (used as a reference for following experiments), 2) perform a Data-Dependent experiment to screen the metabolites, and 3) conduct selective multi-stage MSⁿ experiments to locate more specifically the site of metabolism. Since the samples utilized microsomal preparations fortified with NADPH, only oxidative metabolism occurred. This made the analysis slightly simpler since the most likely metabolic products were an unmodified parent, mono-oxygenated metabolites, and possibly di- or tri-oxygenated metabolites. A list of ions corresponding to the [M+H]⁺ for glyburide and its potential metabolites was entered in the method setup for the analysis in order to prevent the instrument from obtaining spectra on irrelevant, but potentially intense, ions in the samples.

The LCQ was set up to perform the following Data-Dependent experiment. When one of the ions from the list was detected in MS¹ (and above a user-defined threshold), the mass spectrometer automatically acquired a product ion mass spectrum (MS²) for this ion. Next, a second order product ion (MS³) mass spectrum was collected for the base peak from the MS² spectrum. This MS¹/MS²/MS³ sequence was repeated throughout the duration of the chromatographic peak. At the end of the peak, the mass spectrometer returned to MS¹ mode until another ion from the mass list was detected and the cycle was repeated for this new ion.

Having obtained MS¹, MS² and MS³ data, the retention time, molecular weight and significant structural information were obtained in t
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