( cation of benchtop ion trap API mass sp...)In-Vitro Metabolism Studies Using Data-Dependent LC/MSn

cation of benchtop ion trap API mass spectrometry to characterize in-vitro metabolites is discussed. The utility of Data Dependent MS¹/MS²/MS³ analyses, where the mass spectrometer makes real-time decisions about the experiment to be performed, are demonstrated using the characterization of glyburide metabolites as an example.

Experimental Conditions

Microsomal fractions were prepared from rat, dog, monkey, and human liver as described previously.³ Reaction mixtures (250 L) with 5 or 50 M glyburide, 1 mg of microsomal protein/mL, 0.1 M potassium phosphate pH 7.25, 1 mM NADP, 10 mM glucose-6-phosphate, and 1 unit of glucose-6-phosphate dehydrogenase/mL were incubated at 37C for 30 min. They were then quenched by addition of 250 L of acetonitrile and the precipitate was removed by centrifugation. The supernatant was diluted with 500 L of 10 mM ammonium acetate, pH 5.0 before analysis. Metabolic products were separated using a Prodigy 5 mM C8 1502 mm column with a 102 mm guard column. Solvent A was 10 mM ammonium acetate, pH 5.0 and solvent B was acetonitrile. The metabolites were eluted using the following linear gradient: 0 min, 30%B; 30 min, 30%B; 35 min, 60%B; 60 min, 100%B; flow 0.2 mL/min. The mass spectrometer used was an LCQ. The entire 0.2 mL/min flow was directed into the source of the mass spectrometer without splitting, with the first 2.1 min diverted to waste using the built-in automated divert valve. The ion transfer tube was operated at 250C and sheath and auxiliary gases were set to 80 and 25, respectively. A relative collision energy of 25% was used for all MSⁿ experiments with an isolation width of 7.0 u to allow passage of 35Cl and 37Cl isotope peaks within a single scan.

Strategy

An effective strategy for metab
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