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The initial flow rate was 0.5mL min. Mobile phase A was 0.1% formic acid in water and mobile phase B was 100% acetonitrile. Mobile phase B was linearly ramped from 0% to 50% in 3 minutes, held at 50% for 0.5 minutes then immediately stepped back down to 0% at a flow rate of 0.75mL min for reequilibration. Total run time was 4.5 minutes. Multiple reaction monitoring of the + ve substrate metabolite ions with specific collision product ions was monitored using Micromass MassLynx 3.5 software as in Table 1. Control samples with no inhibitor ; were assayed in each analytical run. The amount of metabolite in each sample relative to the control samples ; was plotted versus the concentration of the inhibitor present. IC50 values calculated using Prism GraphPad software, version 3.02 and amaryl.
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