Simultaneous HPLC determination of tolbutamide, phenacetin and their metabolites as markers of cytochromes 1A2 and 2C6/11 in rat liver perfusate

A new, simple, rapid, sensitive, and repeatable reversed-phase HPLC method was developed and validated for the simultaneous determination of tolbutamide, phenacetin and their metabolites in rat liver perfusate. Chlorpropamide was used as an internal standard to ensure the precision and accuracy of this method. Analytes were extracted into diethyl ether using a two-step liquid–liquid extraction. A C18 analytical column and a mobile phase composed of acetonitrile and potassium phosphate buffer were used for the chromatographic separation with UV detection. Limits of detection varied between 20 and 46 ng/mL for phenacetin, tolbutamide and their metabolites. The overall extraction recovery for the analytes varied from 65.4% in paracetamol to 88.0% in tolbutamide for concentrations within the expected range of concentrations from previous experimental samples. In terms of precision, the intra- and inter-day variation at three different concentrations in all analytes never exceeded 7.6 and 11.4%, respectively. This method is applicable for the modeling and description of possible pharmacological interactions on rat cytochromes P450 1A2 and 2C6/11 or can be used for in vitro evaluation of both cytochromes 1A2 and 2C.