Concurrent determination of ezetimibe and its Dhase-l and 11 metabolites by HPLC with UV detection: Quantitative application to various in vitro metabolic stability studies and for qualitative estimation in bile

Simultaneous separation and quantification of ezetimibe (EZM) and its phase-I metabolite i.e., ezetimibe ketone (EZM-K) and phase-II metabolite i.e., ezetimibe glucuronide (EZM-G) in various matrices was accomplished by gradient HPLC with UV detection. The assay procedure involved deproteinization of 500 mu L of either incubation or bile sample containing analytes and internal standard (IS, theophylline) with 75 mu L acetonitrile containing 25% perchloric acid. An aliquot of 100 mu L supernatant was injected onto a C-18 column. The chromatographic separation was achieved by gradient elution consisting of 0.05 M formic acid: acetonitrile: methanol: water at a flow rate of 1.0 mL/min. The detection of analyte peaks were achieved by monitoring the eluate using an UV detector set at 250 nm. Nominal retention times of IS, EZM-G, ezetimibe ketone glucuronide (EZM-KG), EZM and EZM-K were 9.39, 24.23, 27.82, 29.04 and 30.56 min, respectively. Average extraction efficiencies of EZM, EZM-G and IS was > 75-80% and for EZM-K was > 50% from all the matrices tested. Limit of quantitation (LOQ) for EZM, EZM-K and EZM-G was 0.02 mu g/mL. Due to the lack of availability of reference standard of EZM-KG, the recovery and LOQ aspects for this metabolite were not assessed. Overall, the method is suitable for simultaneous measurement of EZM, and its phase-I and phase-II metabolite (EZM-G) in in vitro and in vivo studies. (c) 2007 Elsevier B.V. All rights reserved.