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Link to original content: https://pubmed.ncbi.nlm.nih.gov/17442643
Concurrent determination of ezetimibe and its phase-I and II metabolites by HPLC with UV detection: quantitative application to various in vitro metabolic stability studies and for qualitative estimation in bile - PubMed Skip to main page content
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. 2007 Jun 15;853(1-2):88-96.
doi: 10.1016/j.jchromb.2007.02.053. Epub 2007 Mar 15.

Concurrent determination of ezetimibe and its phase-I and II metabolites by HPLC with UV detection: quantitative application to various in vitro metabolic stability studies and for qualitative estimation in bile

Shaik Jafar Sadik Basha  1 , Shaik Abdul Naveed, Nirbhay Kumar Tiwari, Dhanya Shashikumar, Syed Muzeeb, Thammera Ranjith Kumar, Nyavanandi Vijay Kumar, Nadipalli Prabhakar Rao, Nanduri Srinivas, Ramesh Mullangi, Nuggehally R Srinivas
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Concurrent determination of ezetimibe and its phase-I and II metabolites by HPLC with UV detection: quantitative application to various in vitro metabolic stability studies and for qualitative estimation in bile

Shaik Jafar Sadik Basha et al. J Chromatogr B Analyt Technol Biomed Life Sci. .

Abstract

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 microL of either incubation or bile sample containing analytes and internal standard (IS, theophylline) with 75 microL acetonitrile containing 25% perchloric acid. An aliquot of 100 microL supernatant was injected onto a C18 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 microg/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.

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