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.
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.
Basha
Shaik Jafar Sadik
SJ
Drug Metabolism and Pharmacokinetics, Discovery Research, Dr. Reddy's Laboratories Ltd., Miyapur, Hyderabad 500 049, India.
Naveed
Shaik Abdul
SA
Tiwari
Nirbhay Kumar
NK
Shashikumar
Dhanya
D
Muzeeb
Syed
S
Kumar
Thammera Ranjith
TR
Kumar
Nyavanandi Vijay
NV
Rao
Nadipalli Prabhakar
NP
Srinivas
Nanduri
N
Mullangi
Ramesh
R
Srinivas
Nuggehally R
NR
D000818:Animals; D001384:Azetidines / Q000008:administration & dosage / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D001646:Bile / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000069438:Ezetimibe; D020719:Glucuronides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D008297:Male; D015394:Molecular Structure; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D053719:Tandem Mass Spectrometry
D001384:Azetidines; D020719:Glucuronides; C511436:ezetimibe glucuronide; D000069438:Ezetimibe