228HPLC WITH UV AND MASS SPECTROMETRIC DETECTION FOR QUANTIFYING TOTAL EZETIMIBE IN HUMAN PLASMA: VALIDATION AND APPLICATION IN PHARMACOKINETIC STUDYIn this article, two different methods were developed and compared for quantifying total Ezetimibe (EZM) in human plasma. The total EZM was quantified with liquid chromatography method, after liquid-liquid extraction; one with UV detection (LC-UV) and the other with mass spectrometric detection (LC-MS/MS). Chromatographic separation was achieved on Symmetry Shield C18 column (250 × 4.6 mm i.d., 5 µ) using an isocratic elution with mobile phase composition consisting of Acetonitrile and 1mM Ammonium acetate in the proportion of 60:40 (v/v). For the estimation of total EZM, the glucuronide form of EZM is hydrolyzed by using β-glucuronidase and extracted using t-butyl methyl ether from human plasma samples. 20 µL was loaded onto the system and eluent was monitored at 233 nm. Mass spectrometric data was acquired in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 30 – 500 ng/mL (LC-UV) and 1.02 – 303.07 ng/mL (LC-MS/MS) with correlation coefficients r2 ≥ 0.997 and r2 ≥ 0.999, respectively. The inter-day coefficients of variation (CVs) ranged from 6.51 to 9.56% (LC-UV) and 0.78 to 4.21% (LC-MS/MS) and that of intra-day ranged from 6.78 to 9.81% (LC-UV) and 0.86 to 4.76% (LC-MS/MS) at four different concentrations. The resulting method was successfully applied for the Pharmacokinetic study in healthy human subjectspoonam.gaikwad@nmims.edu.inResearch432013Ezetimibe,β-glucuronidase,HPLC,Pharmacokinetics,Human plasma,LC-MS/MSPoonam Gaikwad,Ashok Bhagwat,Suvarna BhoirSchool of Science, SVKM’s NMIMS (deemed-to-be) University, Vile Parle (w), Mumbai-400056, India.,School of Science, SVKM’s NMIMS (deemed-to-be) University, Vile Parle (w), Mumbai-400056, India.,C. B. Patel Research Centre, Vile Parle (w), Mumbai-400056, India.