Simultaneous determination of clofibrate and its active metabolite clofibric acid in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet absorbance detection

Abstract

A reversed-phase high-performance liquid chromatographic (HPLC) using ultraviolet (UV) absorbance detection method for simultaneous determination of clofibrate ( I) and its major metabolite clofibric acid ( II) in human plasma has been developed to support a clinical study. I, II and internal standard (I.S., III) are isolated from human plasma by 96-well solid-phase extraction (SPE) C 18⋅AR plate and quantified by direct injection of the SPE eluent onto the HPLC with UV detection wavelength at 230 nm. Two chromatographic methods, isocratic and step gradient, have been validated from 1.0 to 100.0 μg/ml and successfully applied to plasma sample analysis for a clinical study. The lower limit of quantitation (LLOQ) is 1.0 μg/ml for both I and II when 500 μl plasma sample is processed. Sample collection and preparation is conducted at 5 °C to minimize the hydrolysis of I to II in human plasma.