Simultaneous quantitative determination of cilostazol and its metabolites in human plasma by high-performance liquid chromatography

Abstract

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cilostazol, a quinolinone derivative, and its known metabolites OPC-13015, OPC-13213, OPC-13217, OPC-13366, OPC-13269, OPC-13326 and OPC-13388 in human plasma was developed and validated. Cilostazol, its metabolites and two internal standards, OPC-3930 and OPC-13112, were extracted from human plasma by a combination of liquid-liquid and liquid-solid phase extractions, with combined organic solvents of n-butanol, methanol, chloroform, methyl-tert.-butyl ether, and a Sep-Pak silica column. The combined extract was then evaporated and the residue was reconstituted in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto a HPLC system and was subjected to reversed-phase HPLC on a 5 microm ODS-80TM column to obtain quality chromatograph and good peak resolution. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the two internal standards were well separated at baseline from each other with resolution factor being 74 and 138. This HPLC method was demonstrated to be specific for all analytes of interest with no significant interference from the endogenous substances of human plasma. The lower limit of quantitation was 20 ng/ml for cilostazol and all metabolites. The method was validated initially for an extended linear range of 20-600 ng/ml for all metabolites and cilostazol, and has been revised later for a linear range of 20-1200 ng/ml for cilostazol and two major and active metabolites OPC-13015 and OPC-13213. The overall accuracy (relative recovery) of this method was established to be 98.5% to 104.9% for analytes with overall precision (CV) being 1.5% to 9.0%. The long-term stability of clinical plasma samples was established for at least one year at -20 degrees C. Two internal standards of OPC-3930 and OPC-13112 were evaluated and validated. However, the data indicated that there was no significant difference for all accuracy and precision obtained by using either OPC-3930 or OPC-13112. OPC-3930 was chosen as the internal standard for the analysis of plasma samples from clinical studies due to its shorter retention time. During the validation standard curves had correlation coefficients greater than or equal to 0.998 for cilostazol and the seven metabolites. These data clearly demonstrate the reliability and reproducibility of the method.