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

A high-performance liquid chromatographic (HPLC) method for the simultaneou s determination of cilostazol, a quinolinone derivative, and its known meta bolites OPC-13015, OPC-13213, OPC-13217, OPC-13366, OPC-13269, OPC-13326 an d OPC-13388 in human plasma was developed and validated. Cilostazol, its me tabolites and two internal standards, OPC-3930 and OPC-13112, were extracte d from human plasma by a combination of liquid-liquid and liquid-solid phas e extractions, with combined organic solvents of rt-butanol, methanol, chlo roform, 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 HPL C system and was subjected to reversed-phase HPLC on a 5 mu m ODS-80TM colu mn to obtain quality chromatograph and good peak resolution. A gradient mob ile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites a nd the two internal standards were well separated at baseline from each oth er with resolution factor being 74 and 138. This HPLC method was demonstrat ed to be specific for all analytes of interest with no significant interfer ence from the endogenous substances of human plasma. The lower limit of qua ntitation was 20 ng/ml for cilostazol and all metabolites. The method was v alidated initially for an extended linear range of 20-600 ng/ml for all met abolites and cilostazol, and has been revised later for a linear range of 2 0-1200 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 i ndicated that there was no significant difference for all accuracy and prec ision obtained by using either OPC-3930 or OPC-13112. OPC-3930 was chosen a s the internal standard for the analysis of plasma samples from clinical st udies due to its shorter retention time. During the validation standard cur ves had correlation coefficients greater than or equal to 0.998 for cilosta zol and the seven metabolites. These data clearly demonstrate the reliabili ty and reproducibility of the method. (C) 1999 Elsevier Science B.V. All ri ghts reserved.