The quantitative determination of cilostazol and its four metabolites in human liver microsomal incubation mixtures by high-performance liquid chromatography

A high-performance liquid chromatography-ultraviolet (HPLC-UV) method for t he quantitation of cilostazol and four of its principal metabolites (i.e. O PC-13015, OPC-13213, OPC-13217 and OPC-13326) in human liver microsomal sol utions was developed and validated. Cilostazol, its metabolites, and the in ternal standard (OPC-3930), were analyzed by protein precipitation followed by reverse-phase HPLC separation on a TSK-Gel ODS-80TM (150 x 4.6 mm, 5 mu m) column and a Cosmil C-18 column (150 x 4.6 mm, 5 mu m) in tandem and UV detection at 254 nm. An 80 min gradient elution of mobile phase acetonitri le in acetate buffer (pH = 6.50) was used to obtain quality chromatography and peak resolution. All the analytes were separated from each other, with the resolution being 2.43-17.59. The components of liver microsomal incubat ion mixture and five metabolic inhibitor probes (quinidine sulfate, diethyl dithiocarbamate (DEDTC), omeprazole, ketoconazole and furafylline) did not interfere with this analytical method. The LOQ was 1000 ng ml(-1) far cilo stazol and 100 ng ml(-1) for each of the metabolites. This method has been validated for linear ranges of 100-4000 ng ml(-1) for OPC-13213, OPC-13217 and OPC-13326; 100-2000 ng ml(-1) for OPC-13015; and 1000-20000 ng ml(-1) f or cilostazol. The percent relative recovery of this method was established to be 81.2-101.0% for analytes, with the precision (% coefficient of varia tion (CV)) being 2.8-7.7%. The autosampler stability of the analytes was ev aluated and it was found that all analytes were stable at room temperature for a period of at least 17 h. This assay has been shown to be precise, acc urate and reproducible. (C) 1998 Elsevier Science B.V. All rights reserved.