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

Abstract

A high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the quantitation of cilostazol and four of its principal metabolites (i.e. OPC-13015, OPC-13213, OPC-13217 and OPC-13326) in human liver microsomal solutions was developed and validated. Cilostazol, its metabolites, and the internal standard (OPC-3930), were analyzed by protein precipitation followed by reverse-phase HPLC separation on a TSK-Gel ODS-80TM (150×4.6 mm, 5 μm) column and a Cosmil C-18 column (150×4.6 mm, 5 μm) in tandem and UV detection at 254 nm. An 80 min gradient elution of mobile phase acetonitrile 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 incubation 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 for cilostazol 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 1000–20 000 ng ml −1 for cilostazol. The percent relative recovery of this method was established to be 81.2–101.0% for analytes, with the precision (% coefficient of variation (CV)) being 2.8–7.7%. The autosampler stability of the analytes was evaluated 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, accurate and reproducible.