Sensitive method for the determination of KC 12291 in rat plasma and urine by high-performance liquid chromatography

A sensitive and selective ion-pair reversed-phase HPLC method has been developed for the quantitative determination of KC 12291 and its major metabolite, KC 13194, in rat plasma and urine. An Ultrasphere ODS column constructed by using a mobile phase of 1 mM 1-octanesulfonic acid containing acetonitrile-0.1 M triethylamine phosphate buffer, pH 2.2 (29:71, v/v in plasma and 27:73, v/v in urine), an internal standard and a fluorescent detector (excitation 265 nm, emission 370 nm) were used for the separation and the quantitative determination, respectively. The plasma samples were made alkaline and both compounds were cleaned up by the use of liquid-liquid extraction. The limit of quantification was 10 ng/ml for KC 12291 in plasma and urine and for KC 13194 and 100 ng/ml in plasma, respectively. The assay has been validated with respect to system suitability, accuracy, precision, recovery, stability and ruggedness. All validated parameters were found to be within the necessary limits.