Abstract
A high-performance liquid chromatographic (HPLC) assay is described for the determination of the new antitumour drug N-benzoylstaurosporine (CGP 41 251; I) and four of its potential metabolites in micro-volumes (100 microliters) of plasma. After addition of an internal standard, the compounds were isolated from plasma by liquid-liquid extraction with diisopropyl ether. Chromatography was carried out using a 5 microns LiChrospher C-18 end-capped column (125 x 4.0 mm i.d.) and binary gradient elution with acetonitrile and a triethylamine-containing phosphate buffer (pH 3.6) as solvents. Fluorimetric detection was performed with excitation and emission wavelengths set at 286 and 386 nm, respectively. The absolute recovery was more than 98% for all of the investigated compounds. The limit of detection (LOD) for I and three metabolites was 0.1 ng ml-1 and the lower limit of quantitation (LLQ) was 0.2 ng ml-1 in 100 microliters of plasma. The LOD and LLQ for the fourth metabolite was 0.25 and 0.5 ng ml-1, respectively. The between-day and within-day precisions were always < 15% for all the analytes. A limited pharmacokinetic study in mice treated and with I demonstrated that the method is appropriate for this purpose.