Liquid chromatographic quantitation of the lactone and the total of lactone and carboxylate forms of 9-nitrocamptothecin in human plasma

Abstract

Simple and sensitive high-performance liquid chromatography (HPLC) assays were developed and validated for the quantitation of the investigational anticancer drug 9-nitrocamptothecin (9-NC) as the lactone form and as the total of the lactone(I) and carboxylate(II) forms in human plasma. For the assay of lactone form (9NC-lac), the analytical method involved a protein precipitation step with adding a mixture of cold acetonitril-chloroform (5:1 (v/v), -20 degrees C) to plasma sample that stabilized the pH-dependent conversion of I to II. After evaporation under gentle stream of nitrogen gas (40 degrees C) the dry extract was dissolved in mobile phase (pH 5.5). For determination of the total of the lactone and carboxylate forms of the drug (9NC-tot), plasma samples were deproteinated with cold acetonitril (-20 degrees C) acidified with perchloric acid (5%), which resulted in the conversion of the carboxylate into the lactone form. After centrifugation the upper solvent was evaporated (nitrogen, 40 degrees C) and the dry extract was dissolved in mobile phase (pH 3.5). All separations were performed on a RP-C(8) column, using a mixture of acetonitril-water as eluent (pH 3.5 for total form and pH 5.5 for lactone form) and UV detection. The presented assay was linear over a concentration range of 25-1500 ng/ml with lower limit of quantitation of 25 ng/ml for both 9NC-tot and 9NC-lac. Within-run and between-run precision was always less than 7.5% in the concentration range of interest. The reported assay method showed good characteristics of linearity, sensitivity, selectivity and precision allowing applying in pharmacokinetic studies.