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

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 °C) to plasma sample that stabilized the pH-dependent conversion of I to II. After evaporation under gentle stream of nitrogen gas (40 °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 °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 °C) and the dry extract was dissolved in mobile phase (pH 3.5). All separations were performed on a RP-C8 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.