A two-step pH-dependent liquid–liquid extraction combined with HPLC-fluorescence method for the determination of 10-hydroxycamptothecin in mouse liver tissue
AbstractContext: Liquid–liquid extraction (LLE) shows high efficiency in the plasma sample preparation. However, this extraction method is not optimal for the biological samples containing complex organic interferences, such as liver and brain tissues. Some plant secondary metabolites can be converted between water-insoluble and water-soluble forms by pH adjustment. Objective: A two-step pH-dependent LLE method was introduced in this study to eliminate both water-soluble and lipidic interferences using the properties of pH-dependent interconvertible forms of analytes during sample preparation. A sensitive and reliable method using a reverse-phase HPLC coupled with a fluorescence detector was developed and validated. Materials and methods: 10-Hydroxycamptothecin (HCPT) with internal standard camptothecin and liver tissues were used as model compounds and biological samples. The lactone form of HCPT was converted to the water-soluble carboxylate form under moderate alkaline conditions, and the water-insoluble interferences were extracted with a nonpolar solvent. Afterward, the water-insoluble lactone form of HCPT was regenerated by acidification and then extracted using an organic solvent in a second LLE step. Results: The calibration curve was linear ( r 2 > 0.999) for HCPT concentrations ranging from 2.5 to 160 ng/mL. The mean recoveries of HCPT were 114.94 ± 3.98, 104.30 ± 2.44 and 95.90 ± 1.40% ( n = 6) at concentrations of 2.5, 10 and 80 ng/mL, respectively. The stability determination data showed that no significant degradation occurred under the experimental conditions. This method was successfully applied to liver tissue distribution study of HCPT in mice. Discussion and conclusion : This two-step LLE can be applied to distribution studies of compounds with pH-dependent interconvertible forms in other biological matrices.