RP-HPLC测定羟基喜树碱脂质体在肿瘤细胞内的聚积

he intracellular accumulation of anti-cancer agents is strongly influence the efficiency of chemotherapy for cancer. In the present study, a simple, rapid, sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated to determine hydroxycamptothecin (HCPT) in Eca109 cells. Cells were treated with HCPT liposomes, and then lysed over ice by sonication. The HCPT in the samples were measured by RP-HPLC with a C18 column after a simple extraction with ethyl acetate. The mobile phase contained 0.1% triethylamine-phosphoric acid buffer (pH 3.0) and acetonitrile (75:25, v/v). The HCPT is monitored with fluorescence detector with excitation and emission wavelengths of 382 and 528 nm, respectively. The calibration curve was linear from 2 to 100ng/ml with correlation coefficient of 0.9999, while the limit of quantification is 2ng/ml. The mean extraction efficiency was 89.76%. The recovery of the assay was between 86.5 and 105.2%. The intra- and inter-day coefficients of variation were less than 10% (R.S.D.). Then the HCPT liposomes were prepared and the validated method was applied to intracellular accumulation study of both HCPT injection and HCPT liposomes in Eca109 cells. HCPT liposomes showed higher intracellular accumulation of HCPT in different incubation times compared with that of conventional HCPT injection.