Zhao Yong-Xing, Gao Jian-Qing, Qiao Hai-Ling, Chen Hai-Liang, Liang Wen-Quan
College of Pharmaceutical Sciences, Zhejiang University, 353 Yan'an Road, Hangzhou, Zhejiang 310031, PR China.
J Pharm Biomed Anal. 2006 Jun 7;41(3):1007-10. doi: 10.1016/j.jpba.2006.01.009. Epub 2006 Feb 23.
The intracellular accumulation of anti-cancer agents strongly influences the efficiency of chemotherapy for cancer. In the present study, a simple, rapid, sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated to determine hydroxycamptothecin (HCPT) in Eca109 cells. HCPT in cellular lysis solution were measured by RP-HPLC with a C18 column after extraction with ethyl acetate. The mobile phase contained 0.1% triethylamine-phosphoric acid buffer (pH 3.0) and acetonitrile (75:25, v/v). Fluorescence detector with excitation and emission wavelengths of 382 and 528 nm was used for determination of HCPT. The calibration curve was linear from 2 to 100 ng/ml with correlation coefficient of 0.9999, while the limit of quantification is 2 ng/ml. The recovery of assay was between 86.5 and 105.2%. The intra- and inter-day coefficients of variation were less than 10% (R.S.D.). Furthermore, the validated method was used to determine the accumulation of HCPT after incubating the liposomal formulation of HCPT and HCPT for injection with the intact cells. HCPT liposomes showed higher intracellular accumulation of HCPT at different incubation times compared with that of conventional HCPT injection.
抗癌药物在细胞内的蓄积对癌症化疗效果有很大影响。在本研究中，开发并验证了一种简单、快速、灵敏的反相高效液相色谱（RP-HPLC）法，用于测定Eca109细胞中的羟基喜树碱（HCPT）。细胞裂解液中的HCPT经乙酸乙酯萃取后，采用C18柱进行RP-HPLC测定。流动相为含0.1%三乙胺-磷酸缓冲液（pH 3.0）和乙腈（75:25，v/v）。采用激发波长和发射波长分别为382和528 nm的荧光检测器测定HCPT。校准曲线在2至100 ng/ml范围内呈线性，相关系数为0.9999，定量限为2 ng/ml。测定回收率在86.5%至105.2%之间。日内和日间变异系数均小于10%（R.S.D.）。此外，采用该验证方法测定了羟基喜树碱脂质体制剂和注射用羟基喜树碱与完整细胞孵育后羟基喜树碱的蓄积情况。与传统的羟基喜树碱注射液相比，羟基喜树碱脂质体在不同孵育时间显示出更高的细胞内羟基喜树碱蓄积量。
