Department of Biology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc 77515, Czech Republic.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Jul 1;879(21):1875-80. doi: 10.1016/j.jchromb.2011.05.008. Epub 2011 May 11.
A sensitive assay for direct determination of intracellular level of daunorubicin (DRN) in resistant leukemia cells with overexpressed P-glycoprotein has been developed. This assay is based on a rapid separation of cells from media and fast cut-off of DRN transportation by centrifugation of cells through a layer of silicone oil. Cell pellets were extracted using 1% (v/v) formic acid in 50% (v/v) ethanol in water. The cell extracts were subsequently analysed by liquid chromatography (HPLC) coupled a low-energy collision tandem mass spectrometer equipped with an electrospray ionization source (ESI-CID-MS/MS) operated in the multiple-reaction monitoring (MRM) mode. Calibration curve was linear from 0.4 to 250nM with correlation coefficient (r²) better than 0.998. The limit of quantitation (LOQ) was 0.4 nM. The assay has been successfully applied to a determination of intracellular content of daunorubicin in sensitive K562 and resistant K562/Dox and K562/HHT300 cells.
已经开发出一种灵敏的分析方法，可用于直接测定过度表达 P-糖蛋白的耐药白血病细胞内柔红霉素（DRN）的细胞内水平。该分析方法基于通过细胞通过硅油层的离心快速将细胞与培养基分离，并快速阻断 DRN 的转运。使用 1%（v/v）甲酸在 50%（v/v）乙醇中的水溶液提取细胞沉淀。随后通过配备电喷雾电离源（ESI-CID-MS/MS）的高效液相色谱（HPLC）分析细胞提取物，ESI-CID-MS/MS 以多反应监测（MRM）模式运行。校准曲线在 0.4 至 250nM 范围内呈线性，相关系数（r²）大于 0.998。定量下限（LOQ）为 0.4 nM。该方法已成功应用于测定敏感 K562 和耐药 K562/Dox 和 K562/HHT300 细胞内柔红霉素的含量。
