Iovel Kutateladze Pharmacochemistry Institute, P. Sarajishvili st 36, Tbilisi 0159, Georgia.
J Pharm Biomed Anal. 2011 Apr 28;55(1):109-13. doi: 10.1016/j.jpba.2010.12.021. Epub 2010 Dec 23.
An accurate, precise and sensitive method was developed and validated for the simultaneous quantification of the flavonoid glycoside robinin, and its algycone kaempferol in human breast cancer MCF-7 cells. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C(18) column using a mobile phase consisting of (A) water with 0.025% formic acid and 1mM ammonium formate and (B) acetonitrile with 0.025% formic acid. Rutin was used as the internal standard for robinin and fisetin as the internal standard for kaempferol. The assay had a limit of detection of 0.1ng/ml for both compounds when present in cell lysate. The calibration curves were linear from 1 to 250ng/ml (r>0.999) for each compound. The intra- and inter-day coefficients of variation were less than 10% and intra- and inter-day accuracies were within 11%. This assay was successfully applied in a robinin cellular uptake study to determine the intracellular concentrations of robinin in MCF-7 cells.
建立并验证了一种准确、精密且灵敏的方法，用于同时定量测定人乳腺癌 MCF-7 细胞中的类黄酮糖苷槲皮素及其苷元山柰酚。采用带 TurboIonspray 接口的液相色谱-串联质谱（LC/MS/MS），在负离子模式下进行多反应监测，对该方法进行了研究。采用 C（18）柱，以含 0.025%甲酸和 1mM 甲酸铵的水（A）和含 0.025%甲酸的乙腈（B）作为流动相，实现了色谱分离。芦丁用作槲皮素的内标，非瑟酮用作山柰酚的内标。当存在于细胞裂解液中时，两种化合物的检测限均为 0.1ng/ml。各化合物的校准曲线在 1 至 250ng/ml 范围内呈线性（r＞0.999）。日内和日间变异系数均小于 10%，日内和日间准确度均在 11%以内。该方法成功应用于槲皮素的细胞摄取研究，以确定 MCF-7 细胞中槲皮素的细胞内浓度。
