中国临床药理学杂志2018，Vol.34Issue(8)：979-982,1004,5.DOI:10.13699/j.cnki.1001-6821.2018.08.018
LC-MS/MS法测定细胞悬液中罗丹明123的浓度
Determination of Rhodamine 123 in cell culture system by LC-MS/MS
摘要
Abstract
Objective To establish a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining the concentration of R123 in MDCK-Pgp cells.Methods The R123 and internal standard Rhodamine 6G (R6G) were extracted from cell lysis with protein precipitation by acetonitrile solution and supernatant separation was performed on Kinetex C18 analytical column (2.1 mm ×75 mm,2.6 μm) with temperature at 40 ℃.A gradient elution with the mobile phase consisting of aqueous solution (0.1％ formic acid) and methanol at a flow rate of 0.3 mL · min-1 were applied for the chromatographic separation and the injection volume was 1 μL.The analytes were detected by electrospray ionization source,multiple reaction monitoring scanning in positive ion mode using triple quadrupole mass spectrometer.The specificity,standard curve and lower limitation of quantitation,precision and recovery rate and matrix effect as well as stability were investigated.Results Good linearity was achieved within the wide concentration range of 0.2-500.0 ng· mL-1 with the calibration curve y =2.98 × 10-3x +2.66(r =0.998 5) and the lowest limit of quantification (LLOQ) of R123 was 0.2 ng · mL-1.The intra-day and inter-day RSD were both less than 10％.The extraction recoveries were between 97.60％-98.29％ and the matrix effects were between 98.44％-99.40％.R123 was stable in long-term,short-term and repeated freeze-thaw conditions.Cyclosporine (5 μ mol · L-1) significantly increased the intracellular amount of R123 approximately by about 66％,153％,46％ and 25％ at 30 min,1 h,2 h and 3 h,respectively in MDCK-Pgp cells.Conclusion The method was shown to be rapid,sensitive,and accurate for determination of R123 in MDCK-Pgp cells and the data presented in this study served as a firm basis for further investigation of P-gp activity and function in cell culture system.关键词
罗丹明123/液质联用法/MDCK-Pgp细胞/P-糖蛋白Key words
Rhodamine 123/liquid chromatography-tandem mass spectrometry/MDCK-Pgp cell/P-glycoprotein分类
医药卫生引用本文复制引用
常媛媛,魏静瑶,魏涵,张佳丽,田鑫..LC-MS/MS法测定细胞悬液中罗丹明123的浓度[J].中国临床药理学杂志,2018,34(8):979-982,1004,5.基金项目
国家自然科学基金资助项目(U1504831) （U1504831）
河南省高等学校青年骨干教师计划基金资助项目(2015GGJS-162) （2015GGJS-162）
