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

目的建立一种测定MDCK-Pgp细胞中罗丹明123(R123)浓度的液质联用(LC-MS/MS)法。方法用罗丹明6G(R6G)作为内标,乙腈沉淀法进行蛋白沉淀。色谱柱:Kinetex C18柱(2.1 mm×75 mm,2.6μm),柱温:40℃,流动相:甲醇-0.1%甲酸水,梯度洗脱,流速:0.30 m L·min^(-1),进样量:1μL。用电喷雾离子源,正离子检测方式,多反应监测扫描方式进行监测。考察该方法的专属性、标准曲线与定量下限、精密度和回收率、基质效应、稳定性。结果 R123的标准曲线方程为y=2.98×10^(-3)x+2.66(r=0.998 5),在0.2~500.0ng·m L^(-1)内线性关系良好,最低定量限为0.2 ng·m L^(-1)。日内与日间精密度均小于10%,提取回收率为97.60%~98.29%,基质效应为98.44%~99.40%,长期、短期及反复冻融稳定性良好。5μmol·L^(-1)环孢素给药后30 min和1,2,3 h分别使MDCK-Pgp细胞内R123浓度增加约66%,153%,46%和25%。结论该方法快速、灵敏、准确,适用于测定细胞的R123的浓度,可为细胞中P-gp活性和功能的评价提供便利且快捷的方法。

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 6 G（ R6 G） were extracted from cell lysis with protein precipitation by acetonitrile solution and supernatant separation was performed on Kinetex C18 analytical column（ 2. 1mm ×75mm,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 m L·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·m L-（-1） with the calibration curve y = 2. 98 × 10-（-3） x ＋ 2. 66（ r = 0. 998 5） and the lowest limit of quantification（ LLOQ） of R123 was 0. 2 ng·m L-（-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 between98. 44%-99. 40%. R123 was stable in long-term,short-term and repeated freeze-thaw conditions. Cyclosporine（ 5 μmol· 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.