HPLC-MS法测定关附甲素及其在Caco-2细胞上的摄取特性

HPLC-MS determination of GFA and its uptake in Caco-2 cells

目的建立关附甲素的LC-MS测定法,研究其在Ca-co-2细胞上的摄取特性。方法采用ShimadzuVP–ODS,C8柱,(250mm×2.1 mm,5μm),流动相:乙腈∶水=30∶70(V/V),用0.2%冰乙酸溶液,加入二乙胺适量调pH至4.0左右,流速:0.2 ml.min-1,电喷雾离子化(ES1)方式,采用选择性离子检测法,检测离子为正离子,关附甲素的选择性离子:[M+H],m/z 430。考察温度、药物浓度、时间和pH值对关附甲素在Caco-2细胞上摄取的影响。结果关附甲素在0.2～50.0μmol.L-1的范围内呈良好的线性关系(r=0.9988),最低定量限0.2μmol.L-1。关附甲素在Caeo-2细胞上的摄取具有一定的浓度、时间和pH值依赖性,其摄取表现为被动扩散。结论该法灵敏、简单,专属性强,关附甲素在Caco-2细胞上具有良好的摄取。

Aim To establish LC-MS assay to determine the uptake status of Guanfu Base A（GFA）in Caco-2 cells.Methods A LC procedure was carried out with a C8,（250 mm×2.1 mm,5 μm,Shimadzu）and an eluate of 0.2% acetic acid-acetonitrile（30 ∶ 70 v/v）at the flow rate of 0.2 ml·min^-1.The MS portion was set with ESI ion source in the positive ion polarity mode and the selective ion monitoring（SIM）.Protonated moleculeswere detected at m/z 430 for GFA.The GFA concentrations in Caco-2 cells at various time,concentration,temperature and pH values were measured.Results The linearity of GFA concentration curve was found in the range of 0.2-50.0 μmin^-1（r=0.9978）.The minimal detection limit was 0.2 μmin^-1.The GFA uptakes in Caco-2 cells obeyed a passive diffusion in a time,concentration and pH-dependent manner.Conclusion This sensitive and specific LC-MS assay is applied to determine GFA concentrations in Caco-2 cells.