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目的:建立灵敏、快速和高效的LC-ESI-MS/MS法测定HepG2细胞中苦参碱浓度,研究苦参碱在HepG2细胞上的摄取转运特性。方法:Hanbon Megres C18色谱柱(4.6 mm×250 mm,5μm),流动相10 mmol·L-1乙酸铵溶液(含0.1%甲酸)-甲醇(35∶65);质谱采用正离子气动辅助电喷雾离子化(ESI),选择性反应监测(SRM)测定药物。以HepG2细胞作为体外模型,考察温度、药物浓度和多特异性有机阳离子转运系统的经典抑制剂对苦参碱在HepG2上摄取的影响。结果:HepG2细胞裂解液中苦参碱在0.05~50.0 nmol·L-1线性关系良好,最低定量限为0.05 nmol·L-1。与37℃相比,苦参碱在4℃时的HepG2细胞摄取量显著下降(P<0.001)。孵育液中药物浓度由0.5μmol·L-1增加到400μmol·L-1时,苦参碱的摄取率显著下降(P<0.01)。与抑制剂吡拉明、维拉帕米、奎尼丁和苯海拉明共孵育时,苦参碱的细胞摄取均显著下降(P<0.001)。结论:该法简便、快速、灵敏,可用于研究苦参碱在HepG2细胞中的摄取特性。苦参碱进入肝细胞以主动转运为主,其中多特异性有机阳离子转运系统介导的摄取转运是主要途径。
OBJECTIVE: To establish a sensitive, rapid and efficient LC-ESI-MS / MS method for the determination of matrine concentration in HepG2 cells and study the uptake and transport of matrine on HepG2 cells. METHODS: Hanbon Megres C18 column (4.6 mm × 250 mm, 5 μm) was used as the mobile phase. The mobile phase consisted of 10 mmol·L -1 ammonium acetate solution (containing 0.1% formic acid) and methanol (35:65) Ionization (ESI), selective reaction monitoring (SRM) assays. HepG2 cells were used as in vitro models to investigate the effects of temperature, drug concentration and classical inhibitors of multispecific organic cation transporters on uptake of matrine at HepG2. Results: The linear relationship of matrine in HepG2 cell lysate was 0.05 ~ 50.0 nmol·L-1, the lowest limit of quantification was 0.05 nmol·L-1. The uptake of HepG2 cells by matrine at 4 ° C was significantly lower than that at 37 ° C (P <0.001). Matrine uptake rate was significantly decreased (P <0.01) when the drug concentration in the incubation solution was increased from 0.5μmol·L-1 to 400μmol·L-1. Incubation with inhibitors Pyrilamine, Verapamil, quinidine, and diphenhydramine significantly decreased matrine cellular uptake (P <0.001). Conclusion: This method is simple, rapid and sensitive and can be used to study the uptake of matrine in HepG2 cells. Matrine into the liver cells to active transport-based, of which multi-specific organic cation transport system-mediated uptake and transport are the main ways.