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目的:建立一种测定Caco-2细胞中斯皮诺素的超高效液相色谱串联质谱(UPLC-MS/MS)检测方法,研究斯皮诺素在Caco-2细胞中的转运方式。方法:采用BEH C_(18)色谱柱(2.1 mm×100 mm,1.7μm),流动相为水—甲醇溶液,梯度洗脱,流速0.30 mL·min~(-1);质谱测定采用电喷雾离子源,负离子模式,多反应监测(MRM)定量离子对m/z 607→427,以Caco-2细胞模型研究斯皮诺素的双向转运,考察斯皮诺素浓度及P-糖蛋白(P-glycoprotein,P-gp)抑制剂对斯皮诺素转运的影响。运用UPLC-MS/MS检测药物浓度,计算其表观渗透系数。结果:UPLC-MS/MS检测斯皮诺素具有良好的线性(r~2>0.999 0)、精密度的RSD≤15%,准确度在88%~115%之间,绝对回收率均在80%以上,斯皮诺素最低检测限为2 ng·mL~(-1)。斯皮诺素在Caco-2细胞中,其转运方向主要是从绒毛面(AP)→基底面(BL),且与浓度相关。斯皮诺素质量浓度在2~40μg·mL~(-1)时,其表观渗透系数P_(app(AP→BL))随浓度的增加而增加,外排率低于0.2,显示被动转运,但在80μg·mL~(-1)时,其外排率为2.84,大于2,显示有P-gp参与外排;加入P-gp抑制剂后,其外排率显著降低。结论:在低浓度时,斯皮诺素在Caco-2细胞中的转运以被动扩散为主,但在高浓度时,受到P-gp糖蛋白的外排作用,吸收降低。
OBJECTIVE: To establish a method for the determination of spinosin in Caco-2 cells by UPLC-MS / MS and investigate the transport mode of spinosin in Caco-2 cells. Methods: BEH C 18 column (2.1 mm × 100 mm, 1.7 μm) was used. The mobile phase consisted of water and methanol. The mobile phase was gradient elution at a flow rate of 0.30 mL · min ~ (-1). The mass spectrometry Source and negative ion mode, multiple reaction monitoring (MRM) quantitative ion pair m / z 607 → 427 were used to study the bidirectional transport of spinosin in Caco-2 cell model. The concentrations of spinosin and P- glycoprotein, P-gp inhibitor on the transport of spinosin. The drug concentration was measured by UPLC-MS / MS and its apparent permeability coefficient was calculated. RESULTS: The results showed that spinosyn with UPLC-MS / MS had good linearity (r ~ 2> 0.999 0), RSD≤15% and accuracy between 88% ~ 115% with absolute recoveries of 80 The minimum detectable limit of spinosyn was 2 ng · mL -1. In Caco-2 cells, spinosad mainly translocates from the apical surface to the basement surface and correlates with the concentration. The apparent permeability coefficient P_ (app (AP → BL)) increased with the increasing of concentration when the concentration of spinosin was 2 ~ 40μg · mL -1, and the efflux rate was lower than 0.2, indicating passive transport , But at 80μg · mL -1, the efflux rate was 2.84, which was greater than 2, indicating that P-gp was involved in the efflux. The efflux rate of P-gp inhibitor was significantly decreased after addition of P-gp inhibitor. CONCLUSIONS: At low concentrations, the transport of spinosin in Caco-2 cells is mainly by passive diffusion, but at high concentrations it is effluxed by the P-gp glycoprotein and its absorption is reduced.