UPLC-MS/MS测定斯皮诺素在Caco-2细胞的转运特征

Transport characteristics of spinosin in Caco-2 cell model by UPLC-MS/MS

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 develop a method for spinosin （ SPI ） determination in Caco-2 cell by ultra high performance liquid chromatography tandem mass spectrometry （ UPLC-MS/MS ）, so as to study the transport mechanism of spinosin. Methods： The chromatographic separation was performed on an Acquity BEH C18 column （2.1 mm×100 mm,1.7μm ） with aqueous methanol as the mobile phase, using quadruple mass speclrometer employing electrtrosparay ionization in gradient elution at a flow rate of 0.3 mL· min^-1. A triplenegative ion mode was developed , and spinosin in Caco-2 cells was determined using multiple reaction monitoring of spinosin → product ion transitions at m/z 607 → 427. Two-way transport of spinosin was studied by using Caco-2 cell model.The effects of drug concentration and inhibitor on the transport of spinosin were investigated. The drug concentrations were determined by uhra-performance liquid chromatography tandem mass spectrometry （ UPLC-MS/MS ） and then the apparent permeability coefficient was calculated. Results： The established method was validated by determining the linearity （ r^2 〉 0. 999 0 ）, and the precision was lower than 15%. The accuracy was ranged between 85% and 115% and the absolute recovery rate were all above 80%. The detection limit for spinosin was 2 ng · mL^-1. The transport of spinosin correlated with the concentration was mainly from the apical side （ AP ） to the base side （ BL ）. When the concentration ranged with in 2-40 μg · mL^-1, the apparent permeability coefficient Papp（AP→ BE）increased with the concentration, and efflux rate （ RE ）of spinosin was below 0.2, which was passive transport. However, when the concentration was 80 μg· mL^-1, the efflux rate of spinosin was more than 2, showing P-gp efflux.Moreover, the efflux rate was significantly reduced when combined with P-gp inhibitors. Conclusion： The established UPLC-MS/MS method is sensitive, reliable and specific, which can be used in the determination of spinosin in Caco-2 cells. Spinosin is transported through the intestinal mucosa via a passive diffusion mechanism primarily at low concentration, and affected by P-gp at high concentration with redacef obsorptien.