UPLC-MS/MS测定斯皮诺素在Caco-2细胞的转运特征

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 C₁₈ 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(r²>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2~40 μg·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80 μg·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,1.7 μm)with aqueous methanol as the mobile phase,using gradient elution at a flow rate of 0.3 mL· min^-1. A triplequadruple mass spectrometer employing electrospray ionization in negative 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 ultra-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(r² >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 P_app(AP→BL) 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.