LC-MS/MS测定扁塑藤素及其在Caco-2细胞模型上的转运特征研究

Intestinal transport of pristimerin across the Caco-2 cell monolayer model and quantification by LC-MS/MS method

目的建立一种快速专一测定扁塑藤素的LC-MS/MS方法,并采用此方法研究扁塑藤素的吸收特性。方法首先采用LC-MS/MS建立扁塑藤素的含量测定方法,采用Waters X-Bridge C18色谱柱(3.0mm×100 mm,3.5μm)在室温条件下进行色谱分离,流动相为水(含0.1%的甲酸)和乙腈(30∶70,v/v),流速为0.4 m L·min-1。采用电喷雾离子源(ESI),多反应监测模式进行检测。扁塑藤素的母离子和子离子分别为m/z 465.3→201.1,内标氢化可的松的检测离子分别为m/z 363.5→120.9。采用聚酯碳酸酯膜连续培养Caco-2细胞21 d,然后对影响扁塑藤素在Caco-2细胞模型上转运特征的因素包括浓度、时间及跨膜转运蛋白[P-糖蛋白(P-gp)、多药耐药蛋白、乳腺癌耐药蛋白]进行了系统的考察。结果扁塑藤素在1~1000 ng·m L^(-1)内线性关系良好(r>0.998),定量限和检测限分别为1 ng·m L^(-1)和0.35 ng·m L^(-1)。扁塑藤素在Caco-2细胞上转运存在一定的浓度及时间依赖性且P-gp介导扁塑藤素在Caco-2细胞上转运。结论扁塑藤素在Caco-2细胞模型上主要以主动转运方式进行吸收,且P-gp参与药物在Caco-2细胞模型上的转运。

Objective To develop a rapid and specific LC-MS/MS method to determine pristimerin and explore the transcellular transport mechanism of pristimerin across Caco-2（the human colon adenocarcia cell lines） monolayer cell transwell model. Methods A sensitive and reliable LC-MS/MS method was developed and applied to determine the concentration of pristimerin in HBSS sample. The chromatographic analysis of pristimerin was performed on a Waters X-Bridge C18 column（3.0 mm×100 mm, 3.5 μm） at room temperature. The mobile phase was water（containing 0.1% formic acid） and acetonitrile（30∶70, v/v） at a flow rate of 0.4 m L·min^（-1）. The mass scan mode was positive MRM mode. The selected monitor ion was m/z 465.3 → 201.1 for pristimerin, and m/z 363.5 → 120.9 for IS. Caco-2 cells were seeded on the permeable polycarbonate membranes and incubated for 21 days, and then the influencing factors were investigated with Caco-2 cell model, including time, concentration, and different membrane transport proteins（P-gp, MRP2 and BCRP）. Results The standard curve for pristimerin in the plasma was linear at 1- 1000 ng·m L^-1 with correlation coefficient 〉0.998. The LLOQ and LLOD were 1 ng·m L^-1 and 0.35 ng·m L^-1. Pristimerin was transported mainly in an active diffusion. The flux of pristimerin was time and concentration-dependent. P-gp was also involved in the transport of pristimerin. Conclusion Pristimerin is transported mainly in an active transport mode, and P-gp is involved in its transport.