大鼠肝微粒体中葛根素的液相色谱-质谱测定法及药物代谢动力学

目的建立大鼠肝微粒体中葛根素及其代谢物的液相色谱质谱测定法,并研究葛根素在大鼠肝微粒体中的药物代谢动力学。方法色谱柱为Waters C18柱(150 mm×4.6 mm,5μm),流动相为甲醇水(体积比为50∶50),通过电喷雾电离源(ESI),以正离子方式进行检测。结果方法的回收率为95.6%~96.8%,其日内、日间的RSD分别为4.9%~7.6%和3.7%~6.2%,葛根素的质量浓度在0.5~20 mg.L-1内与峰面积呈良好的线性关系。在温孵时间0~40 min内、微粒体蛋白质量浓度在0.5~2.0 g.L-1内,葛根素呈线性消除,随着肝微粒体蛋白质量浓度的增大,葛根素呈线性消除,葛根素可被肝微粒体代谢成大豆苷元。其代谢机理为还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)依赖性的氧化代谢。还原型烟酰胺二核苷酸(NADH)对葛根素代谢基本没有催化作用。结论葛根素在大鼠肝微粒体内被迅速代谢,大鼠肝微粒体P450酶参与了葛根素的代谢。

Determination of puerarin in rat liver microsomes by LC-MS and its pharmacokinetics

Objective To develop a LC-MS method for the determination of puerarin and its metabolite and study the kinetics of its metabolism in rat liver microsomes.Methods Chromatography was performed on a Waters C_(18) column(150 mm×4.6 mm,5 μm).A methanol-water(V∶V=50∶50) as the mobile phase was used.A Waters Micromass ZQ detector equipped with an ESI interface was used and operated in the positive ion mode.Results The recovery of of puerarin for the proposed method was 95.6%-96.8%.The relative standard deviation for the within-day and betweenday were 4.9%-7.6% and 3.7%-6.2%.The calibration curve was linear in the range from 0.5 mg·L~(-1) to 20 mg·L~(-1) with r=0.999 5.The elimination of puerarin was linear.Microsomal protein concentration had significant effect on puerarin metabolism.One metabolite of puerarin was found to be daidzein.The metabolism of puerarin exhibited NADPH-dependent oxidation mechanism.NADH as cofactor had no catalytic effect on the metabolism of puerarin.Conclusions Puerarin is rapidly metabolized in rat liver microsomes.The result suggests that CYP450 is involved in the metabolism of puerarin.