HPLC法同时测定口服山楂叶总黄酮提取物后大鼠血浆中3种黄酮的含量及药动学研究

Simultaneous determination of 3 flavonoids contents in rats plasma by HPLC after oral administration of total flavones of hawthorn leaf extract and its application to pharmacokinetic study

目的建立能同时测定口服山楂叶总黄酮提取物(TFHLE)后大鼠血浆中牡荆素-4″-O-葡萄糖苷(VG)、牡荆素-2″-O-鼠李糖苷(VR)和金丝桃苷(HP)含量的HPLC法。方法口服TFHLE后,采集不同时间点大鼠血浆样品,以黄芩苷为内标,甲醇沉淀蛋白。色谱柱为Phenomsil C18(250 mm×4.6 mm,5μm),流动相为甲醇-乙腈-四氢呋喃-4 mL·L^-1醋酸水溶液(6∶1.5∶18.5∶74),检测波长为332 nm。结果VG、VR和HP的线性范围分别为2.5~500,0.2~25和0.25~12.5μg·mL^-1;日内精密度RSD值均在1.2%~13.2%范围内;日间精密度RSD值均在1.5%~12.3%范围内;3种成分和内标物的回收率均不低于87.59%±0.97%。VG、VR和HP的药动学参数AUC0→t与剂量呈正比,t1/2与剂量无相关性,提示VG、VR和HP呈线性动力学特征。结论该方法简便、快速、专属性强,可用于大鼠血浆中VG、VR和HP的含量测定及其药动学研究。

Objective To establish a rapid and sensitive HPLC method for simultaneously determining 3 active compounds including vitexin-4″-O-glucoside(VG),vitexin-2″-O-rhamnoside(VR)and hyperoside(HP)in rat plasma after administrating total flavones of hawthorn leave extract(TFHLE).Methods After oral administration of TFHLE,the plasma samples were collected at different time points,and deproteinized with methanol after addition of internal standard baicalin.The HPLC assay was carried out using a Phenomsil C18(250 mm×4.6 mm,5μm)analytical column with UV detection at 332 nm.The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-4 mL·L^-1 glacial acetic acid(6∶1.5∶18.5∶74).Results The calibration curves were liner over the range of 2.5-500,0.2-25 and 0.25-12.5μg·mL^-1for VG,VR and HP,respectively.The method was reproducible and reliable with the RSDs of 3 concentrations ranged from 1.2%to 13.2%for intra-day assay and from 1.5%to 12.3%for inter-day assay,respectively.The recovery rate of VG,VR and HP at 3 concentrations were no less than 87.59%±0.97%.The area under the plasma concentration-time curve from 0 h to the time of last measurable concentration(AUC0→t)of the 3 analytes in rat plasma increased in direct proportion with the dose,and the terminal half-life(t1/2)had no good correlation with the administered dose,suggesting that the pharmacokinetics of VG,VR and HP in rats obeyed linear processes.Conclusion The method is proved to be simple,rapid and specific,and is suitable for the simultaneous determination of VG,VR and HP in plasma of rats and the pharmacokinetic study.