HPLC测定血浆中荷叶碱的浓度及其在大鼠体内的药代动力学研究

Determination of Nuciferine in Rat Plasma by HPLC and Its Application to Pharmacokinetic Study

目的:建立血浆中荷叶碱的HPLC-UV测定方法,研究大鼠灌胃给予荷叶总生物碱提取物后荷叶碱的药代动力学特征。方法:采用液液萃取法处理血浆样品,采用HPLC-UV法测定,以盐酸巴马汀为内标,色谱柱为Agilent Zorbax-SB C18(4.6mm×150 mm,5μm),流动相为乙腈-水-三乙胺-冰醋酸(27∶70.6∶1.6∶0.78),流速1.0 mL.min-1,检测波长270 nm,柱温25℃,采用WinNolin软件计算药代动力学参数。结果:血浆中荷叶碱在20～1 000μg·L-1线性关系良好(r=0.998 5),方法的日内精密度(RSD)2.24%～3.20%,日间RSD为5.54%～6.95%,准确度(RE)为-7.00%～4.25%,提取回收率为87.2%～104%。大鼠灌胃荷叶总生物碱3个剂量(106,212,319 mg·kg-1)后,t1/2分别为(4.18±2.50),(4.32±1.01),(6.05±3.72)h,tmax分别为(1.50±0.41),(2.25±1.19),(1.88±0.25)h,Cmax分别为(278.75±38.98),(621.75±137.81),(1 146.50±249.44)μg·L-1,AUC0→t分别为(1 796.10±680.87)h.μg-1·L-1,(4 463.49±1 892.42)h.μg-1·L-1和(7 452.08±3 594.24)h.μg-1·L-1。结论:该法灵敏度高,专属性强,准确可靠,操作快速简便,已应用于荷叶碱在大鼠体内的药代动力学研究,阐明了荷叶碱在大鼠体内的药代动力学特征。

Objective： To establish an HPLC method for the determination of nucifefine in rat plasma and to investigate the pharmacokinetics of nucifefine in rats after oral administration of alkaloidal extract of Nelumbo nucifera.Method： The analysis was performed on an Agilent Zorbax-SB C18 column（4.6 mm × 150 mm,5 μm） at 25 ℃ with the mobile phase of acetonitrile-water-triethylamine-glacial acetic acid（27∶ 70.6∶ 1.6∶ 0.78） at a flow rate of 1.0 mL·min-1.The detecting wavelength was set at 270 nm.The plasma samples were collected after oral administration of the alkaloidal extract of Nelumbo nucifera in Wistar rat.The concentrations of nuciferine in plasma were measured by using HPLC method and the pharmacokinetic parameters were calculated by WinNonlin 5.0.1.Result： The calibration curve of nuciferine was linear in the range of 20 ～ 1 000 μg.L-1 with the correlation coefficient of 0.998 3.The intra-day and inter-day precision of this method were 2.24%-3.20%,5.54%-6.95% respectively,and accuracy was 4.25%-7.00%.The extraction recoveries were 87.2%-103.7%.The main pharmacokinetic parameters of nuciferine after adiministration of the alkaloidal etract of N.nuifera to rats were：t1/2（4.18 ± 2.50）,（4.32 ± 1.01）,（6.05 ± 3.72） h,tmax（1.50 ± 0.41）,（2.25 ± 1.19）,（1.88 ± 0.25） h,Cmax（278.75 ± 38.98）,（621.75 ± 137.81）,（1 146.50 ± 249.44） μg.L-1,AUC0→t（1 796.10 ± 680.87）,（4 463.49 ± 1 892.42）,（7 452.08 ± 3 594.24） h.μg-1.L-1.The form of nuciferine in Wistar rat can be described as first-order kinetic model.Conclusion： The method is proved to be sensitive,selective and simple and have been successfully applied to the pharmacokinetic study of nucifefine.