高效液相色谱-荧光法测定大鼠体内金丝桃素血药浓度及其药动学

Study on plasma concentration and pharmacokinetics of hypericin in rats by HPLC-with fuorescence

目的:建立金丝桃素(Hyp)大鼠血浆浓度的HPLC-荧光测定法并应用于大鼠药动学研究。方法:色谱柱:大连依利特Kromasil C18柱(5μm,4.6 mm×200 mm),流动相:甲醇-乙腈-1%磷酸盐缓冲液(65∶30∶5),流速1.0mL·min-1,柱温40℃,激发波长315 nm、检测波长590 nm。大鼠(n=6)经静脉给予100μg·kg-1 Hyp并测定血浆药物浓度,DAS2.0软件进行药动分析。结果:Hyp在5～250 ng·mL-1范围内具有良好线性关系,标准曲线为Y=25997 X-77321,r=0.9991。5、25、250ng·ml-1 Hyp日内变异系数(RSD)分别为1.30%、1.00%、1.02%(n=5),日间变异系数(RSD)分别为4.36%、1.23%、2.31%(n=5),回收率分别为71.9%、74.9%、70.4%。静脉给予100μg·kg-1 Hyp,其药动学特征符合二室模型,浓度-时间曲线平缓,药动学参数为AUC0-t)为(224.16±28.78)μg·L-1·h,t1/2为(4.283±2.68)h,CL为(0.196±0.08)L·h-1·kg-1。结论:本试验方法专属性好,灵敏度高,可靠性强,可用于Hyp药动学分析。

OBJECTIVE To establish an HPLC assay with fluorescence detection for determining the plasma concentration ofhypericin in rats. METHODS The chromatographic conditions were as follows： a Kromasil C18 column （5 μm,4. 6 mm×200mm） was eluted with mobile phase consisted of methanol-acetonitrile-1% phosphate buffer at the column temperature of 40 ℃. The flow rate was 1.0 mL-min^-1 and the fluorescence detector was operated at λex= 315 nm and ;λex = 591） nm. 100μg·kg hypericin was administrated by intravenous injection and plasma concentration were determined by HPI.C assay. RESULTS The method was proved to be linear in the range of 5-250 ng·mL^-1, the standard curve for Y = 25997X- 77321, r = 0. 9991. 5, 25, 250 ng·mL^-1 Hyp The intra-day coefficients of variation were 1.30%, 1.00 %, l. 02% （n = 5） ; the inter-day coefficients of variation were 4. 36G, 1. 23%, 2. 31%（n = 5） ; recoveries were 71.9%, 74. 9%,70.4%. The pharmacokinetic parameter were AUC（0-t）（224.16±28.78）μg·kg^-1.h,t1/2（4.283±2.68） h,CL（0.196±0. 08 L·h^-1·kg^-1 resepctively. CONCLUSION This method was proved to be quick, simple and highly sensitive and can be used for pharmacokinetic research of hypericin.