RP-HPLC法测定大鼠血浆中香柑内酯、欧前胡素和蛇床子素及其药动学研究

Determination of Bergapten,Imperatorin and Osthole in Rat Plasma by RP-HPLC and Their Pharmacokinetic Study

目的:建立测定大鼠血浆中香豆素类成分香柑内酯、欧前胡素和蛇床子素血药浓度的方法,并研究其在大鼠体内的药动学。方法:大鼠灌胃蛇床子提取物(900 mg/kg),分别于给药前及给药后0.25、0.5、0.75、1、2、3、4、6、8、12 h取血,以反相高效液相色谱法测定血浆中香豆素类成分香柑内酯、欧前胡素和蛇床子素的血药浓度。色谱柱为DiamonsilTMC18(150 mm×4.6 mm,5μm),流动相为乙腈-150 mmol/L醋酸铵水溶液(50∶50,V/V,醋酸调pH值至4.3),流速为1.0 ml/min,紫外检测波长为320 nm,进样量为10μl。以非房室模型法(统计矩法)分析药动学参数。结果:香柑内酯、欧前胡素和蛇床子素质量浓度在0.1~5.0、0.5~25.0、2.0~100.0μg/ml范围内与各指标成分和相对应的峰面积积分值比呈良好线性关系。香柑内酯、欧前胡素、蛇床子素精密度试验的RSD≤8.8%,准确度在(91.8±2.01)%^(102.6±0.40)%之间,提取回收率在81.3%~92.8%之间,稳定性试验的RSD<15%。香柑内酯、欧前胡素、蛇床子素的AUC0-∞分别为(5.53±1.07)、(22.67±3.65)、(39.60±4.56)μg·h/ml;t1/2分别为(3.33±0.39)、(2.27±0.55)、(2.24±0.35)h;cmax分别为(1.18±0.22)、(7.03±1.27)、(13.16±1.37)μg/ml。结论:该方法简便、灵敏、专属性好,可用于大鼠体内香柑内酯、欧前胡素和蛇床子素的药动学研究。

OBJECTIVE： To establish a RP-HPLC method for the determination of coumarin components as bergapten, imperatorin and osthole in rat plasma, and to study the pharmacokinetics of them. METHODS： The rats were given osthole extract （900 mg/kg） intragastrically, and the blood samples were collected before medication and 0.25,0.5,0.75, 1,2, 3,4, 6,8 and 12 h after medication. The blood concentrations of bergapten, imperatorin and osthole were determined by RP-HPLC. DiamonsilTM C18 column （150 mm×4.6 mm, 5μm） was used with mobile phase consisted of acetonitrile-ammonium acetate （50：50, V/V, pH adjusted to 4.3 using acetic acid） at the flow rate of 1.0 ml/min. UV detection wavelength was set at 320 nm, and sample size was 10μl. The pharmacokinetic parameters were calculated with non-compartmental method （statistical moment）. RESULTS： The linear ranges of bergapten, imperatorin and osthole were 0.1-5.0 μg/ml, 0.5-25 μg/ml and 2.0-100.0 μg/ml, respectively. RSDs of inta-day and inter-day were all lower than 8.8%, and the accuracy was calculated as （91.8 ± 2.01）%-（102.6 ± 0.40）%. The average recoveries were 81.3%-92.8% ; RSD of stability test was lower than 15%. The pharmacokinetic parameters of ergapten, imperatorin and osthole were as follows： AUC0-∞ were （5.53 ± 1.07）μg.h/ml, （22.67 ± 3.65）μg-h/ml and（39.60 ± 4.56）μg.h/ml; t1/2 were （3.33 ± 0.39） h, （2.27 ± 0.55）h and（2.24 ± 0.35）h; cmax were（1.18 ± 0.22）μg/ml, （7.03 ± 1.27）μg/ml and（13.16 ± 1.37）μg/ml. CONCLUSIONS： The method is convenient, sensitive and specific, and can be used for pharmacokinetic study of bergapten, imperatorin and osthole.