高效液相色谱法测定大鼠血浆中欧前胡素与异欧前胡素的浓度及药动学研究

目的：建立利用高效液相色谱法检测大鼠血浆内欧前胡素及异欧前胡素的方法，并进行灌胃及静脉注射后药动学研究。方法：血浆样品经乙醚萃取，吸取上层液置于氮气流下吹干，甲醇溶解后取20 μL进行检测。采用Agilent 1100型液相色谱仪，Hypersil ODS2（250 mm×4.6 mm，5 μm）色谱柱，Agilent C₁₈（12.5 mm×4.6 mm）预柱，流动相为甲醇-水（70：30），流速为1.0 mL·min^-1，紫外波长为300 nm，进样体积为20 μL，蛇床子素作为内标物质。大鼠灌胃和静脉注射欧前胡素与异欧前胡素，定时剪尾取血测定血浆中药物浓度，药动学参数以3p97统计学软件处理。结果：欧前胡素及异欧前胡素浓度均为0.05~36.45 μg·mL^-1时，线性较好（r=0.999 9）；定量限均为0.05 μg·mL^-1；日内、日间精密度RSD<15%；方法回收率均在80%~90%之间；低、中、高3个系列规格的血浆样品在4℃保存7 d、-20℃及-80℃各保存2个月后结果均保持稳定。灌胃给予含欧前胡素与异欧前胡素剂量均为30 mg·kg^-1混合溶液，房室模型均拟合为一室模型：欧前胡素t_1/2Ke（74.7±18.15）min，t_max（8.7±1.54） min，C_max（1.15±0.16） μg·mL^-1，AUC（124.42±44.74） μg·min·mL^-1，CL/F（s）（0.25±0.1） L·kg^-1·min^-1，V/F（c）（24.44±3.55） L·kg^-1；异欧前胡素t_1/2Ke（60.48±14.22）min，t_max（8.6±1.21）min，C_max（0.3±0.04）μg·mL^-1，AUC（27.27±5.46） μg·min·mL^-1，CL/F（s）（1.14±0.25）L·kg^-1·min^-1，V/F（c）（96.42±12.96）L·kg^-1。静脉给予含欧前胡素与异欧前胡素剂量均为20 mg·kg^-1混合溶液，房室模型均拟合为二室模型：欧前胡素t_1/2β（85.64±23.25） min，AUC（1 152.02±555.25） μg·min·mL^-1，CL（s）（0.022 8±0.014 2） L·kg^-1·min^-1，V（c）（0.73±0.31）L·kg^-1；异欧前胡素t_1/2β（89.28±29.02） min，AUC（210.26±75.76） μg·min·mL^-1，CL（s）（0.105 4±0.034 1） L·kg^-1·min^-1，V（c）（2.74±0.6） L·kg^-1。欧前胡素与异欧前胡素灌胃给药，其绝对生物利用度经过计算结果为7.20%与8.65%。结论：经方法学验证，此方法具有操作简单、专属性强和准确率高等特点，能满足大鼠体内中欧前胡素与异欧前胡素含量检测及药动学研究。

Study on concentrations and pharmacokinetics of imperatorin and isoimperatorin in rat plasma by HPLC

OBJECTIVE To establish a HPLC method for determination of imperatorin and isoimperatorin in rat plasma and perform pharmacokinetic study after intragastric and intravenous administration.METHODS Blood plasma samples were extracted with ether.The supernatant was dried under a stream of nitrogen.The analysis was carried out after extract was dissolved in methanol and a 20 μL aliquot was injected into the chromatographic system.Agilent 1100 liquid chromatography was performed on a Hypersil ODS2 (250 mm×4.6 mm,5 μm) column,an Agilent C₁₈ (12.5 mm×4.6 mm) precolumn,using a mobile phase of methanol-water (70:30) at a flow rate of 1.0 mL·min^-1.The detecting wavelength was 300 nm,the injection volume was 20 μL,with osthole as the internal standard.The plasma samples were collected after intragastric and intravenous administration of imperatorin and isoimperatorin in rats.The concentrations of drugs in plasma were measured by HPLC.Compartment model,pharmacokinetic parameters of imperatorin and isoimperatorin in rats were calculated with the pharmacokinetic software of 3p97 for statistical analysis.RESULTS The good linear range of imperatorin and isoimperatorin was 0.05-36.45μg·mL^-1 with a correlation coefficients of 0.999 9.The LOQ was 0.05 μg·mL^-1.The intra-day and inter-day precision RSD were both less than 15%.The method recovery was 80%-90%.The plasma samples at low,middle and high concentrations were stable at 4℃ for seven days,at -20℃ for two months and at -80℃ for two months.The drug compartment model in plasma was fitted with one compartment model after intragastric administration of the mixed solution of 30 mg·kg^-1 imperatorin and 30 mg·kg^-1 isoimperatorin in rats,with the following main pharmacokinetic parameters:imperatorin,t_1/2Ke=(74.7±18.15) min,t_max=(8.7±1.54) min,C_max=(1.15±0.16) μg·mL^-1,AUC=(124.42±44.74) μg·min·mL^-1,CL/F(s)=(0.25±0.1) L·kg^-1·min^-1,V/F(c)=(24.44±3.55) L·kg^-1;isoimperatorin,t_1/2Ke=(60.48±14.22) min,t_max=(8.6±1.21) min,C_max=(0.3±0.04) μg·mL^-1,AUC=(27.27±5.46) μg·min·mL^-1,CL/F(s)=(1.14±0.25) L·kg^-1·min^-1,V/F(c)=(96.42±12.96) L·kg^-1.The compartment model in plasma was fitted with two compartment model after intravenous administration of the mixed solution of 20 mg·kg^-1 imperatorin and 20 mg·kg^-1 isoimperatorin in rats,with the following main pharmacokinetic parameters:imperatorin,t_1/2β=(85.64±23.25) min,AUC=(1 152.02±555.25) μg·min·mL^-1,CL(s)=(0.022 8±0.014 2) L·kg^-1·min^-1;V(c)=(0.73±0.31) L·kg^-1;isoimperatorin,t_1/2β=(89.28±29.02) min,AUC=(210.26±75.76) μg·min·mL^-1,CL(s)=(0.105 4±0.034 1) L·kg^-1·min^-1,V(c)=(2.74±0.6)L·kg^-1.The absolute bioavailability values of imperatorin and imperatorin were 7.20% and 8.65% respectively after intragastric administration of imperatorin and isoimperatorin.CONCLUSION Method validation indicates that the method is simple,stable and accurate.It is suitable for the pharmacokinetic study and determination of imperatorin and isoimperatorin in plasma.