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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20μL进行检测。采用Agilent 1100型液相色谱仪,Hypersil ODS2(250 mm×4.6 mm,5μm)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1.0mL·min~(-1),紫外波长为300 nm,进样体积为20μL,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0.05~36.45μg·mL~(-1)时,线性较好(r=0.999 9);定量限均为0.05μg·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t1/2 Ke(74.7±18.15)min,tmax(8.7±1.54)min,Cmax(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t1/2 Ke(60.48±14.22)min,tmax(8.6±1.21)min,Cmax(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t1/2β(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t1/2β(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。
OBJECTIVE: To establish a method for the determination of imperatorin and isoimperatorin in rat plasma by high performance liquid chromatography (HPLC) and to study its pharmacokinetics after gavage and intravenous injection. Methods: The plasma samples were extracted with ether. The supernatant was aspirated and dried under a stream of nitrogen. After the methanol was dissolved, 20 μL was taken for detection. An Agilent 1100 liquid chromatograph, Hypersil ODS2 (250 mm × 4.6 mm, 5 μm) column and an Agilent C18 (12.5 mm × 4.6 mm) precolumn was used. The mobile phase consisted of methanol-water mL · min ~ (-1), UV wavelength of 300 nm, injection volume of 20 μL, osthole as internal standard substance. Rats were orally and intravenously injected with imperatorin and isoimperatorin, and blood samples were taken from time to time to tail vein to determine the drug concentration in the plasma. Pharmacokinetic parameters were determined by 3p97 statistical software. Results: The linearity of Imperatorin and isoimperatorin was 0.05 ~ 36.45μg · mL -1 (r = 0.999 9), and the limits of quantitation were 0.05μg · mL -1 ). The RSD <15% of intra-day and inter-day precision was obtained. The recovery rates of the methods were between 80% and 90%. The plasma samples of three series of low, medium and high standard were stored at 4 ℃ for 7 days, The results remained stable after 2 months of storage at 80 ° C. Oral imperatorin and isoimperatorin were all mixed at a dose of 30 mg · kg -1. The atrioventricular model was fitted as a one-compartment model: Imperatorin t1 / 2 Ke (74.7 (18.15) min, tmax (8.7 ± 1.54) min, Cmax (1.15 ± 0.16) μg · mL -1, AUC (124.42 ± 44.74) μg · min · mL -1, CL / F ) (0.25 ± 0.1) L · kg -1 · min -1, V / Fc 24.44 ± 3.55 L · kg -1, isoimperatorin t1 / 2 (P <0.05), Ke (60.48 ± 14.22) min, tmax (8.6 ± 1.21) min, Cmax (0.3 ± 0.04) μg · mL -1, AUC (27.27 ± 5.46) μg · min · mL -1, F (s) (1.14 ± 0.25) L · kg -1 (-1) min -1 and V / F (96.42 ± 12.96) L · kg -1. Intravenous administration of imperatorin and isoimperatorin were both 20 mg · kg -1. The atrioventricular model was fitted to a two-compartment model: Imperatorin t1 / 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 (0.73 ± 0.31) L · kg -1, isoimperatorin t1 / 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 and V c 2.74 ± 0.6 L · kg -1. Imperatorin and isoimperatorin administered orally, the absolute bioavailability calculated by 7.20% and 8.65%. Conclusion: This method has the characteristics of simple operation, strong specificity and high accuracy, which can meet the requirements of the detection and the pharmacokinetics of midoprene and isoimperatorin in rats.