高效液相色谱法测定小鼠血浆中8-甲氧基补骨脂素及其药代动力学研究

Determination of 8-methoxypsoralen in mouse plasma by high performance liquid chromatography and its application to pharmacokinetic study

目的:建立小鼠血浆中8-甲氧基补骨脂素(8-methoxypsoralen,8-MOP)的高效液相色谱(high performance liquid chromatography,HPLC)测定方法,并应用于8-MOP在小鼠体内的药代动力学研究。方法:以5-甲氧基补骨脂素为内标,Waters Symmetry~C_(18)柱(250 mm×4.6 mm,5μm)分离,等度洗脱,流动相为甲醇-水(体积比55∶45),流速为1.0 m L/min,荧光检测器检测,激发波长与发射波长分别为334 nm和484 nm,内标法定量。60只雄性健康的ICR小鼠随机分为12组,对照组以1%(质量分数)吐温80灌胃给药,其余11组小鼠灌胃给药8-MOP(40 mg/kg),HPLC法测定给药后不同时间点小鼠血浆中8-MOP的浓度,DAS 2.0软件计算药代动力学参数。结果:小鼠血浆中8-MOP在0.05~10.00 mg/L范围内线性良好(r=0.999 3),检出限为0.015 mg/L;高、中、低3种浓度8-MOP的加标回收率为92.5%~100.6%;日内精密度为3.3%~8.2%,日间精密度为3.4%~6.7%;提取回收率为90.9%~92.0%;小鼠血浆样品在-80℃下至少可以保存15 d。小鼠灌胃给药后5 min即可在小鼠血浆中检出8-MOP(1.4 mg/L),给药2 h血药浓度达到最高峰值,且给药后24 h仍可检出(1.1 mg/L)。t_(1/2)为(39.21±3.65)h,C_(max)为(2.31±0.02)mg/L,t_(max)为(2.00±0.00)h,AUC_(0-t)为(33.34±1.19)(h·mg)/L。结论:该方法准确、简便,适用于小鼠体内8-MOP药代动力学研究。

Objective： To establish a high performance liquid chromatography （HPLC） method for the determination of 8-methoxypsoralen （8-MOP） in mouse plasma and apply it to a pharmacokinetic study of8-MOP. Methods： 8-MOP was separated on a Waters Symmetry ~C_（18） column （250 mm × 4. 6 mm,5μm） and determined by HPLC using isocratic elution,and 5-methoxypsoralen was used as internal standard. The mobile phase consisted of methanol-water （55 ∶ 45,V/V） at a flow rate of 1. 0 m L/min. The excitation and emission wavelength of fluorescence detector were set at 334 nm and 484 nm respectively,and the internal standard method was used for quantitative analysis. In the study,60 healthy ICR male mice were randomly divided into twelve groups. The mice in control group were administered intragastrically with 1% Tween 80,and the mice in the other eleven groups were administered intragastrically with8-MOP （40 mg/kg）. Plasma concentrations of 8-MOP in the mice at different time points after treatment were determined by HPLC. Pharmacokinetic parameters were calculated by DAS 2. 0 software. Results：The calibration curve of 8-MOP was linear with a correlation coefficient of 0. 999 3 over the concentration range of 0. 05 to 10 mg/L,and the limit of detection was 0. 015 mg/L. The average recoveries of 8-MOP at three different concentrations （0. 10,0. 50,2. 5 mg/L） were from 92. 5% to 100. 6%. The intra-day precision of 8-MOP was from 3. 3% to 8. 2%,while the inter-day precision was from 3. 4% to 6. 7% at three spiked concentration levels. The extraction recoveries of 8-MOP were from 90. 9% to 92. 0%,and the plasma samples could be stored at-80℃ for 15 days at least at three spiked concentration levels. 8-MOP could be detected in mouse plasma 5 min after intragastrical administration to the mice （1. 4 mg/L）. The concentration of 8-MOP in the mouse plasma reached a _（max） imum 2 h after administration,and 8-MOP could still be detected 24 h after administration （1. 1 mg/L）. t_（1/2） was （39. 21 ± 3. 65） h,C_（max） was （2. 31 ±0. 02） mg/L,t_（max） was （2. 00 ± 0. 00） h,and AUC_（0-t） was （33. 34 ± 1. 19） （h·mg）/L. Conclusion： The proposed method is accurate and simple,suitable for pharmacokinetics of 8-MOP in mice.