RP-HPLC法测定大鼠血浆中槲皮素含量

Determination of quercetin concentration in rat plasma by RP-HPLC

目的建立大鼠血浆中槲皮素的反相高效液相色谱（RP-HPLC）测定方法,研究菟丝子水提取液中槲皮素在正常大鼠血浆中的药物代谢动力学过程。方法给予大鼠灌胃菟丝子水提取液后,于不同时间点采血。血浆样品经酸化后乙酸乙酯萃取,RP-HPLC法进行检测,采用Hypersil ODS柱（250mm×4.6mm,5μm）,流动相为甲醇-0.5%磷酸水（60∶40）,流速1.0ml/min,柱温25℃,检测波长为360nm。应用药物代谢动力学软件DAS 2.1.1拟合房室模型,并计算药物代谢动力学参数。结果槲皮素在0.5-6μg/ml线性关系良好,最低检测限为0.5μg/ml,高、中、低（含槲皮素5、2.5和1μg/ml）3种浓度的平均回收率均为90%-105%,日内和日间相对标准偏差均〈5%。槲皮素的血药浓度-时间曲线符合二室模型,槲皮素的消除半衰期为4.589h,0-t时刻药时AUC为13.498μg/（L·h）,药时总AUC为20.765μg/（L·h）。结论建立了测定菟丝子水提取液中槲皮素在血浆的RP-HPLC测定方法,此方法简便、灵敏、准确、稳定,适用于测定槲皮素的血药浓度及在大鼠体内的药物代谢动力学研究。

Objective To establish a RP- HPLC method for determination of quercetin in rat plasma and to investigate its pharmacokinetics after oral administration of dodder extract in rats. Methods After oral administration of dodder extract, rat blood samples were collected at different time points. Plasma samples were extracted by ethyl acetate, the analysis was performed on a Hypersil ODS column, the mobile phase was composed of methanol- 0.5% phosphoric acid water（60 ： 40） with the flow rate of 1.0ml/min and the column temperature of 25℃ and UV wave length of 360 nm. The data obtained were fitted with DAS 2.1.1 program and the pharmacokinetic parameters were calculated. Results Linear calibration curve obtained with the peak area of quercetin versus drug concentration was found to be linear between the ranges of 0.5 -6μg/ml. The lowest detectable concentration was 0.5μg/ml. The average recoveries were 90%-105%. The inter- and intra- day RSDs were all less than 5%. The plasma concentration- time data fit to a two- compartment model. Elimination half lives of quercetin was 4.589 h. The area under the concentration- time curve（AUC0- t） was 13.498μg/（L·h）, and the total area under the concentration- time curve（AUC0- ∞） was 20.765μg/（L·h）. Conclusion A validated reversed- phase HPLC method has been developed for determining quercetin in rat plasma. The assay method is proved to be simple, sensitive, precise and reliable enough for the pharmacokinetics study of quercetin in rats after oral administration.