高效液相色谱法测定大鼠血浆中总槲皮素浓度及其药动学的应用

Determination of total quercetin in rat plasma by HPLC and its application in pharmacokinetics study

目的:建立高效液相色谱法测定大鼠血浆中总槲皮素的浓度,并应用于其药动学研究。方法:血浆样本在酸性条件下经酶水解后,经乙酸乙酯进行萃取,以乙腈-0.2%磷酸水溶液(30∶70)为流动相,阿魏酸作内标,采用Inertsil ODS-SP(4.6 mm×150 mm,5μm)色谱柱进行分离,检测波长366 nm,进样体积20μL,流速1.0mL·min-1,柱温30℃。8只SD雄性大鼠单次灌胃槲皮素50 mg·kg-1后采集血样,以高效液相色谱法测定槲皮素浓度,采用DAS3.0软件计算药动学参数并进行分析。结果:槲皮素在0.10～10.07μg·mL^(-1)范围内线性关系良好,方法定量下限为0.10μg·mL^(-1),回收率为88.85%～101.66%,日内精密度为1.11%～2.79%,日间精密度为2.60%～3.64%。大鼠单次灌胃50 mg·kg-1的槲皮素后的Cmax=(1.97±0.41)μg·mL^(-1),t1/2=(6.10±2.84)h,tmax=(8.63±3.89)h,AUC0-∞=(29.07±8.24)μg·h·mL^(-1)。结论:该方法专属性强、灵敏度高,能准确地定量大鼠血浆中槲皮素的总浓度,适用于槲皮素的药代动力学研究。

OBJECTIVE To develop a HPLC method for the determination of quercetin in the plasma of rats.METHODSThe acidified sample was extracted by ethyl acetate.The analytical column was Inertsil ODS-SP（4.6×150 mm,5μm）.The mobile phase was composed of acetonitrile-0.2%phosphoric acid aqueous solution（30∶70）and the internal standard was ferulic acid.The flow rate was 1.0 mL·min-1.The UV detector was set at 366 nm and the column temperature was maintained at30℃.The sample injection volume was 20μL.The pharmacokinetic characteristics of quercetin were investigated in8 male SD rats.After single oral dose administration of quercetin（50 mg·kg-1）,the serial blood samples were collected and analyzed by HPLC established.RESULTS Quercetin was linear in the range of 0.10-10.07μg·mL-1 and the lowest detection limit was0.01μg·mL-1.The recovery range of method was from88.85%to 101.66%.The intra-day precision was 1.11%-2.79%,and the inter-day precision was 2.60%-3.64%.After the rats received a single oral dose of quercetin（50 mg·kg-1）,the main pharmacokinetic parameters were as follows：Cmax=（1.97±0.41）μg·mL-1,t1/2=（6.10±2.84）h,tmax=（8.63±3.89）h,AUC0-∞=（29.07±8.24）μg·h·mL-1.CONCLUSION This method has high specificity and sensitivity,can accurately quantify total concentration of quercetin in rats plasma,and is suitable for the pharmacokinetic study of quercetin.