HPLC法同时测定大鼠灌胃给予葛根芩连微丸血浆中的3种有效成分的含量

Simultaneous analysis of puerarin,baicalin and three effective constituents in rat plasma after intragastric administration of Gegen Qinlian pill by HPLC

目的建立大鼠灌胃给予葛根芩连微丸后血浆中葛根素、黄芩苷和小檗碱含量的测定方法,测定单次给药后血药浓度的经时变化。方法血浆样品预处理后采用2.5倍乙腈沉淀蛋白,HPLC测定血药浓度。色谱柱为铂金ODS柱(250 mm×4.6 mm,5μm),流动相为乙腈-水(含体积分数0.025%H3PO4+体积分数0.035%三乙胺),梯度洗脱,流速为1 mL.min-1,检测波长为270 nm,柱温为30℃。结果葛根素、黄芩苷、小檗碱的质量浓度分别在0.079 5～31.795 8、0.084 8～33.915 2、0.055 5～22.200 0μg.mL-1的范围内与峰面积呈良好的线性关系。葛根素、黄芩苷和小檗碱的定量限分别为0.079 5、0.084 8、0.055 5μg.mL-1。各成分的低、中、高质量浓度溶液的日内和日间精密度(RSD)均小于15%,回收率均在75%～120%之间,RSD均小于15%。结论该方法简便、准确,可作为葛根芩连微丸血中葛根素、黄芩苷和小檗碱血药浓度同时测定的定量分析方法。

Objective To establish a HPLC method for simultaneous qualitative determination of puerarin,baicalin and berberine in rat plasma after intragastric administration of Gegen Qinlian pill for determination of plasma concentration time-dependent reliability by single-dose.Methods The plasma samples were precipitated with 2.5 times acetonitrile and plasma concentration was determined by using HPLC.PLATISILTM-ODS（250 mm×4.6 mm,5 μm） column was used with a mobile phase consisted of acetonitrile-water（containing 0.025% phosphoric acid＋0.035% triethylamine）（gradient elution） at a flow rate of 1 mL·min-1.The detection wavelength was set at 270 nm and the column temperature was at 30 ℃.Results The puerarin,baicalin and berberine showed good linearity respectively in the range of 0.079 5-31.795 8,0.084 8-33.915 2 and 0.055 5-22.200 0 μg·mL-1.The LOQ of puerarin,baicalin and berberine hydrochloride were 0.079 5,0.084 8 and 0.0555 μg·mL^-1,respectively.The intraday and interday precision（RSD） of each component at low,middle and high concentrations were less than 15%.The extraction recoveries were between 75%-120%,and the RSD were less than 15%.Conclusion The method is easy and accurate,which can be used to analyze quantitatively the plasma concentration of Gegen Qinlian pill.