HPLC法同时测定灌胃黄芩水煎剂后大鼠血浆中黄芩苷和汉黄芩苷的质量浓度及药动学研究

Simultaneous determination of baicalin and wogonoside by HPLC in rat plasma administered with Huangqin Decoction and its pharmacokinetic study

目的建立大鼠ig黄芩水煎剂后血浆中黄芩苷和汉黄芩苷的HPLC测定方法及药动学研究.方法大鼠ig黄芩水煎剂后,不同时间眼底静脉丛取血,制备血浆.血浆样品经甲醇沉淀蛋白,HPLC-UV测定血药浓度.色谱柱为Shim-pack ODS(250 mm×4.6 mm,5μm);流动相采用梯度洗脱,体积流量:1.5 mL/min;检测波长:276nm.结果黄芩苷的线性范围为0.156～10μg/mL,汉黄芩苷的线性范围为0.109～7 μg/mL,定量下限(LLOQ)分别为0.156和0.109μg/mL,日内和日间精密度(RSD)均小于15%,准确度(RE)为-6.82%～3.26%.大鼠ig黄芩水煎剂后,血浆中黄芩苷和汉黄芩苷血药浓度-时间曲线均存在双峰:黄芩苷的tmax1和tmax2分别为(12.0±4.5)min和(7.2±1.79)h;Cmax1和Cmax2分别为(5.29±1.96)和(4.49±2.12)μg/mL,汉黄芩苷的tmax1和tmax2分别为(14.0±9.0)min和(6.8±1.1)h;Cmax1和Cmax2分别为(1.38±0.16)和(1.62±0.71)μg/mL;黄芩苷的CL/F为(4.72±1.68)L/h,汉黄芩苷的CL/F为(3.04±0.98)L/h.结论该方法经考察符合生物样品的测定要求,可应用于大鼠体内黄芩苷和汉黄芩苷血药浓度的测定和药动学研究.大鼠ig黄芩水煎剂后血浆中黄芩苷和汉黄芩苷质量浓度存在双峰现象,黄芩苷的口服清除率大于汉黄芩苷.

Objective To develop an HPLC method with ultraviolet detection for simultaneous determination of baicalin and wogonoside in rat plasma after ig Huangqin Decoction (HQD) and study its pharmacokinetics. Methods Rats were given HQD by ig, plasma were collected at designed times from oculi chorioidea vein. Following protein precipitation by the mixture of methanol and acetonitrile (1∶1), the blood concentration was determined by HPLC-UV. The analytes were separated on a Shim-pack ODS (250 mm×4.6 mm, 5 μm) column using a gradient mobile phase consisting of acetonitrile-phosphate buffer. The flow-rate was set at 1.5 mL/min and the eluent was detected at 276 nm. Results The linear range was 0.156—10 μg/mL and 0.109—7 μg/mL for baicalin and wogonoside, respectively. The lower limit of quantification was 0.156 μg/mL and 0.109 μg/mL for baicalin and wogonoside, respectively. The intra-day and inter-day variations of RSD were less than 15%, with relative error (RE) ranging from -6.82% to 3.26%. The existence of double peaks were demonstrated in the blood concentration-time curves of baicalin and wogonoside after ig HQD. For baicalin, t_(max1) and t_(max2) were (12.0±4.5) min and (7.2±1.79) h, C_(max1) and C_(max2) were (5.29±1.96) and (4.49±2.12) μg/mL. For wogonoside, t_(max1) and t_(max2) were ((1.40±)9.0) min and (6.8±1.1) h; C_(max1) and C_(max2) were (1.38±0.16) and (1.62±0.71) μg/mL. Oral clearances (CL/F) were (4.72±1.68) L/h for baicalin and (3.04±0.98) L/h for wogonoside, (respectively.) Conclusion This method should be specific, accurate, and sensitive enough to be applied to the pharmacokinetic studies of baicalin and wogonoside. Plasma concentration-time curves of baicalin and wogonoside show the existence of double peaks after ig HQD. The oral clearances of baicalin is higher than that of wogonoside.