RP-HPLC法测定活血化瘀方中7种有效成分

RP- HPLC in determination of seven active ingredients in blood-invigorating and stasis-removing prescription

目的建立RP-HPLC法测定活血化瘀方(blood-invigorating and stasis-removing prescription,BSP)中丹参素、原儿茶醛、芍药苷、葛根素、阿魏酸、丹参酮ⅡA及黄芪甲苷含量的方法。方法采用Waters Symmetry ShieldTMRP C18(150mm×4.6mm,3.5μm)色谱柱,以甲醇-0.25%(体积比)冰醋酸水溶液为流动相,流速为0.8mL/min,柱温30℃。丹参素、原儿茶醛、芍药苷、葛根素、阿魏酸和丹参酮ⅡA采用紫外检测器,检测波长为280nm;黄芪甲苷采用蒸发光散射检测器,飘移管温度90℃,气流量2.8L/min(压缩空气)。结果丹参素、原儿茶醛、芍药苷、葛根素、阿魏酸、丹参酮ⅡA和黄芪甲苷的线性范围分别为0.01～0.80μg(r=0.999 8),0.005～0.4μg(r=0.999 7),0.05～4μg(r=0.999 8),0.005～0.4μg(r=0.999 7),0.006～0.5μg(r=1),0.005～0.4μg(r=1),0.031～2.46μg(r=0.999 3);加样回收率均在97.0%～101.0%之间,且RSD均小于2%。测定了3批样品中丹参素、原儿茶醛、芍药苷、葛根素、阿魏酸、丹参酮ⅡA和黄芪甲苷的含量(平均值)分别为1.15、0.13、4.48、0.80、0.72、0.31、3.12mg/g。结论该方法简便、准确、灵敏,为测定BSP中的有效成分的含量提供参考。

Objective To develop an RP-HPLC method for determination of tanshinol, protocatechualdehyde, paeoniflorin, puerarin, ferulic acid, tanshinone ⅡA and astragaloside in blood-invigorating and stasis-removing prescription （BSP）. Methods The analysis was performed with a column of Waters Symmetry ShieldTM RP C18 （150 mm × 4.6 mm, 3.5 μm）, and the mobile phase consisted of methanol-0.25% acetic acid. The flow rate was 0.8 mL/min and the column temperature was 30℃. UV was employed to determine the contents of tanshinol, protocatechualdehyde, paeoniflorin, puerarin, ferulic acid and tanshinone ⅡA, and the detection wavelength was set at 280 nm. Evaporative light scattering detection （ELSD） was employed to determine the contents of astragaloside. The temperature of drift tube was 90℃ and the gas flow was 2.8 L/min （compressed air）. Results The linearity was obtained over 0.01-0.80 μg （r=0.999 8） for tanshinol, 0.005-0.4 μg （r=0.999 7） for protocatechualdehyde, 0.05-4 μg （r=0.999 8） for paeoniflorin, 0.005-0.4 μg （r=0.999 7） for puerarin, 0.006-0.5 μg （r=1） for ferulic acid, 0.005-0.4 μg （r=1） for tanshinone ⅡA, and 0.031-2.46 μg （r=0.999 3） for astragaloside. The recoveries were all between 97.0%-101.0%, and RSDs were all less than 2%. The contents （mean） of tanshinol, protocatechualdehyde, paeoniflorin, puerarin, ferulic acid, tanshinone ⅡA and astragaloside in three batches of samples were 1.15, 0.13, 4.48, 0.80, 0.72, 0.31 and 3.12 mg/g, respectively. Conclusion The method in our study is convenient, accurate and sensitive, and it provides a reference for the determination of active ingredients in BSP.