HPLC波长切换法同时测定补肾活血方中丹参素、葛根素、大豆苷和丹酚酸B的含量

目的: 建立HPLC 波长切换法同时测定补肾活血方中丹参素、葛根素、大豆苷、丹酚酸B 含量。方法: 采用Inertsil ODS-SP C₁₈(4.6 mm×250 mm,5 μm)色谱柱,以乙腈(A)-0.2% 磷酸水溶液(B)为流动相梯度洗脱,柱温30 ℃,波长切换(0~12 min,282 nm,检测丹参素;13~22 min,250 nm,检测葛根素、大豆苷;23~36 min,286 nm,检测丹酚酸B)。结果: 丹参素、葛根素、大豆苷、丹酚酸B 线性范围分别为0.205 6~2.056 μg(r=0.999 7)、0.1~1.0 μg(r=0.999 6)、0.050 1~0.501 μg(r=0.999 4)、1.0~10.0 μg(r=0.999 5);平均回收率(n=6)分别为99.3%、100.7%、99.3% 和98.8%,RSD 分别为1.1%、1.6%、1.6% 和1.6%。含量测定结果(n=3)分别为3.328 4~4.707 0、0.919 6~1.631 4、0.455 1~0.610 8、29.478 4~31.116 9 mg·g^-1,RSD 分别为1.6%~2.2%、0.4%~1.1%、1.2%~2.0%、0.8%~1.3%。结论: 方法学验证结果表明,本法能为补肾活血方的质量标准研究提供参考。

Objective: To develop an HPLC method for simultaneously determination of 4 components including danshensu,puerarin,daidzin and salvianolic acid B in Bushen Huoxue prescription. Methods: The Inertsil ODS-SP C₁₈(4.6 mm×250 mm,5 μm)was adopted. The mobile phase consisted of acetonitrile(A)-0.2% aqueous phosphoric acid(B)in a linear gradient elution mode. The column temperature was 30 ℃. The detection wavelength was set at 282 nm for danshensu in the first 12 min,then changed to 250 nm for puerarin and daidzin between 13 and 22 min,and changed to 286 nm for salvianolic acid B in the final 14 min. Results: The linear ranges of danshensu,puerarin,daidzin and salvianolic acid B fell within the ranges of 0.205 6-2.056 μg(r=0.999 7),0.1-1.0 μg(r=0.999 6),0.050 1-0.501 μg(r=0.999 4),1.0-10.0 μg(r=0.999 5),respectively. The recoveries (n=6)of danshensu,puerarin,daidzin and salvianolic acid B were 99.3%,100.7%,99.3%,98.8%,and the relative standard deviations were 1.1%,1.6%,1.6%,1.6%,respectively. The results of content assay(n=3)were 3.328 4-4.707 0,0.919 6-1.631 4,0.455 1-0.610 8,29.478 4-31.116 9 mg·g^-1,separately,and the relative standard deviations were 1.6%-2.2%,0.4%-1.1%,1.2%-2.0%,0.8%-1.3%,respectively. Conclusion: This established method was proved by methodology validation that it can provide a reference for quality standard of Bushen Huoxue prescription.