建立HPLC法同时测定复方黑骨藤中9种有效成分的含量

目的：建立HPLC 法同时测定复方黑骨藤中落干酸、6'-O-β-D- 葡萄糖基龙胆苦苷、獐芽菜苦苷、龙胆苦苷、獐芽菜苷、杠柳苷、延胡索乙素、四氢小檗碱、延胡索甲素9 种有效成分的含量。方法：采用KromasilC18 色谱柱（250 mm×4.6 mm，5 μm），以乙腈（A）-0.05% 三乙胺+ 乙酸水溶液（B，pH=6.0）为流动相，梯度洗脱（0~5 min，5%A → 8%A；5~5.5 min，8%A → 11%A；5.5~20 min，11%A；20~30 min，11%A → 30%A；30~40min，30%A → 40%A；40~42 min，40%A → 55%A；42~53 min，55%A → 75%A），流速1.2 mL·min^-1，柱温25 ℃，检测波长为206 nm（延胡索乙素、四氢小檗碱、延胡索甲素）、225 nm（落干酸和杠柳苷）、243 nm（獐牙菜苦苷和獐牙菜苷）和275 nm（6'-O-β-D- 葡萄糖基龙胆苦苷和龙胆苦苷），进样量10 μL。结果：落干酸、6'-O-β-D- 葡萄糖基龙胆苦苷、獐芽菜苦苷、龙胆苦苷、獐芽菜苷、杠柳苷、延胡索乙素、四氢小檗碱和延胡索甲素的质量浓度在一定范围内，与峰面积呈良好的线性关系；平均回收率（n=6）分别为92.7%、90.9%、91.2%、94.9%、97.4%、94.6%、96.1%、90.9% 和104.4%，RSD 分别为2.1%、1.3%、1.1%、1.6%、0.7%、0.6%、1.8%、2.0% 和1.4%；样品中上述9 种成分含量范围分别为3.464~3.535、1.133~1.178、0.373~0.382、13.598~14.142、0.109~0.112、0.499~0.510、0.114~0.119、0.027~0.028 和0.163~0.174 mg·g^-1。结论：经方法学验证，本方法可用于黑骨藤复方药物中9 种有效成分的分析和含量测定，为复方黑骨藤药物的质量控制奠定基础。

Objective: To establish an HPLC analysis method for simultaneous determination of logannic acid,6'-O-β-D-glucosylgentiopicroside,swertiamarin,gentiopicroside,sweroside,glucoperiplocymarin,tetrahydropalmatine, tetrahydroberberine, and corydaline in compound Heiguteng.Methods: The HPLC analysis was performed on a Kromasil C18 column (250 mm×4.6 mm, 5 μm) with the mobile phase consisted of acetonitrile (A)-0.05% trimethylamine and glacial acetic acid(B,pH=6.0) in gradient mode (0-5 min,5%A → 8%A;5- 5.5 min,8%A → 11%A;5.5-20 min,11%A;20-30 min,11%A → 30%A;30-40 min,30%A → 40%A;40- 42 min,40%A → 55%A;42-53 min,55%A → 75%A) at the flow rate of 1.2 mL· min^-1 .The column temperature was 25 ℃ and the detection wavelengths were 206 nm( tetrahydropalmatine, tetrahydroberberine and corydaline ), 225 nm( logannic acid, glucoperiplocymarin), 243 nm (swertiamarin, sweroside) and 275 nm (6'-O-β-D-glucosylgentiopicroside, gentiopicroside).Results: In a certain range, the mass concentrations of logannic acid, 6'-O-β-Dglucosylgentiopicroside, swertiamarin, gentiopicroside, sweroside, glucoperiplocymarin, tetrahydropalmatine, tetrahydroberberine, corydaline and the peak areas had a good linear relationship.The average recoveries (n=6) of nine components were 92.7% with RSD of 2.1%, 90.9% with RSD of 1.3%, 91.2% with RSD of 1.1%, 94.9% with RSD of 1.6%, 97.4% with RSD of 0.7%, 94.6% with RSD of 0.6%, 96.1% with RSD of 1.8%, 90.9% with RSD of 2.0%, and 104.4% with RSD of 1.4%, respectively.The content ranges of above ingredients in compound Heiguteng were 3.464-3.535 mg·g^-1 for logannic acid, 1.133-1.178 mg·g^-1 for 6'-O-β-Dglucosylgentiopicroside, 0.373-0.382 mg·g^-1 for swertiamarin, 13.598-14.142 mg·g^-1 for gentiopicroside, 0.109-0.112 mg·g^-1 for sweroside, 0.499-0.510 mg·g^-1 for glucoperiplocymarin, 0.114-0.119 mg·g^-1 for tetrahydropalmatine, 0.027-0.028 mg·g^-1 for tetrahydroberberine and 0.163-0.174 mg·g^-1 for corydaline. Conclusion: It is verified that the established method can be used for content determination of the nine effective components in compound Heiguteng.