贯叶金丝桃药材中萘骈二蒽酮类成分的分光光度法和HPLC法定量分析

Quantitative determinations of naphthodianthrones of herba Hypericum perforatum L.by UV spectrophotometry and HPLC method

目的:建立测定贯叶金丝桃药材中萘骈二蒽酮类成分的紫外-可见分光光度法和HPLC法,比较两者测定结果的差异与定量可靠性。方法:紫外-可见分光光度法以金丝桃素为指标性成分,在590nm处测定。HPLC法以DiamonsilC18柱(150mm×4.6mm,5μm为色谱柱,甲醇-10mmol/L CH3COONH4水溶液(pH值为6.2)(90∶10)为流动相,流速1.0ml/min,检测波长590nm,柱温30℃。经紫外光谱与质谱确认其中主要色谱峰成分为伪金丝桃素和金丝桃素,并以伪金丝桃素、金丝桃素为对照品进行定量,萘骈二蒽酮大类成分含量以伪金丝桃素、金丝桃素含量之和来表征。结果:紫外-可见分光光度法定量金丝桃素在2.44～19.52μg/ml范围内线性关系良好(r=0.999 6,n=3),测得贯叶金丝桃药材中萘骈二蒽酮类成分以金丝桃素计为(0.990 2±0.006 0)μg/mg。HPLC法定量伪金丝桃素在0.402～8.032μg/ml范围内线性关系良好(r=0.999 5,n=3)),定量金丝桃素在0.488～9.760μg/ml范围内线性关系良好(r=0.999 7,n=3)),测得贯叶金丝桃药材中伪金丝桃素和金丝桃素含量分别为(0.387 2±0.001 4)、(0.220 2±0.000 7)μg/mg,两者的总量为(0.605 5±0.001 2)μg/mg。结论:对贯叶金丝桃药材中萘骈二蒽酮类成分的定量分析,紫外-可见分光光度法和HPLC法测定结果差异明显;即使化学结构与光谱特性相似的大类成分,其紫外-可见分光光度法的定量准确性仍需要验证与评价。

Objective： To investigate and compare the quantitative determination of naphthodianthrones of Hypericum perforaturn L. by UV speetrophotometry and HPLC method. Methods： Hypericin was used as the reference compound for UV spectrophotometry,and the detection wavelength was 590 nm. HPLC was performed on a Diamonsil C18 analytical column （150 mm×4.6 mm,5 μm）with mobile phase consisted of CH3OH-10 mmol/L CH3COONH4 （90 ： 10, pH 6.2） at the flow rate of 1.0 ml/min. The detection wavelength was 590 nm and column temperature was 30 ℃. The two peaks in HPLC chro matograms of herba Hypericum perforaturn were identified as hypericin and pseudohypericin by UV spectra and negative ion mass spectra. The content of naphthodianthrones of Hypericum perforatum herba was determinated and estimated based on the sum of hypericin and pseudohyperiein. Results： The linear range of hypericin was 2.44-19.52 μg/ml for UV spectra（r=- 0. 999 6, n = 3）. The content of naphthodianthrones of Hypericum perforaturn herba calculated with reference to hypericin was （0. 990 2±0. 006 0） μg/mg. For HPLC analysis, pseudohypericin had good linearity in the range of （0. 402 8. 032） μg/ml （r=0. 999 5,n=3） and hypericin also had good linearity in the range of （0. 488-9. 760） μg/ml（r=0. 999 7,n=3）. The con tents of pseudohypericin and hypericin from Hypericum perforatum herba calculated with its standard references were （0. 387 2±0. 001 4） μg/rag and （0. 220 2±0. 000 7） fig/rag, respectively. The total content of naphthodianthrones of Hyperi cure perforatum herba determined by HPLC with references to pseudohypericin and hypericin was （0. 605 5±0. 001 2） μg/mg. Conclusion： The contents of naphthodianthrones from Hypericum perforatum herba determined by UV spectrophotometry and HPLC are significantly different. This suggests that the accuracy of UV spectrophotometry is suspectable of determing the content of naphthodianthrones in Hypericum perforatum although the chemical structure and UV spectra are similar.