化橘红中香豆素类物质的高效液相色谱法测定

Determination of Coumarins in Tomentose Pummelo Peel by HPLC

采用高效液相色谱(HPLC)法测定化橘红中的多种香豆素成分,并建立10种香豆素混合标准品的分离方法。这10种香豆素标准品是羟甲香豆素、香豆素、7-甲氧基香豆素、呋喃香豆素、6-甲基香豆素、5,7-二甲氧基香豆素、佛手内酯、欧前胡素、异欧前胡素和香柠檬亭。10种香豆素的分离条件:分离柱NucleosilC18反相柱(150mm×4.6mm,3μm),流动相水-甲醇-乙腈,三元梯度洗脱,起始流速0.3ml/min,流速0.3～1ml/min,柱温35℃,分离波长315nm。标准品质量浓度范围为0.078～15.000μg/ml,回归方程的相关系数为0.999～1.000,线性关系极好。标准品连续5次进样和在48h内多次进样,保留时间和峰面积的相对标准偏差均不高于3.466%。对采自广东省茂名市化州市10个不同栽培地点的化橘红样品分析,除6-甲基香豆素和5,7-二甲氧基香豆素未在样品分离中出现外,化橘红样品含有其余的香豆素成分。10个样品间在香豆素含量上存在显著差异,其中7号样品含量最高,香豆素达1380μg/g干粉,异欧前胡素为1226μg/g干粉,分别达0.138%和0.123%。各样品中基本以香豆素和异欧前胡素这两种香豆素含量为主。

A high-performance liquid chromatography （HPLC） method for separating and determining 10 standard coumarins was developed, and was used to determine coumarins in Tomentose Pummelo Peel （Exocarpium Citri Grandis） collected from ten areas in Huazhou County. Ten standard coumarins were as follows： hymecromone, coumarin, 7-hydroxycoumarin, psoralen, 6-methylcoumarin, 5,7-dimethoxycoumarin, bergapten, imperatorin, isoimperatorin and bergamottin. Samples were chromated using reversed-phase Nucleosil C18 column （150 mm×4.6 mm, 3 μm）, and the mobile phases A, B, C were water, methanol and acetonitrile respectively. Triple gradient elution started at 0.3 ml/min and proceeded at 0.3 - 1.0 ml/min. Operation conditions were set as follows： column temperature 35 ℃, detection at 315 nm. Results showed that linear calibration of ten standard samples were within 0.078 - 15.000 μ g/ml with correlation （r） 0.999 - 1.000. Relative standard deviation （RSD） of retention time and peak area were both below 3.466% in five consecutive tests or repeated tests during 48 h. Except 6-methylcoumarin and 5,7-dimethoxycoumarin, totally 8 coumarins were detected in all of the ten Tomentose Pummelo Peel samples with significant difference. No.7 sample was the richest in coumarins with coumarin content at 0.138% dried mass （DM） and isoimperatorin content at 0.123% DM. It indicated that coumarin and isoimperatorin were main coumarins in Tomentose Pummelo Peel.