固相萃取/液相色谱-质谱/质谱法测定山银花中5种主要黄酮苷元的含量

该文通过含有盐酸的乙醇溶液回流水解并提取,HLB固相萃取柱净化,液相色谱-质谱/质谱法检测,建立了山银花中槲皮素、木犀草素、山萘酚、芹菜素和黄芩素5种黄酮苷元含量的测定方法。实验以芦丁、木犀草苷、紫云英苷、野漆树苷和黄芩苷5种黄酮苷为代表开展研究,山银花样品经50%的乙醇溶液(含10%浓盐酸)回流2 h水解黄酮苷,同时对黄酮苷元进行提取,HLB固相萃取柱净化,采用Mightysil RP-18色谱柱分离,液相色谱-质谱/质谱法检测(电喷雾离子源、多反应监测模式、负离子扫描),外标法定量测定水解后的5种黄酮苷元含量。方法的定量下限(S/N=10)为0.005 g/kg(槲皮素),0.01 g/kg(木犀草素和芹菜素)和0.05 g/kg(山萘酚和黄芩素)。在0~1.0 g/kg范围内,5种黄酮苷元的线性相关系数均大于0.995;在山银花样品中对待测物进行3种加标水平的回收实验(加标水平相当于水解后槲皮素和木犀草素含量为:0.10、0.20、0.40 g/kg,山萘酚、芹菜素和黄芩素含量为:0.05、0.10、0.20 g/kg),方法的平均回收率70.4%~104%;相对标准偏差为4.0%~12%。该方法实现了山银花中多种主要黄酮苷元含量的同时测定,且对研究山银花药效及与黄酮类化合物的关系具有重要意义。

Simultaneous Determination of 5 Main Flavone Aglycones in Lonicerae Flos by Solid Phase Extraction Purification and Liquid Chromatography Tandem Mass Spectrometry

In this paper,an analytical method were developed for the determination of 5 main flavone aglycones including quercetin,luteolin,kaempferol,apigenin and baicalein,in Lonicerae Flos by solid phase extraction and liquid chromatography tandem mass spectrometry(SPE/LC-MS/MS).In Lonicerae Flos samples,rutin,cynaroside,astragalin,rhoifolin and baicalin were hydrolyzed and extracted by 50%ethanol solution(contained 10%hydrochloric acid),refluxed for 120 min,and finally cleaned up with an HLB solid phase extraction column.The solution was separated on a Mightysil RP-18 column(150 mm×4.6 mm(i.d),3μm),and determined by LC-MS/MS with negative electrospray ionization(ESI-)in multiple reaction monitoring(MRM)mode.The external standard method was used for the quantitation analysis of 5 main flavone aglycones.The limits of quantitation(LOQs,S/N=10)were 0.005 g/kg for quercetin,0.01 g/kg for luteolin and apigenin,and 0.05 g/kg for kaempferol and baicalein.Single-laboratory method validation data were determined,and the calibration standard curves for 5 flavone aglycones were linear in the range of 0-1.0 g/kg with correlation coefficients more than 0.995.The recoveries,at there spiked levels of 0.1,0.2,0.4 g/kg for quercetin and luteolin,0.05,0.1,0.2 g/kg for kaempferol,apigenin and baicalein after hydrolysis,ranged from 70.4%to 104%with relative standard deviations of 4.0%-12%.This method was suitable for the simultaneous determination and confirmation of flavone aglycones in Lonicerae Flos.