液相色谱-串联质谱法测定山银花中有机酸和黄酮类化合物的含量

Simultaneous Determination of Organic Acids and Flavonoids in Lonicerae flos by Liquid Chromatography-Tandem Mass Spectrometry

摘要:

本文通过甲醇溶液超声提取，HLB固相萃取柱净化，液相色谱-串联质谱法检测，建立了山银花中4种有机酸（绿原酸、咖啡酸、异绿原酸A和异绿原酸C）和14种黄酮类化合物（槲皮素、芦丁、木犀草素、木犀草苷、山奈酚、紫云英苷、芹菜素、野漆树苷、黄芩素、黄芩苷、槲皮素-3-甲基醚、香叶木素、白杨素和山奈素）含量测定的方法。山银花样品经50%的甲醇溶液超声提取40分钟，HLB固相萃取柱净化，采用Mightysil RP-18色谱柱分离，液相色谱-串联质谱法进行检测（电喷雾离子源，多反应监测模式，负离子扫描），外标法定量进行测定。方法定量限（以S/N大于10计）0.005 g/kg-0.05 g/kg，配制0.05、0.10、0.20、0.50和1.00 g/kg线性工作曲线，线性相关系数大于0.993。在山银花样品中进行添加回收试验（添加水平绿原酸10.0、20.0和40.0 g/kg，咖啡酸1.0、2.0和4.0 g/kg，异绿原酸A和异绿原酸C 5.0、10.0和20.0 g/kg，其他化合物0.05、0.10和0.20 g/kg），方法总体回收率69.2%~116%，相对标准偏差3.3%~12.0%。方法实现了山银花中多种主要有机酸和黄酮类化合物含量的同时测定。

Abstract:

In this paper, an analytical method for the determination of 4 organic acids (chlorogenic acid, caffeic acid, isochlorogenic acid A and isochlorogenic acid C) and 14 flavonoids (quercetin, rutin, luteolin, cynaroside, kaempferol, astragalin, apigenin, rhoifolin, baicalein, baicalin, quercetin-3-methylether, diosmetin, chrysin and kaempferide) in Lonicerae flos were developed and validated by solid phase extraction and liquid chromatography with tandem mass spectrometry (LC-MS/MS). In Lonicerae flos samples, the organic acids and flavonoids were extracted by methanol solution (concentration: 50%), ultrasonication for 40 min, and cleaned up with HLB column by solid phase extraction. The methanol eluent was ?ltered through nylon membrane and analysis by LC-MS/MS. The solution was separated by Mightysil RP-18 column (150 mm × 4.6 mm (i.d), 3 μm) and the quantitative detection was performed on LC-MS/MS by multiple reaction monitoring (MRM) mode under negative electrospray ionization (ESI-). External standard method was used for quantitation analysis. The limits of quantification (LOQs, S/N>10) were 0.005 g/kg~0.05 g/kg. Single-laboratory method validation data were determined, the calibration standard curves concentrations were 0.05~1.00 g/kg and the correlation coefficient of calibration standard curves was more than 0.993. The recoveries were found in the range of 69.2%~116% and the relative standard deviations were 3.3%~12.0% at the respiked levels (10.0, 20.0 and 40.0 g/kg for chlorogenic acid; 1.0, 2.0 and 4.0 g/kg for caffeic acid; 5.00, 10.0 and 20.0 g/kg for isochlorogenic acid A and isochlorogenic acid C; 0.05, 0.10 and 0.20 g/kg for other compounds). This method was suitable for simultaneous determination and confirmation of 4 organic acid and 14 flavonoids in Lonicerae flos. Key words: Lonicerae flos; organic acid; flavonoids; high performance liquid chromatography with tandem mass spectrometry of quantification (LOQs, S/N>10) were 0.005 g/kg~0.05 g/kg. Single-laboratory method validation data were determined, the calibration standard curves concentrations were 0.05~1.00 g/kg and the correlation coefficient of calibration standard curves was more than 0.993. The recoveries were found in the range of 69.2%~116% and the relative standard deviations were 3.3%~12.0% at the respiked levels (10.0, 20.0 and 40.0 g/kg for chlorogenic acid; 1.0, 2.0 and 4.0 g/kg for caffeic acid; 5.00, 10.0 and 20.0 g/kg for isochlorogenic acid A and isochlorogenic acid C; 0.05, 0.10 and 0.20 g/kg for other compounds). This method was suitable for simultaneous determination and confirmation of 4 organic acid and 14 flavonoids in Lonicerae flos.