芹菜籽是芹菜的种子,《中华本草·维吾尔药卷》将芹菜籽列为维药的一种常用药材[1]。芹菜籽中含有丰富的功能性成分,如黄酮、挥发性成分、脂肪酸和氨基酸等[2-5],具有降血压、抗氧化、抗肿瘤和降血脂等多种药理功效[6-9]。近年来,芹菜籽开始作为一种保健产品在市场上兴起,前景广阔。

黄酮是植物体内产生的一类次生代谢产物,泛指通过中央三碳原子连接2个含酚羟基的苯环而形成的化合物。其基本结构母核为2-苯基色原酮,主要可分为黄酮类、黄酮醇类、二氢黄酮类等。芹菜苷、芹菜素、芦丁、木犀草素、槲皮素、柯伊利素等是芹菜籽中的代表性黄酮类成分[10-12]。研究表明,黄酮具有抗肿瘤[13]、抗氧化[14]、镇静[15]、降血压血脂[16]等作用。

目前,常见的黄酮检测方法有紫外分光光度法、薄层色谱法、高效液相色谱法及液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry, LC-MS/MS)等[17-18]。其中,紫外分光光度法一般测定样品中总黄酮含量,不能用于痕量分析；薄层色谱法准确度较低,难以区分相似化合物[19]；高效液相色谱法存在对多种黄酮分离难度大,分析时间长,专属性差等不足[20]；LC-MS/MS具有高灵敏度,强选择性,定量准确,黄酮成分分离及结构分析过程简单等优点[21]。因此,本文采用LC-MS/MS建立了同时测定芹菜籽中9种黄酮含量的方法,同时对前处理和检测条件进行优化,为芹菜籽中黄酮的研究提供技术支持。

1 材料与方法

1.1 材料与试剂

芹菜籽(实芹籽、冬芹籽、西芹籽),天津市购；芹菜苷、芦丁、黄芩苷、黄芩素、汉黄芩素、芹菜素、柯伊利素,中国食品药品检定研究院；木犀草素、槲皮素,中国药品生物制品检定所。

Al(NO3)3、NaNO2,均为分析纯,天津市凯通化学试剂有限公司；NaOH(分析纯),天津市华东试剂厂；甲醇(质谱纯)、乙腈(质谱纯),德国Merck公司；甲酸(色谱纯),CNW.EU公司；纯净水,广州屈臣氏食品饮料有限公司；AB-8、D3520、X-5、NKA-9型大孔树脂、无水乙醇(色谱纯),天津市光复精细化工研究所。

1.2 仪器与设备

1200 series液相色谱-6410三重四极杆串联质谱仪,美国Agilent公司；TU-1900紫外-可见分光光度计,北京普析通用仪器公司；KQ-5200E型超声清洗机,昆山市超声仪器有限公司；FW135中草药粉碎机,天津市泰斯特仪器有限公司；RE-52A旋转蒸发仪,广东昆山仪器有限公司。

1.3 实验方法

1.3.1 芹菜籽中黄酮提取

以实芹籽为原料对芹菜籽中黄酮提取、纯化方法进行优化。实芹籽除杂、粉碎,过40目筛,石油醚脱脂,40 ℃干燥至恒重,备用。单因素试验结合正交试验优化芹菜籽中总黄酮的提取工艺：称取脱脂后的芹菜籽粉1.0 g,超声提取,3 000 r/min离心10 min,过滤,取上清液NaNO2-Al(NO3)3-NaOH显色[22],紫外-可见分光光度计测吸光度,按公式(1)计算总黄酮提取率:

总黄酮提取率

(1)

式中：C,样品中黄酮含量相当于标准芦丁浓度,mg/mL；V,提取液体积,mL；D,稀释倍数；m,样品质量,g。

1.3.2 粗黄酮纯化

按1.3.1优化后条件提取芹菜籽中黄酮,取过滤后上清液旋蒸,除去乙醇,冻干样品,得到黄酮粗品,配制1.0 mg/mL的黄酮水溶液,待用。按冯靖等[23]的方法,对4种型号大孔树脂(AB-8、D3520、X-5、NKA-9)进行预处理。称取大孔树脂3 g,加入30 mL 1.0 mg/mL的黄酮溶液,100 r/min下摇床振荡,使吸附平衡,取样测吸光度,按公式(2)计算吸附率。称取吸附平衡的大孔树脂3 g,加入30 mL 70%体积分数乙醇水溶液,100 r/min下摇床振荡,使解吸平衡,取样测吸光度,按公式(2)计算解吸率。

吸附率(解吸率)

(2)

式中：ρ0,吸附或解吸前溶液黄酮质量浓度,mg/mL；ρt,吸附或解吸后溶液黄酮质量浓度,mg/mL。

1.3.3 LC-MS/MS检测

按1.3.1优化后条件提取、纯化芹菜籽中黄酮,取解吸平衡后溶液旋蒸、冻干,用甲醇溶解并定容至10 mL,过0.22 μm有机滤膜,液相色谱-串联质谱仪检测。

1.3.4 标准溶液的配制

分别用甲醇将芹菜苷、芦丁、黄芩苷、黄芩素、木犀草素、槲皮素、汉黄芩素、芹菜素、柯伊利素9种黄酮标准品溶解,定容得到1.0 mg/mL的标准储备液,再用甲醇将上述储备液稀释成不同质量浓度梯度的标准工作液。

1.3.5 液相色谱条件

色谱柱ZORBAX Eclipse XDB-C18(4.6 mm×150 mm,3.5 μm)；柱温30 ℃；进样量10 μL；流动相V(乙腈)∶V(0.1%体积分数甲酸水溶液)=3∶7；流速0.4 mL/min。

1.3.6 质谱条件

电喷雾离子源,负离子扫描方式,多反应监测(multiple reaction monitoring,MRM)模式,离子源温度350 ℃,毛细管电压4.0 kV,N2流速10 L/min,雾化气压力40 psi。优化后的母离子、子离子、去簇电压以及碰撞电压具体参数见表1。

2 结果与分析

2.1 色谱柱的选择

实验比较了ZORBAX Eclipse XDB-C18 (4.6 mm×150 mm,3.5 μm)、ZORBAX Eclipse Plus C18(2.1 mm×100 mm,3.5 μm)和HyPMRITY C18 (4.6 mm×250 mm,5.0 μm) 3种色谱柱对9种黄酮的分离效果。结果表明,HyPMRITY C18柱和Eclipse Plus C18柱无法分离开9种黄酮,响应信号低；XDB-C18柱对9种黄酮出峰明显且能很好分离,响应值高,见图1。因此,实验选用ZORBAX Eclipse XDB-C18色谱柱。

2.2 流动相及其比例的优化

2.2.1 流动相的优化

考察甲醇-水、乙腈-水、甲醇-0.1%甲酸水、乙腈-0.1%甲酸水4种流动相对9种黄酮的分离效果,在前3种流动相条件下,9种黄酮分离效果不佳。因此选择乙腈-0.1%甲酸水溶液作为本方法的流动相,结果见图2。

2.2.2 流动相比例的优化

比较不同体积比例(2∶8、3∶7、4∶6)流动相乙腈-0.1%甲酸水对9种黄酮分离效果的影响。当流动相比例为V∶V=3∶7时,9种黄酮分离较好而且响应值较高,如图3所示。

2.3 质谱条件优化

通过优化去簇电压和碰撞电压,得到芹菜苷、芦丁、黄芩苷、黄芩素、木犀草素、槲皮素、汉黄芩素、芹菜素、柯伊利素9种黄酮的二级质谱图,见图4。

2.4 前处理过程优化

2.4.1 芦丁标准曲线的绘制

无水乙醇溶解芦丁标准品,NaNO2-Al(NO3)3-NaOH显色处理[22],紫外-可见分光光度计测吸光度,绘制芦丁标准曲线方程为y=21.157 1x+0.002 9(R2=0.999 5)。

2.4.2 提取工艺条件的优化

如表2所示,正交试验结果表明RB>RC>RA,料液比是影响芹菜籽中黄酮提取的最主要因素,其次是提取时间,最后是乙醇体积分数。根据K值分析,选择A2B3C3,即70%体积分数乙醇水溶液,料液比1∶20(g∶mL),提取时间60 min,作为超声辅助提取芹菜籽中黄酮的最佳工艺条件。通过3次重复性验证试验,得到芹菜籽中总黄酮平均提取率为3.78%,高于此前王文君等[24]的提取率1.67%,与徐斌等[25]的提取率13.54%存在差异,原因可能是芹菜籽品种和产地的不同,以及后者测定的是脱脂脱蛋白后芹菜籽中黄酮提取率。

2.4.3 大孔树脂选择

4种大孔树脂吸附和解吸平衡时的吸附率、解吸率见表3。

由表3可知,AB-8大孔树脂的吸附率和解吸率均高于其他3种大孔树脂,故选择AB-8大孔树脂进行纯化。在林薇薇等[26]的方法上进行修改,上样液质量浓度1.0 mg/mL、pH 6.0、上样液流速2 BV/h、洗脱液乙醇体积分数70%、洗脱液流速2 BV/h,黄酮纯度由3.4%提高到25.2%。

2.5 线性范围、标准曲线和检出限

分别配制质量浓度0.5、1.0、2.0、5.0、10.0、20.0 μg/mL的9种黄酮标准混合液,采用LC-MS/MS检测。以质量浓度为横坐标,MRM模式下的峰面积作为纵坐标,绘制标准曲线,得到线性方程和线性相关系数R2。检出限按信噪比S/N=3计算[27],结果见表4。

由表4可知,9种黄酮在0.5～20.0 μg/mL的线性范围内线性关系良好,线性相关系数R2均>0.999 0。S/N=3时,检出限为10～66 μg/kg。

2.6 精密度、稳定性和加标回收率

取同一质量浓度的9种黄酮标准品混合溶液重复进样6次,记录峰面积,计算精密度相对标准偏差(relative standard deviation,RSD)。9种黄酮精密度RSD均<2%,仪器精密度较好。

取1.3.3条件下制备的同一黄酮样品溶液,LC-MS/MS检测,共进样6次,每次间隔4 h,计算峰面积RSD。9种黄酮的RSD为1.4%～4.0%,表明芹菜籽黄酮样品溶液在24 h内稳定性较强。

将9种黄酮混合标准溶液以低、中、高3个水平分别加入到1.0 g芹菜籽样品中,每个水平平行测定3次,计算平均回收率和RSD。结果见表5。

由表5可知,9种黄酮的加标回收率为88.0%～106.2%,RSD为0.7%～4.9%,满足方法学要求。

2.7 样品测定

按1.3.5和1.3.6中液相色谱、质谱条件对天津实芹籽样品进行检测,得到总离子流图和MRM图,见图5。

利用试验建立的液相色谱串联质谱法对天津市售的实芹籽、冬芹籽、西芹籽3种芹菜籽中9种黄酮的含量进行测定,结果见表6。实芹籽、冬芹籽、西芹籽3种芹菜籽中9种黄酮含量差异较小,可能是由于来自同一产地天津,气温、海拔等生长环境条件相同。其中实芹籽中均含有9种黄酮,与文献[28-29]报道较为一致。冬芹籽中芹菜苷和黄芩苷未检出,西芹籽中柯伊利素未检出,这种差异可能是品种不同造成的。

3 结论

本文成功建立了一种同时测定芹菜籽样品中芹菜苷、芦丁、黄芩苷、黄芩素、木犀草素、槲皮素、汉黄芩素、芹菜素、柯伊利素9种黄酮含量的液相色谱串联质谱法,对芹菜籽中黄酮的提取、纯化、液相色谱以及质谱等条件进行了优化。该方法操作简便,纯化效果好,灵敏度、精密度高,重复性好,各项参数均能达到日常检测的技术要求,能够满足测定芹菜籽中9种黄酮含量的需要。

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Determination of nine flavonoids in celery seed by liquid chromatography-tandem mass spectrometry

HUANG Huang,WANG Fengling*,ZHAO Wei,WANG Xueqing,ZHANG Tingting

(Tianjin Key Laboratory of Food Biotechnology,College of Biotechnology and Food Science,Tianjin University of Commerce,Tianjin 300134,China)

ABSTRACT A laboratory method for the detection of the concentration of nine flavonoids (apigenin, rutin, baicalin, baicalein, luteolin, quercetin, wogonin, apigenin, coylidin) in celery seeds were established based on liquid chromatography-tandem mass spectrometry. The relevant parameters such as sample extraction, purification, liquid phase separation and tandem mass spectrometry were optimized. The sample was extracted with a material-to-liquid ratio of 1∶20 (g∶mL), 70% volume fraction of ethanol aqueous solution by ultrasonic for 60 min, and purified by AB-8 macroporous resin. Using acetonitrile-0.1% formic acid water solution as mobile phase, the sample was separated by ZORBAX Eclipse XDB-C18 chromatographic column and detected by electrospray negative ions, MRM mode. The results showed that the nine flavonoids had a good linear relationship in the mass concentration range of 0.5-20.0 μg/mL, the correlation coefficient r2≥0.999 0, and the recovery rates of the nine flavonoids were all 88.0%-106.2% (relative standard deviation was 0.7%-4.9%), the detection limits of nine flavonoids were 10-66 μg/kg. This method is simple, and the results is accurate and reliable and with good stability. It is suitable for the simultaneous detection of nine flavonoids in celery seeds.

Key words celery seed; flavonoids; macroporous resin purification; liquid chromatography-tandem mass spectrometry (LC-MS/MS)

第一作者：硕士研究生(王凤玲高级实验师为通讯作者，E-mail：wfling@tjcu.edu.cn)

收稿日期：2020-12-02,改回日期：2021-01-21

DOI:10.13995/j.cnki.11-1802/ts.026351

引用格式:黄璜,王凤玲,赵薇,等.液相色谱-串联质谱法测定芹菜籽中九种黄酮含量[J].食品与发酵工业,2021,47(11):259-265.HUANG Huang,WANG Fengling,ZHAO Wei,et al.Determination of nine flavonoids in celery seed by liquid chromatography-tandem mass spectrometry[J].Food and Fermentation Industries,2021,47(11):259-265.