CN115015428B - 一种黄酒中多酚类化合物的检测方法 - Google Patents
一种黄酒中多酚类化合物的检测方法 Download PDFInfo
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- CN115015428B CN115015428B CN202210695893.0A CN202210695893A CN115015428B CN 115015428 B CN115015428 B CN 115015428B CN 202210695893 A CN202210695893 A CN 202210695893A CN 115015428 B CN115015428 B CN 115015428B
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Abstract
本发明公开了一种黄酒中多酚类化合物的检测方法,包括:(1)配制多酚类化合物混合标准工作液,采用液相色谱‑质谱联用仪检测,拟合标准曲线,计算检出限和定量限;(2)样品前处理:a.浓盐酸调节酒样pH至2.0;乙酸乙酯萃取样品,离心,重复一次,合并上清液;b.乙醚再次萃取、离心,并重复一次,合并上清液;c.通过真空平行浓缩仪将上清液样品浓缩至近干,乙腈水溶液溶解残渣,过滤膜,制得待测样品;(3)液相色谱‑质谱法上机检测。本发明建立了一种能够同时测定黄酒中多酚类成分的简便、快速、准确、高灵敏度的定性定量分析方法,为推广黄酒的营养价值、引导人们进行健康消费提供数据支持,利于推动黄酒产业的发展。
Description
技术领域
本发明涉及化学成分定量检测技术领域,特别是涉及一种黄酒中多酚类化合物的检测方法。
背景技术
黄酒是世界上最古老的酒类之一,它以稻米、泰米、小米、玉米、小麦、水等为主要原料,添加酒曲和糖化剂,在众多微生物作用下酿造而成。黄酒保留了发酵过程中产生的营养和健康活性物质,历来以营养丰富、保健养生著称。黄酒中除富含多种氨基酸、维生素、低聚糖、矿物质微量元素外,还含有对人类健康非常重要的功能性营养成分,如黄酮、类黄酮、单宁等这类分子中具有多个酚羟基的多酚类物质。黄酒中的多酚类化合物具有很强的活性氧以及氧自由基的清除能力,可以抑制和隔断链式自由基的氧化反应,还可以与金属离子螯合,进而降低金属离子对氧化反应的催化作用,对自由基所引起的生物大分子损伤起较强的保护作用。黄酒多酚除了具有抗氧化作用外,还具有保护心血管、防止动脉硬化等作用。
当前黄酒中多酚类物质的检测主要采用高效液相色谱法,该方法是采用高压输液系统,以液体为流动相,将流动相泵入装有固定相的色谱柱,在柱内完成各成分的分离,进入检测器进行检测,从而实现对试样的分析。文献中公开的用于黄酒中多酚类物质检测的条件大致为:C18色谱柱,流动相由乙酸、水、乙腈组成,一般使用紫外检测器的检测波长为240nm-450nm。但由于黄酒中结构相近的多酚类物质在色谱行为上不易区分且存在其他杂质干扰,无法进行准确定性,致使黄酒的营养价值不能被充分挖掘和宣传,导致黄酒产品竞争力弱,推广难度大。
发明内容
本发明的目的在于克服上述现有技术的不足,提供一种黄酒中多酚类化合物的检测方法,建立了一种能够同时测定黄酒中多酚类成分的简便、快速、准确、高灵敏度的定性定量分析方法,为推广黄酒的营养价值、引导人们进行健康消费提供数据支持,利于推动黄酒产业的发展。
为了实现上述目的,本发明采用如下技术方案:
一种黄酒中多酚类化合物的检测方法,包括以下步骤:
(1)以50wt%乙腈水溶液配制不同浓度梯度的含20种多酚类化合物的混合标准工作液,采用液相色谱-质谱联用仪进行检测,拟合标准曲线,以性噪比S/N≥3和S/N≥10计算出20种多酚的方法检出限和定量限;
(2)样品前处理:
a.采用浓盐酸调节酒样pH至2.0,混匀;取pH至2.0的酒样2.0mL于离心管中,加入10mL乙酸乙酯震荡萃取,离心,取上清液于浓缩瓶中;重复萃取离心一次,合并上清液;
b.向步骤a所得的合并上清液中加入10mL乙醚震荡萃取,离心,取出上清液,重复萃取、离心一次,合并上清液;
c.将步骤b所得的合并上清液置于真空平行浓缩仪中,35℃以下浓缩至近干,采用10.0mL 50wt%乙腈水溶液溶解残渣,过滤膜,制得待测样品,待进样;
(3)液相色谱-质谱法检测:采用液相色谱-质谱联用仪对待测样品进行检测,记录HPLC色谱图和峰面积,根据不同多酚化合物的标准曲线,计算获得样品中不同多酚化合物的含量。
步骤(1)中,混合标准工作液具体配制步骤为:分别取20种多酚类化合物各5mg,采用50wt%乙腈配制500mg/L的单一标准储备液并于-20℃保存;取上述标准储备液,采用50wt%乙腈水溶液稀释,配制成5、10、50、100、250、500μg/L混合标准工作液。
所述20种多酚类化合物包括没食子酸、儿茶素、香草酸、咖啡酸、丁香酸、对香豆酸、阿魏酸、槲皮素、香豆酸、原儿茶素、山奈酚、绿原酸、牡荆素、槲皮苷、金丝桃苷、芦丁、表儿茶素、鞣花酸、肉桂酸、芥子酸。
步骤(2)中,每次离心的离心速率为6000r/min,离心时间为2min。
色谱条件为:Waters ACQUITY HSS T3色谱柱,规格为2.1×100mm,1.8μm;流动相A:1%乙酸水,流动相B:甲醇;流速:0.4mL/min;进样量:1μL;柱温:40℃;梯度洗脱。
梯度洗脱程序为:0min,A相95%、B相5%,瞬间达到设定比例;0-0.5min,保持A相95%、B相5%;0.5-3min,两种流动相均速变化到A相50%、B相50%;3-6min,两种流动相均速变化到A相5%、B相95%;6-8min,保持A相5%、B相95%;8.1min,瞬时调整为A相95%、B相5%;8.1-10min,保持A相95%、B相5%;上述均为体积百分比。
本发明的有益效果是:
应用高效液相色谱串联三重四级杆质谱(HPLC-QQQ)技术,采用多反应监测模式检测,建立了一种能够同时测定黄酒中多酚类成分的简便、快速、准确、高灵敏度的定性定量分析方法,解决了采用现有高效液相色谱法检测时存在的结构相近的多酚类物质在色谱行为上不易区分且受其他杂质干扰的问题,将其应用于多种黄酒产品的检测,总结归纳黄酒中多酚类物质的种类和含量,能够为推广黄酒的营养价值、引导人们进行健康消费提供数据支持,从而推动黄酒产业的发展。
附图说明
图1为本发明20种多酚化合物中8种多酚的MRM色谱图;
图2为本发明20种多酚化合物中7种多酚的MRM色谱图;
图3为本发明20种多酚化合物中5种多酚的MRM色谱图;
图4为本发明没食子酸的色谱图;
图5为本发明儿茶素的色谱图;
图6为本发明香草酸的色谱图;
图7为本发明咖啡酸的色谱图;
图8为本发明丁香酸的色谱图;
图9为本发明对香豆酸的色谱图;
图10为本发明阿魏酸的色谱图;
图11为本发明槲皮素的色谱图;
图12为本发明香豆酸的色谱图;
图13为本发明原儿茶素的色谱图;
图14为本发明山奈酚的色谱图;
图15为本发明绿原酸的色谱图;
图16为本发明牡荆素的色谱图;
图17为本发明槲皮苷的色谱图;
图18为本发明金丝桃苷的色谱图;
图19为本发明芦丁的色谱图;
图20为本发明表儿茶素的色谱图;
图21为本发明鞣花酸的色谱图;
图22为本发明肉桂酸的色谱图;
图23为本发明芥子酸的色谱图;
图24本发明不同提取方式下20种多酚化合物中10种多酚的提取效果对比图;
图25本发明不同提取方式下20种多酚化合物中另10种多酚的提取效果对比图。
图1中,1:原儿茶酸、2:咖啡酸、3:对香豆酸、4:牡荆素、5:芦丁、6:金丝桃苷、7:槲皮苷、8:肉桂酸;图2中,9:没食子酸、10:香豆酸、11:绿原酸、12:表儿茶素、13:香草酸、14:丁香酸、15:阿魏酸;图3中,16儿茶素、17:芥子酸、18:鞣花酸、19:槲皮素、20:山奈酚;
图4-23中,(Ⅰ)表示混合标准溶液中其中一种多酚的定量离子色谱标准图,(Ⅱ)表示混合标准溶液中其中一种多酚的定性离子色谱图;
图24中,a:没食子酸、b:儿茶素、c:香草酸、d:咖啡酸、e:丁香酸、f:对香豆酸、g:阿魏酸、h:槲皮素、i:香豆酸、j:原儿茶素;图25中,k:山奈酚、l:绿原酸、m:牡荆素、n:槲皮苷、o:金丝桃苷、p:芦丁、q:表儿茶素、r:鞣花酸、s:肉桂酸、t:芥子酸。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步描述:
实施例
1、一种黄酒中多酚类化合物的检测方法
(1)以50wt%乙腈水溶液配制不同浓度梯度的含20种多酚类化合物的混合标准工作液,采用液相色谱-质谱联用仪进行检测,拟合标准曲线,以性噪比S/N≥3和S/N≥10计算出20种多酚的方法检出限和定量限;
混合标准工作液具体配制步骤为:分别取20种多酚类化合物各5mg,采用50wt%乙腈配制500mg/L的单一标准储备液并于-20℃保存;取上述标准储备液,采用50wt%乙腈水溶液稀释,配制成5、10、50、100、250、500μg/L混合标准工作液。
20种包括多酚类化合物包括没食子酸、儿茶素、香草酸、咖啡酸、丁香酸、对香豆酸、阿魏酸、槲皮素、香豆酸、原儿茶素、山奈酚、绿原酸、牡荆素、槲皮苷、金丝桃苷、芦丁、表儿茶素、鞣花酸、肉桂酸、芥子酸。
(2)样品前处理:
a.采用浓盐酸调节酒样pH至2.0,混匀;取pH至2.0的酒样2.0mL于离心管中,加入10mL乙酸乙酯震荡萃取,6000r/min离心2min,取上清液于浓缩瓶中;重复萃取离心一次,合并上清液;
b.向步骤a所得的合并上清液中加入10mL乙醚震荡萃取,6000r/min离心2min,取出上清液,重复萃取、离心一次,合并上清液;
c.将步骤b所得的合并上清液置于真空平行浓缩仪中,35℃以下浓缩至近干,采用10.0mL 50wt%乙腈水溶液溶解残渣,过滤膜,制得待测样品,待进样;
(3)液相色谱-质谱法检测:采用液相色谱-质谱联用仪对待测样品进行检测,记录HPLC色谱图和峰面积,根据不同多酚化合物的标准曲线,计算获得样品中不同多酚化合物的含量。样品中多酚化合物含量计算公式为:多酚含量(μg/L)=上机结果(μg/L)×定容体积(mL)/取样体积(mL)。
上述黄酒中多酚类化合物的检测操作中,色谱条件为:Waters ACQUITY HSS T3色谱柱,规格为2.1×100mm,1.8μm;流动相A:1%乙酸水,流动相B:甲醇;流速:0.4mL/min;进样量:1μL;柱温:40℃;梯度洗脱。
梯度洗脱程序为:0min,A相95%、B相5%,瞬间达到设定比例;0-0.5min,保持A相95%、B相5%;0.5-3min,两种流动相均速变化到A相50%、B相50%;3-6min,两种流动相均速变化到A相5%、B相95%;6-8min,保持A相5%、B相95%;8.1min,瞬时调整为A相95%、B相5%;8.1-10min,保持A相95%、B相5%;上述均为体积百分比。见表1。
表1多酚测定洗脱条件
步骤 | 时间(min) | A(%V/V) | B(%V/V) |
0 | 0 | 95 | 5 |
1 | 0.5 | 95 | 5 |
2 | 3.0 | 50 | 50 |
3 | 6.0 | 5 | 95 |
4 | 8.0 | 5 | 95 |
5 | 8.1 | 95 | 5 |
6 | 10.0 | 95 | 5 |
质谱检测在ESI负离子模式下,采用多反应离子监测模式进行测定,采用外标法,以质量数和保留时间定性,以峰面积定量。多酚质谱检测条件见表2。
表2多酚质谱检测条件
2、流动相的优化
多酚类物质通常具有带1个或多个羟基的芳环结构,在负离子模式下检测可获得较强的质谱信号。依据负离子模式检测时物质离子化的特点,若流动相中含酸会抑制目标物的离子化,所以以水为流动相。但以甲醇-水为流动相时,多酚类物质在色谱柱上的峰形严重拖尾,而且基线噪声很强,而加入少量乙酸后,峰形明显改善。为兼顾多酚类物质的峰形和响应值,分别在流动相中添加0.1wt%、0.5wt%、1.0wt%、1.5wt%的乙酸进行测定,结果表明,以含1.0wt%乙酸的水溶液与甲醇为流动相时多酚类物质的响应最佳。多酚定量离子色谱标准图(图1-3)和定性离子色谱图(图4-23)。
3、提取方式的优化
本发明采用乙酸乙酯-乙醚混合提取多酚类化合物。考察了固相萃取小柱(混合型阴离子MAX/亲水亲油平衡材料HLB/安捷伦Bond Elute Plexa)和液液萃取(乙酸乙酯(2×10mL)-乙醚(2×10mL))对黄酒中多酚类物质的提取效率,提取过程如上样品处理过程,各种固相萃取和液液萃取提取效率见图24、图25。由图可知,采用乙酸乙酯-乙醚液液萃取法进行前处理,黄酒中多酚类物质除个别稍低以外,其它多酚回收率均可达80%以上。因此,乙酸乙酯-乙醚提取效率最高,这与多酚在pH 2.0呈分子状态,更易溶于乙酸乙酯和乙醚有关。
4、线性关系和方法检出限
以50%乙腈配制20种多酚类化合物的标准工作液,进行液相色谱质谱联用仪检测,拟合标准曲线,以性噪比S/N≥3和S/N≥10计算出20种多酚的方法检出限和定量限,结果见表3。
表3 20种多酚的线性方程、检出限和定量限
5、回收率和精密度
由于空白基质的样品不存在,并且黄酒中某些多酚类物质含量较高,所以选择15%乙醇做加标试验,在标准曲线范围内,选取三个梯度浓度对样品进行加标测试,并计算精密度,结果见表4-23。多酚的回收率在70~120%,RSD(n=6)≤8%,所建立的方法能满足黄酒中多酚的分析要求。
表4没食子酸、儿茶素的回收率和精密度
表5香草酸、咖啡酸的回收率和精密度
表6丁香酸、对香豆酸的回收率和精密度
表7阿魏酸、槲皮素的回收率和精密度
表8香豆酸、原儿茶素的回收率和精密度
表9山奈酚、绿原酸的回收率和精密度
表10牡荆素、槲皮苷的回收率和精密度
表11金丝桃苷、芦丁的回收率和精密度
表12表儿茶素、鞣花酸的回收率和精密度
表13肉桂酸、芥子酸的回收率和精密度
选取不同年份古越龙山黄酒样品,采用本发明检测方法测定其中的多酚含量,检出多酚种类及含量结果见表14。从表14可知,所检不同年份黄酒中检出多酚的种类在5-13种,组加、加饭两种类型的黄酒多酚的种类较少,为5-7种,其他品种黄酒检出多酚种类在9种以上。
表14不同年份古越龙山黄酒中多酚含量(μg/L)
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种黄酒中多酚类化合物的检测方法,其特征在于:包括以下步骤:
(1)以50wt%乙腈水溶液配制不同浓度梯度的含20种多酚类化合物的混合标准工作液,采用液相色谱-质谱联用仪进行检测,拟合标准曲线,以性噪比S/N≥3和S/N≥10计算出20种多酚的方法检出限和定量限;
(2)样品前处理:
a.采用浓盐酸调节酒样pH至2.0,混匀;取pH至2.0的酒样2.0mL于离心管中,加入10mL乙酸乙酯震荡萃取,离心,取上清液于浓缩瓶中;重复萃取离心一次,合并上清液;
b.向步骤a所得的合并上清液中加入10mL乙醚震荡萃取,离心,取出上清液,重复萃取、离心一次,合并上清液;
c.将步骤b所得的合并上清液置于真空平行浓缩仪中,35℃以下浓缩至近干,采用10.0mL 50wt%乙腈水溶液溶解残渣,过滤膜,制得待测样品,待进样;
(3)液相色谱-质谱法检测:采用液相色谱-质谱联用仪对待测样品进行检测,记录HPLC色谱图和峰面积,根据不同多酚化合物的标准曲线,计算获得样品中不同多酚化合物的含量。
2.如权利要求1所述一种黄酒中多酚类化合物的检测方法,其特征在于:步骤(1)中,混合标准工作液具体配制步骤为:分别取20种多酚类化合物各5mg,采用50wt%乙腈配制500mg/L的单一标准储备液并于-20℃保存;取上述标准储备液,采用50wt%乙腈水溶液稀释,配制成5、10、50、100、250、500μg/L混合标准工作液。
3.如权利要求1或2所述一种黄酒中多酚类化合物的检测方法,其特征在于:所述20种多酚类化合物包括没食子酸、儿茶素、香草酸、咖啡酸、丁香酸、对香豆酸、阿魏酸、槲皮素、香豆酸、原儿茶素、山奈酚、绿原酸、牡荆素、槲皮苷、金丝桃苷、芦丁、表儿茶素、鞣花酸、肉桂酸、芥子酸。
4.如权利要求1所述一种黄酒中多酚类化合物的检测方法,其特征在于:步骤(2)中,每次离心的离心速率为6000r/min,离心时间为2min。
5.如权利要求1所述一种黄酒中多酚类化合物的检测方法,其特征在于:色谱条件为:Waters ACQUITY HSS T3色谱柱,规格为2.1×100mm,1.8μm;流动相A:1%乙酸水,流动相B:甲醇;流速:0.4mL/min;进样量:1μL;柱温:40℃;梯度洗脱。
6.如权利要求5所述一种黄酒中多酚类化合物的检测方法,其特征在于:梯度洗脱程序为:0min,A相95%、B相5%,瞬间达到设定比例;0-0.5min,保持A相95%、B相5%;0.5-3min,两种流动相均速变化到A相50%、B相50%;3-6min,两种流动相均速变化到A相5%、B相95%;6-8min,保持A相5%、B相95%;8.1min,瞬时调整为A相95%、B相5%;8.1-10min,保持A相95%、B相5%;上述均为体积百分比。
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