CN114527211A - 一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素a的方法 - Google Patents
一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素a的方法 Download PDFInfo
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Abstract
一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，包括如下步骤：步骤一：将赭曲霉毒素A含量分别为1‑100μg/kg的空白样品，进行液相色谱检测；步骤二：以空白样品中赭曲霉毒素A的浓度(x,μg/kg)为横坐标，对应色谱峰峰面积(y)为纵坐标，绘制基质标准工作曲线，进行回归计算，其线性方程为y＝0.1621x+0.3173，线性范围为1‑100μg/kg，线性相关系数r为0.9994；步骤三：取待测葡萄样品进行液相色谱检测，将待测葡萄样品检测后得到的赭曲霉毒素A的色谱峰面积代入回归方程，得到样品中赭曲霉毒素A的浓度。本发明得出能够准确检测出葡萄中赭曲霉毒素A方法，从而填补了现有领域的空白，为以后葡萄中赭曲霉毒素A的准确检测提供了方法。
Description
技术领域
本发明属于葡萄中赭曲霉毒素A领域，具体地说是一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法。
背景技术
赭曲霉毒素A(Ochratoxin A,OTA)是由多种曲霉属和青霉属真菌产生的一种次级代谢产物，主要污染粮谷类农作物，在多种粮食及制品中都检出过赭曲霉毒素A。人和动物进食被赭曲霉毒素A污染的食物后，会导致体内赭曲霉毒素A的蓄积，而且代谢缓慢。赭曲霉毒素A主要危及人和动物肾脏，具有高度致癌、致畸、致突变的作用，并被认为与人类的巴尔干肾病和泌尿系统肿瘤有关，国际癌症研究机构已将其定位2B类致癌物。联合国粮农组织和世界卫生组织建议赭曲霉毒素A的周摄入量不超过100ng/kg体重，我国目前还没有关于人群赭曲霉毒素A膳食摄入量的评价研究，但食品安全国家标准规定了谷物等粮食及制品中赭曲霉毒素A的限量标准为5ug/kg。现有赭曲霉毒素A的国标检测中有关于葡萄酒的相关标准，但是对于葡萄中的赭曲霉毒素A的检测方法缺少相关统一标准，目前我方培育出的葡萄品种在送样到山东省农业科学院农业质量标准与检测技术研究所进行检测分析时，对于葡萄中赭曲霉毒素A的检测方法采用的是检测辣椒中赭曲霉毒素A的国标检测方法，但是葡萄和辣椒中的成分存在巨大差异，葡萄采用辣椒检测的前处理方式以及检测方法能会出现误差，影响最终检测结果的准确性。
发明内容
本发明提供一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，用以解决现有技术中的缺陷。
本发明通过以下技术方案予以实现：
一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，包括如下步骤：
步骤一：将赭曲霉毒素A含量分别为1-100μg/kg的空白样品，进行液相色谱检测；
步骤二：以空白样品中赭曲霉毒素A的浓度(x,μg/kg)为横坐标，对应色谱峰峰面积(y)为纵坐标，绘制基质标准工作曲线，进行回归计算，其线性方程为y＝0.1621x+0.3173，线性范围为1-100μg/kg，线性相关系数r为0.9994；
步骤三：取待测葡萄样品进行液相色谱检测，将待测葡萄样品检测后得到的赭曲霉毒素A的色谱峰面积代入回归方程，得到样品中赭曲霉毒素A的浓度。
如上所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，所述的步骤一和步骤三中的液相色谱检测具体操作包括如下步骤：
步骤一：将葡萄样品破碎，称取10.0g破碎后的葡萄样品置于50mL离心管，加入10mL乙腈甲酸混合溶液，手动震荡1min，再依次加入1.0g氯化钠、4.0g无水硫酸镁，手动震荡1min，然后在8000r/min的离心速度下离心处理5min，取1mL上清液移入另一装有150mg无水硫酸镁和80mg石墨粉的2mL离心管中，手动震荡1min，然后再12000r/min的离心速度下离心处理5min，用带有过滤器的1mL注射器抽取上清液过滤，得待检测液；
步骤二：待检测液进行HPLC检测，HPLC检测条件为：色谱柱为：3.0mm×150mm，2.7μm的Poroshell120 EC-C18色谱柱；流动相：流动相A：0.1％甲酸水溶液，流动相B：乙腈，采用梯度洗脱；柱温：30℃；进样量：10μL；检测器：FLD；激发波长Ex：334nm；发射波长Em：460nm。
如上所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，所述的步骤一中乙腈甲酸混合溶液中乙腈和甲酸的体积比为99：1。
如上所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，所述的步骤一种的过滤器中的滤膜为0.22μm滤膜。
如上所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，所述的步骤二梯度洗脱的操作为：0-5min：流动相A：50％，流动相B：50％，流量为0.5mL/min；5-10min：流动相A：30％，流动相B：70％，流量为0.5mL/min；10-15min：流动相A：10％，流动相B：90％，流量为0.5mL/min；15-18min：流动相A：50％，流动相B：50％，流量为0.5mL/min。
如上所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，标准储备液：所述的取赭曲霉毒素A标准品1.0mg置于10mL容量瓶中，用甲醇溶解并定容，配制成浓度为100mg·L-1的赭曲霉毒素A标准溶液，4℃下避光保存。
如上所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，所述的空白样品的选取：选取健康的阳光玫瑰葡萄作为空白样品，经检测该样品中无待测样品，使用此空白样品进行加标回收试验和基质标准工作曲线的绘制。
本发明的优点是：本发明通过对葡萄破碎后的处理方法以及对液相色谱条件的摸索，选择效果优异且价格相对低廉的石墨粉作为净化材料，并摸索出相应的液相色谱条件，并通过采用基质标准工作曲线定量的方式，消除了基质效应的影响，最终得出能够准确检测出葡萄中赭曲霉毒素A方法，从而填补了现有领域的空白，为以后葡萄中赭曲霉毒素A的准确检测提供了方法。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1是本发明实施例的样品测定色谱图；
图2是本发明实施例的提取溶剂的优化选择对比图；
图3是本发明实施例的提取溶剂的优化选择对比图；
图4是本发明实施例的提取溶剂的优化选择对比图；
图5是本发明绘制的基质标准工作曲线。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例
1.1主要材料与设备
Agilent1260 InfinityⅡ液相色谱仪及工作站，配备真空脱气装置，二元泵，自动进样装置，柱温箱及荧光检测器(美国安捷伦公司)，Centrifuge 5424R离心机(德国艾本德公司)，万分之一电子天平(梅特勒-托利多仪器上海有限公司)，乙腈(色谱级)(美国Sigma公司)，甲酸(分析纯)(阿拉丁试剂有限公司)，超纯水，分析标准品：赭曲霉毒素A(购于青岛普瑞邦生物工程有限公司)
1.2标准储备液的配制
标准储备液：取赭曲霉毒素A标准品1.0mg置于10mL容量瓶中，用甲醇溶解并定容，配制成浓度为100mg·L-1的赭曲霉毒素A标准溶液，4℃下避光保存。
1.3HPLC检测条件
Poroshell120 EC-C18色谱柱(3.0mm×150mm，2.7μm)，流动相A：0.1％甲酸水溶液，B：乙腈，采用梯度洗脱，流速0.50mL·min-1，柱温30℃，进样量10μL，检测器FLD，激发波长Ex：334nm，发射波长Em：460nm。
1.4试验过程
称取10.0g经料理机破碎的葡萄样品置于50mL离心管，加入10mL乙腈(1％甲酸)，手动震荡1min，再依次加入1.0g氯化钠、4.0g无水硫酸镁，手动震荡1min，再8000r/min离心5min，取1mL上清液移入另一装有150mg无水硫酸镁和80mg石墨粉的2mL离心管中，手动震荡1min，12000r/min离心5min，用1mL注射器抽取上清液过0.22μm滤膜，待HPLC检测。
空白样品的选取：选取健康的阳光玫瑰葡萄作为空白样品，经检测该样品中无待测样品，使用此空白样品进行加标回收试验和基质标准工作曲线的绘制。
2.1样品前处理方法的优化
2.1.1提取
本发明考察了乙腈，乙腈+1％甲酸，甲醇和乙醇4种提取溶剂，试验结果表如图2所示，由图2可知乙腈+1％甲酸的提取效果比纯乙腈的提取效果略好，但远好于甲醇和乙醇，此外，为了跟流动相尽可能保持一致，因此，本发明提取剂选择乙腈+1％甲酸。
本发明，考察了氯化钠和无水硫酸镁的2个添加比例，MgSO4:NaCl＝4:1和MgSO4:NaCl＝3:2，结果如图3所示，由图3可知MgSO4:NaCl＝4:1提取效果最优。
本发明考察了14种净化包，其对应的检测结果如图4所示，由图4可知，以峰面积大小来说，以硅胶，石墨粉和C18组成的净化包的净化效果最好，但是，由于C18相比硅胶和石墨粉来说，价格昂贵，因此，本发明不选择C18，硅胶与石墨粉相比较，硅胶去除色素的效果很差，考虑到对色谱柱寿命的影响，因此本发明选择石墨粉与无水硫酸镁作为净化剂。本发明还考察了50mg石墨粉+150mg MgSO4与80mg石墨粉+150mg MgSO4的净化效果，试验表明，两个组合的色谱峰面积无明显差异，对于去除色素方面80mg石墨粉+150mg MgSO4更优，因此，本发明选择80mg石墨粉+150mg MgSO4作为净化剂。
3.方法学验证
3.1基质标准工作曲线的制定、方法检出限和定量分析
本发明考虑到葡萄中多种化合物基质的影响，采用基质标准工作曲线定量的方式，消除了基质效应的影响，在本发明选定的最佳条件下，外标法定量。将赭曲霉毒素A含量分别为1-100μg/kg的空白样品，按照1.4的方法试验。以空白样品中赭曲霉毒素A的浓度(x,μg/kg)为横坐标，对应色谱峰峰面积(y)为纵坐标，绘制基质标准工作曲线，进行回归计算，其线性方程为y＝0.1621x+0.317，线性范围为1-100μg/kg，线性相关系数r为0.9994，说明赭曲霉毒素A在1-100μg/kg的浓度范围内与其色谱峰面积呈现良好的线性关系，见图5。将葡萄检测后得到的赭曲霉毒素A的色谱峰面积代入回归方程，得到样品中赭曲霉毒素A的浓度。以3倍信噪比(S/N＝3)计算得到赭曲霉毒素A的检出限为0.063μg/kg，以10倍信噪比(S/N＝10)计算得到赭曲霉毒素A的定量限为0.21μg/kg。
3.2方法准确度与精密度
以阴性葡萄作为空白基质，在3个不同添加水平下，每个水平重复3次。结果表明：3种阴性葡萄中赭曲霉毒素A的添加回收率在87.4％～108.3％之间，相对标准偏差(RSD)(n＝3)在3.2％～4.5％之间。该方法有较高的准确度和良好的稳定性，可用于葡萄中生物毒素赭曲霉毒素A的检测。
添加赭曲霉毒素A含量为10μg/kg的阳光玫瑰葡萄的高效色谱图，见图1。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (6)
1.一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，其特征在于：包括如下步骤：
步骤一：将赭曲霉毒素A含量分别为1-100μg/kg的空白样品，进行液相色谱检测；
步骤二：以空白样品中赭曲霉毒素A的浓度(x,μg/kg)为横坐标，对应色谱峰峰面积(y)为纵坐标，绘制基质标准工作曲线，进行回归计算，其线性方程为y＝0.1621x+0.3173，线性范围为1-100μg/kg，线性相关系数r为0.9994；
步骤三：取待测葡萄样品进行液相色谱检测，将待测葡萄样品检测后得到的赭曲霉毒素A的色谱峰面积代入回归方程，得到样品中赭曲霉毒素A的浓度；
所述的步骤一和步骤三中的液相色谱检测具体操作包括如下步骤：
步骤一：将葡萄样品破碎，称取10.0g破碎后的葡萄样品置于50mL离心管，加入10mL乙腈甲酸混合溶液，手动震荡1min，再依次加入1.0g氯化钠、4.0g无水硫酸镁，手动震荡1min，然后在8000r/min的离心速度下离心处理5min，取1mL上清液移入另一装有150mg无水硫酸镁和80mg石墨粉的2mL离心管中，手动震荡1min，然后再12000r/min的离心速度下离心处理5min，用带有过滤器的1mL注射器抽取上清液过滤，得待检测液；
步骤二：待检测液进行HPLC检测，HPLC检测条件为：色谱柱为：3.0mm×150mm，2.7μm的Poroshell120 EC-C18色谱柱；流动相：流动相A：0.1％甲酸水溶液，流动相B：乙腈，采用梯度洗脱；柱温：30℃；进样量：10μL；检测器：FLD；激发波长Ex：334nm；发射波长Em：460nm。
2.根据权利要求2所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，其特征在于：所述的步骤一中乙腈甲酸混合溶液中乙腈和甲酸的体积比为99：1。
3.根据权利要求1所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，其特征在于：所述的步骤一种的过滤器中的滤膜为0.22μm滤膜。
4.根据权利要求1所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，其特征在于：所述的步骤二梯度洗脱的操作为：0-5min：流动相A：50％，流动相B：50％，流量为0.5mL/min；5-10min：流动相A：30％，流动相B：70％，流量为0.5mL/min；10-15min：流动相A：10％，流动相B：90％，流量为0.5mL/min；15-18min：流动相A：50％，流动相B：50％，流量为0.5mL/min。
5.根据权利要求1所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，其特征在于：所述的标准储备液：取赭曲霉毒素A标准品1.0mg置于10mL容量瓶中，用甲醇溶解并定容，配制成浓度为100mg·L-1的赭曲霉毒素A标准溶液，4℃下避光保存。
6.根据权利要求1所述的一种以石墨粉为净化材料的检测葡萄中赭曲霉毒素A的方法，其特征在于：所述的空白样品的选取：选取健康的阳光玫瑰葡萄作为空白样品，经检测该样品中无待测样品，使用此空白样品进行加标回收试验和基质标准工作曲线的绘制。
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