CN114965818A - 一种降尿酸、痛风类功能食品中非法添加药物的检测方法 - Google Patents
一种降尿酸、痛风类功能食品中非法添加药物的检测方法 Download PDFInfo
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Abstract
本发明涉及一种降尿酸、痛风类功能食品中非法添加药物的检测方法,该方法建立一种超高效液相色谱-串联三重四极杆质谱法快速定性和定量筛查降尿酸类功能性食品中的69种非法添加药物的方法。方法采用QuEhERS前处理技术对样品进行净化,运用Waters BEHC18色谱柱对69种非法添加药物进行分离,乙腈-水作为流动相进行梯度洗脱,正离子、负离子同时扫描,多反应监测(multiple response monitoring,MRM)模式检测,外标法定量。建立的方法线性良好,69种物质的相关系数均大于0.99,检出限为0.03~0.1μg/g,平均回收率为63.2%~90.5%,相对标准偏差(relative standard deviations,RSDs)为1.26%~8.32%。该方法稳定、灵敏度高,适用于快速定性和定量筛查宣称降尿酸、痛风类功能性食品中的69种非法添加药物。
Description
技术领域
本发明属于检测技术领域,具体为一种降尿酸、痛风类功能食品中非法添加药物的检测方法。
背景技术
高尿酸血症是嘌呤代谢紊乱引起的代谢异常综合征。血尿酸超过其在血液或组织液中的饱和度可在关节局部形成尿酸钠晶体并沉积,诱发局部炎症反应和组织破坏,即痛风〔1〕(中国高尿酸血症与痛风诊疗指(2019)。治疗此类疾病的药物主要有一别嘌醇和非布司他等抑制尿酸生成药物,二苯溴马隆等促进尿酸排泄药物三糖皮质激素等抗痛消炎药物,市场上销售的痛风西药主要有3大类:第一类是非甾体抗炎类,主要有双氯芬酸钠和保泰松片等,具有解热、镇痛和抗炎等作用;第二类是糖皮质激素类,主要有强的松、萘普生和地塞米松等,具有抗炎、抗毒和抗休克等作用;还有生物碱,主要是秋水仙碱,具有降温、消炎和抑制等作用。
近年来,不法商家将上述药物添加于宣称降尿酸或痛风类功能性食品中,欺骗消费者,增加销售量,消费者在不知情的情况下,持续、大量服用含有非法添加药物的功能性食品,可能产生不可预知的不良反应,出现中毒现象,对社会造成极大的危害。
目前,降尿酸、痛风类药物的检测方法有高效液相色谱法,高效液相色谱-串联质谱法,且基质普遍集中于降尿酸或痛风药物含量的测定,针对处于保健食品27功能外的降尿酸痛风这类功效性食品研究基本处于空白,本发明QuEhERS前处理技术对样品进行净化,超高效液相色谱-串联三重四极杆质谱法测定市场上宣称降尿酸或痛风类的功能性食品中非法添加药物的含量,对此类产品中缺失的非法添加药物检验方法进行了补充。
发明内容
本发明针对目前相关检测方法的空白,提供了一种针对性强,灵敏度高,检测种类多的检测方法,具体是通过超高效液相色谱-串联三重四极杆质谱法快速定性和定量筛查降尿酸类功能性食品中的69种非法添加药物的方法。
为了解决上述问题,实现本发明的发明目的,本发明提供以下技术方案:
一种降尿酸、痛风类功能食品中非法添加药物的检测方法,其特征在于,包括如下步骤:S1、准备工作:混合标准储备溶液:称取69种标准品各10mg于同一10mL容量瓶中,加甲醇溶解并定容至刻度,摇匀,即得质量浓度为1.0mg/mL的混合标准储备溶液,使用时用90%乙腈溶液稀释混合标准储备溶液至所需浓度;样品制备:所有固体样品使用粉碎机粉碎后使用,胶囊样品取出内容物进行使用,备用;
S2、样品处理:称取1g粉碎试样于50mL塑料离心管中,加入5mL水,准确加入乙腈10.0mL,盖上盖子,摇匀后超声提取30分钟,加入QuEChERS分散SPE试剂盒(Part No:5982-4950),涡旋提取2min,5000转/分钟离心3分钟,吸取2mL上层清液即乙腈层置于QuEChERS净化试剂管(Part No:5982-4956CH)中,涡旋30s,3000转/分钟离心3分钟,取1.0mL于氮吹管中,45℃氮气吹干,准确加入1.0mL甲醇溶液溶解,过0.22μm有机相滤膜,待测;
S3、液相色谱-串联质谱仪测定,采用正负离子同时检测模式,色谱柱:ACQUITYBEH C18;柱温:40℃;进样量:2μL;进行梯度洗脱;质谱条件为:毛细管电压:5.5kV;离子源温度:150℃;脱溶剂气温度:500℃;脱溶剂气流量:800L/hr;MRM模式;碰撞气体:氦气;
S4、根据测量结果进行定性和定量判定;所述定性判定方法为:在相同试验条件下测定供试品溶液和筛查用混合标准溶液,记录供试品溶液和标准溶液中待测组分的色谱保留时间及定性离子的相对丰度比;若试样中检出与筛查用混合标准溶液中待测组分保留时间一致的色谱峰,且其定性离子与浓度相当的标准溶液中相应的定性离子的相对丰度相比,偏差不超过规定的范围,则可以确定试样中检出对应的成分;所述定量判定方法为:取基质混合标准工作溶液依次测定,以待测组分的系列浓度为横坐标,待测组分的峰面积为纵坐标,进行线性回归,绘制基质标准曲线,其线性相关系数应大于0.99,取供试品溶液测定,将对应的定量离子色谱峰面积代入线性回归方程;按计算公式计算样品中待测组分的含量即可定量。
进一步的,所述梯度洗脱条件为:
进一步的,其中质谱检测时69种唑类监测离子对及相关参数设定如下:
*为推荐的定量离子。
进一步的,所述69种非法添加药物的线性范围、回归方程、工作曲线、检出限及相对标准偏差如下:
与现有技术相比,本发明的有益效果为:本发明采用固相萃取方法,建立了超高效液相色谱-串联三重四极杆质谱(ultra performance liquid chromatography-tandemtriple quadrupole mass spectrometry,UPLC-MS/MS)方法对宣称降尿酸、痛风类功能性食品中69种非法添加药物的检测方法。本方法简单方便、易于操作、灵敏度高、检出限低,能够满足对宣称降尿酸、痛风类功能性食品中69种非法添加药物的快速检测和确证的要求,为此类功能性食品的有效监管提供了可靠方法。
附图说明
图1示出了非法添加1-4后总离子色谱图;
图2示出了非法添加5-8后总离子色谱图;
图3示出了非法添加9-12后总离子色谱图;
图4示出了非法添加13-16后总离子色谱图;
图5示出了非法添加17-20后总离子色谱图;
图6示出了非法添加21-24后总离子色谱图;
图7示出了非法添加25-28后总离子色谱图;
图8示出了非法添加29-32后总离子色谱图;
图9示出了非法添加33-36后总离子色谱图;
图10示出了非法添加37-40后总离子色谱图;
图11示出了非法添加41-44后总离子色谱图;
图12示出了非法添加45-48后总离子色谱图;
图13示出了非法添加49-52后总离子色谱图;
图14示出了非法添加53-56后总离子色谱图;
图15示出了非法添加57-60后总离子色谱图;
图16示出了非法添加61-64后总离子色谱图;
图17示出了非法添加65-68后总离子色谱图;
图18示出了非法添加69-70后总离子色谱图。
具体实施方式
结合实施例和附图对本发明作进一步说明,不能把它们理解为对本发明保护范围的限定。
1.仪器与试剂
AB Triple Quad 4500超高效液相色谱-串联质谱仪(美国Waters公司);JP-C300超声波清洗机(广州吉普超声波电子设备有限公司);3-3KS高速冷冻离心机(美国SCIEX公司);DT-502A电子天平(常熟市金羊砝码仪器有限公司);EYELA SB-1100旋转蒸发仪(日本东京理化公司);IKA MS3漩涡混合器(德国IKA公司);Milli-Q去离子水发生器(美国Millipore公司)。QuEChERS分散试剂盒(安捷伦)。
乙腈、甲醇(色谱纯,美国Fisher公司);甲酸(优级纯,德国CNW公司);乙腈、甲醇(分析纯,天津市永大化学试剂有限公司);实验用水为二级超纯水(美国Millipore公司);氮气(纯度大于99.99%,佛山顺德区东顺气体有限公司)。乙酸铵(色谱纯,德国TEDIA公司)。对乙酰氨基酚、酮咯酸、美洛昔康、秋水仙碱、非布司他、别嘌醇、布洛芬、双氯芬酸钠、苯溴马隆、丙磺舒标准品(德国Dr.Ehrenstorfer公司,纯度不少于98%),60种糖皮质激素混标(上海安谱实验科技股份有限公司)
2.1样品前处理
样品制备:所有固体样品使用粉碎机粉碎后使用,胶囊样品取出内容物进行使用。
称取1g粉碎试样(精确至0.01g)于50mL塑料离心管中,加入5mL水,准确加入乙腈10.0mL,盖上盖子,摇匀后超声提取30分钟,加入QuEChERS分散SPE试剂盒(Part No:5982-4950),涡旋提取2min,5000转/分钟离心3分钟,吸取2mL上层清液(乙腈层)置于QuEChERS净化试剂管(Part No:5982-4956CH)中,涡旋30s,3000转/分钟离心3分钟,取1.0mL于氮吹管中,45℃氮气吹干,准确加入1.0mL甲醇溶液溶解,过0.22μm有机相滤膜,待测。
2.2标准品的配制
混合标准储备溶液:称取69种标准品各10mg(精确到0.00001g)于同一10mL容量瓶中,加甲醇溶解并定容至刻度,摇匀,即得质量浓度为1.0mg/mL的混合标准储备溶液。使用时用90%乙腈溶液稀释混合标准储备溶液至所需浓度。
2.3仪器条件
(1)LC-MS/MS液相条件
表1流动相与梯度洗脱条件
(2)LC-MS/MS质谱条件
采用正负离子同时检测模式,毛细管电压:5.5kV;离子源温度:150℃;脱溶剂气温度:500℃;脱溶剂气流量:800L/hr;MRM模式;碰撞气体:氦气。质谱参考条件见表2。
表2质谱参考条件
注:*为定量离子
3.结果与分析
3.1仪器条件优化
69种非法添加药物种类多,结构复杂,选择在电喷雾正、负离子模式下分别对宣称降尿酸或痛风功能性食品中69种非法添加药物标准溶液进行质谱全扫描,得到69种非法添加药物的一级母离子,通过二级质谱扫描得到信号稳定、信号强度较大的特征碎片离子,选择无干扰、灵敏度高、相对丰度最高的离子作为定量离子,相对较弱的作为定性离子,并优化69种非法添加药物的母离子和子离子所需的最佳去簇电压和碰撞能量,以多反应监测模式进行扫描,得到69种非法添加药物优化后总离子流色谱图。
3.2色谱条件的选择
为达到最佳分离度效果,实验对3种不同品牌的色谱柱Shim-pack ODS-Ⅱ(2.0mm×75mm,1.6μm)、Agilent SB-C18(2.1mm×100mm,1.8μm)以及ACQUITYBEH C18(2.1mm×100mm,1.7μm)进行了比较,结果发现试验涉及的69种非法添加药物Shim-pack ODS-Ⅱ柱的分离度较低且负离子峰形色谱峰较宽;Agilent SB-C18和ACQUITYBEH C18色谱柱分离得到的色谱峰峰型较好,且分离度良好,但是Agilent SB-C18倍他米松和地塞米松未实现完全分离,故本试验选择ACQUITYBEH C18柱进行下述试验。
试验考察了甲醇和乙腈作为流动相的差异,发现糖皮质激素在色谱柱上的保留较强,乙腈洗脱能力较甲醇强,能将69种非法添加药物较快速洗脱,且分离效果较好,为了得到更好的分离效果以及更好的峰形,试验考察了在0.1%甲酸溶液、10mmol乙酸铵溶液2种流动相体系,结果表明2种流动相体系中69种非法添加药物中地塞米松醋酸酯和倍他米松双丙酸酯两种标准物质不能实现分离,参考文献()发现纯水作为流动相能分离上述物质,综合比较发现乙腈-水作为流动相能较好地分离69种非法添加药物,故确定该实验的最佳梯度洗脱条件如表1。
3.3样品前处理条件的确定
宣称降尿酸、痛风类功能性食品主要以菊苣栀子茶、芹菜籽亚麻籽油、葛酸茶等各种茶饮为主,成分多样、基体复杂、干扰严重且含有大量植物色素,固相萃取技术能有效去除基质干扰提高富集倍数,但是本试验在净化阶段遇到多次堵塞固相萃取柱现象,使试验难以进行。QuEChERS前处理技术具有回收率高、分析范围广、分析速度快的优点且能解决传统固相萃取柱的净化过程慢且堵塞固相萃取柱的缺陷,本发明采用QuEChERS前处理技术对宣称降尿酸、痛风类功能性食品中的n种非法添加药物处理,并对提取液进行了优化。最终确定称取样品后,加入5mL水,准确加入乙腈10.0mL,盖上盖子,摇匀后超声提取30分钟,加入QuEChERS分散SPE试剂盒后涡旋提取2min,吸取2mL上层清液(乙腈层)置于QuEChERS净化试剂管中,取1.0mL于氮吹管中,45℃氮气吹干,准确加入1.0mL甲醇溶液溶解,过0.22解,有机相滤膜后进液相色谱-质谱联用仪进行检测。
3.4检出限、定量限、标准曲线、线性范围
69种非法添加药物在6-100ng/mL的质量浓度范围内线性关系良好,相关系数均在0.99以上,以峰面积为纵坐标Y,标样的质量浓度为横坐标X,绘制线性回归方程,得到各化合物的线性回归方程。在空白样品中添加标准物质溶液,以3倍信噪比、10倍信噪比分别计算方法检出限、定量限,结果见表3。在最优条件下得到离子图见图1。
表3 69种非法添加药物的线性范围、回归方程、工作曲线、检出限及相对标准偏差
3.5加标回收率及精密度
选择不含69种非法添加药物的宣称降尿酸、痛风类功能性食品中添加标准物质0.03、0.06、0.3μg/g的标准溶液,每个浓度做6组平行,计算平均回收率,平均回收率在63.2%~90.5%之间,相对标准偏差均小于10.0%(n=6),表明该方法测定准确,回收率好。具体结果见表3。
3.6实际样品的检测结果
在网络及实体店采购宣称降尿酸、痛风类功能性食品50批次,采用本方法对50批次样品进行处理,检测69种非法添加物质,2批次样品检出别嘌醇,浓度为36μg/g和11μg/g,检出率为4.0%,说明宣称降尿酸、痛风类功能性食品存在较严重的非法添加西药的问题。
以上所述,仅为本发明的较佳实施例,并不用以限制本发明,本发明的专利保护范围以权利要求书为准,凡是运用本发明的说明书内容所作的等同结构变化,同理均应包含在本发明的保护范围内。
Claims (4)
1.一种降尿酸、痛风类功能食品中非法添加药物的检测方法,其特征在于,包括如下步骤:
S1、准备工作:混合标准储备溶液:称取69种标准品各10mg于同一10mL容量瓶中,加甲醇溶解并定容至刻度,摇匀,即得质量浓度为1.0mg/mL的混合标准储备溶液,使用时用90%乙腈溶液稀释混合标准储备溶液至所需浓度;样品制备:所有固体样品使用粉碎机粉碎后使用,胶囊样品取出内容物进行使用,备用;
S2、样品处理:称取1g粉碎试样于50mL塑料离心管中,加入5mL水,准确加入乙腈10.0mL,盖上盖子,摇匀后超声提取30分钟,加入QuEChERS分散SPE试剂盒(Part No:5982-4950),涡旋提取2min,5000转/分钟离心3分钟,吸取2mL上层清液即乙腈层置于QuEChERS净化试剂管(Part No:5982-4956CH)中,涡旋30s,3000转/分钟离心3分钟,取1.0mL于氮吹管中,45℃氮气吹干,准确加入1.0mL甲醇溶液溶解,过0.22μm有机相滤膜,待测;
S3、液相色谱-串联质谱仪测定,采用正负离子同时检测模式,色谱柱:ACQUITYBEH C18;柱温:40℃;进样量:2μL;进行梯度洗脱;质谱条件为:毛细管电压:5.5kV;离子源温度:150℃;脱溶剂气温度:500℃;脱溶剂气流量:800L/hr;MRM模式;碰撞气体:氦气;
S4、根据测量结果进行定性和定量判定;所述定性判定方法为:在相同试验条件下测定供试品溶液和筛查用混合标准溶液,记录供试品溶液和标准溶液中待测组分的色谱保留时间及定性离子的相对丰度比;若试样中检出与筛查用混合标准溶液中待测组分保留时间一致的色谱峰,且其定性离子与浓度相当的标准溶液中相应的定性离子的相对丰度相比,偏差不超过规定的范围,则可以确定试样中检出对应的成分;所述定量判定方法为:取基质混合标准工作溶液依次测定,以待测组分的系列浓度为横坐标,待测组分的峰面积为纵坐标,进行线性回归,绘制基质标准曲线,其线性相关系数应大于0.99,取供试品溶液测定,将对应的定量离子色谱峰面积代入线性回归方程;按计算公式计算样品中待测组分的含量即可定量。
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CN117368390A (zh) * | 2023-12-08 | 2024-01-09 | 济南辰欣医药科技有限公司 | 一种利用高效液相色谱法测定依碳酸氯替泼诺原料药杂质物质的方法 |
CN117368390B (zh) * | 2023-12-08 | 2024-02-20 | 济南辰欣医药科技有限公司 | 一种利用高效液相色谱法测定依碳酸氯替泼诺原料药杂质物质的方法 |
CN118211748A (zh) * | 2024-03-19 | 2024-06-18 | 青岛中信源食品科技有限公司 | 一种食品添加剂配方安全性判定方法、介质及系统 |
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