CN111693711B - 一种超高压液相色谱串联质谱同时检测鲜蛋中103种抗生素残留的方法 - Google Patents
一种超高压液相色谱串联质谱同时检测鲜蛋中103种抗生素残留的方法 Download PDFInfo
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Abstract
本发明公开了一种鲜蛋中103种抗生素的同时检测方法,主要包括将样品经过溶液萃取、用Prime HLB固相萃取柱净化富集,经反相色谱柱进行分离后采用超高效液相色谱串联质谱联用仪在多反应监测模式下进行检测,确立各种抗生素的色谱峰保留时间、母离子/子离子对,然后进行定性和定量分析。本发明可以对鲜鸡蛋,鲜鸭蛋、鲜鹌鹑蛋等样品动物源性食品中的喹诺酮类,磺胺类,大环内酯类,内酰胺类,四环素类,抗真菌类和硝基咪唑类等103种抗生素同时检测。本发明的检测方法灵敏准确快速,仪器上的分析时间仅需18分钟就可同时进行103种抗生素进行定性和定量分析。而且本发明的检测方法对其他抗生素的检测也具有一定的参考价值。
Description
技术领域
本发明属于抗生素检测领域,涉及到同时检测鲜蛋中103种抗生素残留超高压液相色谱串联质谱法。
背景技术
抗生素是由微生物(包括细菌、真菌、放线菌属)或高等动植物在生活过程中所产生的具有抗病原体或其他活性的一类次级代谢产物,能干扰其他生物细胞发育功能的化学物质。根据发表在PNAS期刊上的研究报告,从2000年至2015年15年期间,全球抗生素使用量激增65%,增加多数源自中低收入国家对抗生素使用的增多。研究显示,2000年人均抗生素消耗量大的多为如法国、新西兰、中国香港、美国等发达国家或地区,但至2015年土耳其、坦桑尼亚、阿尔及利亚和罗马尼亚4个中等或低收入国家挤入人均抗生素消耗量排名的前六位。其中更有报道显示中国消耗了世界上近一半的抗生素,其中一大半用在饲养家禽家畜中,家禽家畜的排泄物会污染水源及土壤,使得一些未吃相关处方药的人在尿样中测试出抗生素。大量使用抗生素无疑是对致病菌抗药能力的“锻炼”,在绝大多数普通细菌被杀灭的同时,原先并不占优势的具有抗药性的致病菌却存留了下来,并大量繁衍。而且由于药物长期刺激,使一部分致病菌产生变异、成为耐药菌株。这种耐药性既会被其他细菌所获得,也会遗传给下一代。“超级细菌”很大程度上就是抗菌药物滥用催生出来的。如果这种情况继续恶化下去,很可能使人类面临感染时无药可用的境地。
鲜蛋中抗生素含量的检测为建立国家风险监控数据库和揭示其暴露水平提供有效的科学依据,也为企业开展质量监控提供技术手段。而目前国家或行业等相关标准检测方法多集中于单一种类抗生素化合物,缺点在于前处理复杂,流程多,耗时长等,随着分析科学技术和质谱技术的发展,高灵敏、高通量、高选择性等特点为抗生素的检测提供了新的思路,固相萃取技术的发展为复杂样品的处理提供有力的支持,尤其是Oasis Prime HLB这种通用型固相萃取柱解决了对于多兽药残留化合物种类多,化合物结构及性质差异大,与动物体内结合方式多样,尤其对鲜蛋样品中小分子的磷脂的干扰等问题。目前已成为常用的样品前处理方法。近年来,国内外有不少对糖肽类抗生素的高效液相色谱质谱检测的研究多集中于鲜蛋样品中单类兽药残留化合物居多,也有少量关于多兽药残留的报道,但针对鲜蛋样品中单一抗生素的多残留化合物的分析研究并没有报道过。尤其是对基于超高压液相色谱串联质谱质谱(UPLC-MS/MS)平台利用Prime HLB固相萃取柱净化同时检测鲜蛋样品中103抗生素的方法研究目前未见报道。
发明内容
针对现有技术中存在的问题,本发明的目的在于提供一种同时检测鲜蛋中103种抗生素残留的超高压液相色谱串联质谱法。
一种同时检测鲜蛋中103种抗生素残留超高压液相色谱串联质谱法,主要包括以下步骤:
(1)对鲜蛋样品进行均质处理;
(2)经过匀浆的样品进行有机溶剂提取;
(3)对步骤2得到的提取液进行富集净化:采用正己烷和Oasis PrimeHLB固相萃取柱进行净化,净化液浓缩,用0.1%甲酸水/乙腈溶液定容上机;
(4)将步骤(3)所得溶液经反相色谱柱在柱温为35℃,流速为0.3mL/min采用0.1%甲酸水与乙腈进行梯度洗脱;再采用超高压液相色谱串联质谱联用仪用多反应监测模式下在优化的色谱和质谱分析条件下进行检测;
(5)定性和定量分析:建立抗生素质谱检测库(包括色谱峰保留时间、母离子/子离子对),用样品溶液中出现的色谱峰保留时间和母离子/子离子对进行定性分析;制作103种抗生素的标准曲线,用基质外标法分别对样品溶液中103种抗生素的含量进行定量计算。
进一步的,所述103种抗生素包括喹诺酮类,磺胺类,大环内酯类,四环素类,内酰胺类,抗真菌类和硝基咪唑类。
进一步的,步骤(1)对鲜蛋样品进行均质操作主要包括:取200g鲜蛋液以2000r/min的速度匀浆均质混匀5min。
进一步的,步骤(2)中对样品进行有机相萃取主要包括:称取2.00g(精确至0.01g)均质后鲜蛋液置于离心管中,加入20mL乙腈旋涡混合,于振荡器上提取15min,再用冷冻离心机在温度低于4℃的条件下8000r/min离心5min,取上清液于另一离心管中备用。
进一步的,步骤(3)中所述固相萃取柱净化操作包括:将步骤(2)取得的备用液中加入20mL乙腈/水(v/v:4/1)饱和的正己烷,涡旋混合2min,再用冷冻离心机在温度低于4℃的条件下8000r/min离心2min,转移下层清液至Oasis PrimeHLB固相萃取柱,控制流速为2~3mL/min,收集滤液,45℃下氮气浓缩至干,最后用0.1%甲酸水/乙腈溶液(v/v:95/5)定容至1.0mL,过0.22μm PTFE滤膜上机。
上述的鲜蛋包括鸡蛋,鸭蛋和鹌鹑蛋。
进一步的,步骤(4)中梯度洗脱为:0~1min时0.1%甲酸水与乙腈体积比为95:5,1.0-16min0.1%甲酸水的比例逐渐将为25%,16.1min0.1%甲酸水的比例变为95%,16.1~18min0.1%甲酸水与乙腈体积比为95:5。
进一步的,步骤(4)中优化的色谱和质谱条件的选择的具体方法为:用103种抗生素的标准溶液进行子离子扫描,在103抗生素的碎片离子中选择响应较好的两组离子对作为定性离子对,选择其中响应较好的一组作为定量离子对,优化各离子对的碎裂电压和碰撞能量以得到最佳的相应值。
进一步的,步骤(5)所述外标法进行定量计算主要包括:取阴性基质样品进行加标,制备混合标准溶液,经步骤(1)-(4)的样品前处理,在步骤(4)所述优化的色谱和质谱分析条件下测103种抗生素的线性方程、线性范围、相关系数。
进一步的,所述103种抗生素浓度检测范围为1.0.0μg/kg到10.0μg/kg。
与现有技术相比,采用正己烷和Oasis PrimeHLB固相萃取柱净化富集,来进行同步分离富集,相对于分别处理,其鲜蛋液中的脂肪,蛋白和磷脂的清除效率提高,降低了基质干扰,提高了分析物的回收率;反相色谱柱进行分离后采用超高效液相色谱串联质谱联用仪对鲜蛋样品中103种抗生素进行同时检测。本发明的检测方法灵敏准确快速,在18min内同时对103种抗生素进行定性和定量分析。本发明的检测方法对鲜蛋中其他多兽药残留方法的开发也具有一定的参考价值。
附图说明
下面结合附图和实例对本发明进一步说明。
图1 Dimetridazole(硝基咪唑类)的标准曲线及图谱
图2 Nalidixicacid(喹诺酮类)的标准曲线及图谱
图3 Bifonazole(抗真菌类)的标准曲线及图谱
图4 Sulfadiazine(磺胺类)的标准曲线及图谱
图5 Cephradine(内酰胺类)的标准曲线及图谱
图6 Doxycycline(四环素类)的标准曲线及图谱
图7 Oleandomycin(大环内酯类)的标准曲线及图谱
图8 Dimetridazole(硝基咪唑类)的加标图谱(加标水平2.0μg/kg)
图9 Nalidixicacid(喹诺酮类)的加标图谱(加标水平2.0μg/kg)
图10 Sulfadiazine(磺胺类)的加标图谱(加标水平2.0μg/kg)
图11 Bifonazole(抗真菌类)的加标图谱(加标水平2.0μg/kg)
图12 Cephradine(内酰胺类)的加标图谱(加标水平2.0μg/kg)
图13 Doxycyline的加标图谱(四环素类)(加标水平2.0μg/kg)
图14 Oleandomycin(大环内酯类)的加标图谱(加标水平2.0μg/kg)
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合实施例对本发明作进一步地详细描述。
实施例1
一种同时检测鲜蛋中103种抗生素残留超高压液相色谱串联质谱法,其中103种抗生素包括喹诺酮类,磺胺类,四环素类,大环内酯类,抗真菌类,硝基咪唑类,内酰胺类,主要包括以下步骤:
称取均质试样2.00g,置于50mL聚丙烯离心管中,加入20mL乙腈,于振荡器上提取15min,再用冷冻离心机在温度低于4℃的条件下8000r/min离心5min,取上清液于另一离心管中备用。
将取得的备用液中加入20mL乙腈/水(v/v:4/1)饱和的正己烷,涡旋混合2min,再用冷冻离心机在温度低于4℃的条件下8000r/min离心2min,转移下层清液至OasisPrimeHLB固相萃取柱,控制流速为1.0mL/min,收集滤液,40℃下氮气浓缩至小于2mL,最后用0.1%甲酸水/乙腈(v/v:95/5)溶液定容至1.0mL,过0.22μm PTFE滤膜上机。
色谱分析条件如表1所示:
表1梯度洗脱程序
Table 1Program of gradient elution
*A:0.1%甲酸;B:乙腈
沃特世公司三重四级杆质谱Xevo TQ-XS Triple Quadrupole MassSpectrometer。
进行自动调谐在上述质谱分析条件下用各组分的标准溶液进行子离子扫描,在各组分的碎片离子中选择响应较好的两组离子对作为定性离子对,选择其中响应较好的一组作为定量离子对,优化各离子对的碎裂电压和碰撞能量以得到最佳的相应值。在上述优化的色谱和质谱分析条件下,103种抗生素的定量离子对、保留时间、线性、检出限、加标回收(低浓度)和相对偏差如表2所示。
表2 103种抗生素的母/子离子对,线性、检出限、加标回收和相对偏差
注:*表示定量离子
制作各组分的标准曲线,用基质外标法进行定量计算。
取阴性基质样品进行加标,制备混合标准溶液,经上述样品前处理步骤操作,在上述色谱分离和质谱分析条件下测定103种抗生素的线性方程、线性范围、相关系数,其结果如表3所示。从表3可以得知组分的103种抗生素在线性范围为X:1.0μg/kg到100.0μg/kg。再将浓度降低就会导致质谱信号的减弱,难以得到稳定性比较好的峰,从而是定性定量不准。将浓度线性上限提高的话会导致离子抑制,线性相关性变差。对于高浓度点可以将其稀释进行测试。一般液质不建议直接上样高浓度,容易造成残留。当样品中测得组分的浓度在线性范围之内时,按照线性方程计算样品浓度;当样品中测得组分的浓度在线性范围之外时,将样品稀释至线性范围以内再按照线性方程计算样品浓度。得到各大类典型代表的UPLC-MS/MS色谱图如图1-7所示。
将试样中的103种抗生素分别制备成1.0、2.0、5.0、10.0和20.0g/kg五个浓度,然后进行五个浓度的加标回收实验,结果如表2所示,从表2中可以看出回收率在62.4%-119.0%,对于小于100.0ug/kg水平加标,回收率完全满足要求。浓度为10μg/kg的103种抗生素中喹诺酮类,磺胺类,四环素类,大环内酯类,抗真菌类,硝基咪唑类,内酰胺类代表性组分的色谱如图8-14所示。
本实施例中的分析样品可以为鲜鸡蛋,鲜鸭蛋和鲜鹌鹑蛋。
103种抗生素包括喹诺酮类,磺胺类,大环内酯类,四环素类,内酰胺类,抗真菌类和硝基咪唑类一种或者多种时也可以采用相似的分析方法。
Claims (9)
1.一种鲜蛋中103种抗生素的同时检测方法,其特征在于,主要包括以下步骤:
(1)对鲜蛋样品进行液相萃取;
(2)对步骤(1)得到的提取液进行富集净化:采用Prime HLB固相萃取柱净化富集,经洗脱后,将洗脱液吹干,复溶于0.1%甲酸水溶液中;
(3)将步骤(2)所得溶液经反相色谱柱在柱温为35℃、流速为0.3mL/min,采用0.1%甲酸水与乙腈进行梯度洗脱;再采用超高效液相色谱串联质谱联用仪在多反应监测模式下在优化的色谱和质谱分析条件下进行检测;
(4)定性和定量分析:建立抗生素质谱检测库(包括色谱峰保留时间、母离子/子离子对),用样品溶液中出现的色谱峰保留时间和母离子/子离子对进行定性分析;制作103种抗生素的标准基质曲线,用基质外标法分别对样品溶液中103种抗生素的含量进行定量。
2.根据权利要求1所述鲜蛋中103种抗生素的同时检测方法,其特征在于,所述103种抗生素包括特喹诺酮类,磺胺类,四环素类,内酰胺类,硝基咪唑类,抗真菌类和大环内酯类内一种或者几种。
3.根据权利要求1所述鲜蛋中103种抗生素的同时检测方法,其特征在于,步骤(1)中对样品进行液相萃取主要包括:称取经过均质的鲜蛋样液置于离心管中,加入乙腈旋涡混合,于振荡器上提取15min,在用冷冻离心机在温度低于5℃的条件下离心5min,取上清液,用快速滤纸过滤。
4.根据权利要求1所述鲜蛋中103种抗生素的同时检测方法,其特征在于,步骤(2)中所述固相萃取包括:无需活化平衡;将步骤(1)所得提取液以2~3mL/min的速度过柱,直接接收滤液于玻璃试管中,置于45℃下氮气吹干。
5.根据权利要求1所述鲜蛋中103种抗生素的同时检测方法,其特征在于,所述本文所述的基质为鲜鸡蛋,鲜鸭蛋和鲜鹌鹑蛋。
6.根据权利要求1所述鲜蛋中103种抗生素的同时检测方法,其特征在于,步骤(3)中梯度洗脱为:0-1.0min时0.1%甲酸水与乙腈含量比为95:5,1.0-16.0min时0.1%甲酸水与乙腈含量比为25:75,16.0-16.1min时0.1%甲酸水与乙腈含量比为95:5,16.1-20.0min时0.1%甲酸水与乙腈含量比为95:5。
7.根据权利要求1所述鲜蛋中103种抗生素的同时检测方法,其特征在于,步骤(3)中优化的色谱和质谱条件的选择的具体方法为:用103种抗生素的标准溶液进行子离子扫描,在103种抗生素的碎片离子中选择响应较好的两组离子对作为定性离子对,选择其中响应较好的一组作为定量离子对,优化各离子对的碎裂电压和碰撞能量以得到最佳的相应值。
8.根据权利要求1所述鲜蛋中103种抗生素的同时检测方法,其特征在于,步骤(4)所述基质外标法进行定量计算主要包括:取阴性基质样品进行加标,制备混合标准溶液,经步骤(1)-(3)的样品前处理,在步骤(3)所述优化的色谱和质谱分析条件下测103种抗生素的线性方程、线性范围、相关系数。
9.根据权利要求1所述鲜蛋中103种抗生素的同时检测方法,其特征在于,所述103种抗生素浓度检测限范围为1.0μg/kg到10.0μg/kg。
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