CN116381087A - 一种同时检测4类17种热加工危害物的方法及其应用 - Google Patents
一种同时检测4类17种热加工危害物的方法及其应用 Download PDFInfo
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Abstract
本发明提供一种同时检测4类17种热加工危害物的方法,包括:在样品中加入正己烷,混匀后丢弃正己烷相,用二甲基酮和超声辅助提取丙烯酰胺、5‑羟甲基糠醛和游离杂环胺,收集上清液,净化除杂;同时将沉淀物转移到另一管中,加入四氢硼酸钠还原,加入氢氯酸水解,释放结合态杂环胺和晚期糖基化末端产物,进行UPLC‑MS/MS分析。本发明首次提出一种用两步法提取,用UPLC‑MS/MS在7分钟内同时测定4类17种热加工危害物的方法,并对方法的准确度、精密度、检测限、定量限和回收率等进行验证,各项测试指标都符合要求。该方法适用于高淀粉、高蛋白、高脂和高糖这4类食品基质的热加工危害物测定。
Description
技术领域
本发明属于检测技术领域,具体涉及一种同步检测热加工食品中4类17种热加工危害物的检测方法。
背景技术
在食品热加工过程中,美拉德反应赋予食品颜色和风味。同时,也容易产生热加工伴生危害物,威胁人类健康,如丙烯酰胺(acrylamide,AA)、5-羟甲基糠醛(5-hydroxymethylfurfural,HMF)、杂环胺(heterocyclic amines,HAs)和晚期糖基化末端产物(advanced glycation end products,AGEs)(Potentially harmful Maillardreaction products in food and herb medicines.Li等,Journal of Food Quality,2021.https://doi.org/10.1155/2021/1798936)。AA通常存在于富含碳水化合物的食品中,在高蛋白和高糖食品中也有检出。AA具有神经毒性和遗传毒性,已被国际癌症研究机构(International Agency for Research on Cancer,IARC)列为可能的人类致癌物(2A类)(Acrylamide from Maillard reaction products.Stadler等,Nature,419(6906),449–450.https://doi.org/10.1038/419449a)。HMF在干果、焦糖和面包中含量较高,在体内的代谢物具有致突变性和致癌性(Evolution of5-hydroxymethylfurfural(HMF)andfurfural(F)in fortified winessubmitted to overheating conditions.Pereira等,Food ResearchInternational,44(1),71–76.https://doi.org/10.1016/j.foodres.2010.11.011)。HAs通常在加热(高于150℃)的高蛋白质食品中产生。到目前为止,已经在食品中发现了25种不同的HAs。其中9种被IARC列为可能的人类致癌物(2B类),1种被列为2A类。烘烤和油炸食品中存在高含量的AGEs。长期摄入高AGEs的食品,会促进氧化应激和炎症的发生,诱发多种慢性疾病,如糖尿病、代谢障碍和癌症等。这些威胁人类健康的热加工伴生危害物往往同时存在于食品中。因此,需要一种检测方法能够在复杂食品基质中同步检测AA、HMF、HAs和AGEs这些热加工伴生危害物,以监测食品中可能存在的安全风险。
同步提取是同步检测的前提。HAs和AGEs存在自由态和蛋白结合态两种状态。结合态HAs和AGEs在食品中占比很高,但不能直接被检测,必须在前处理过程中转换为自由态。在转换时,需要兼顾HAs和AGEs的差异,避免含量高估或者低估。再者,这些物质的极性差异大,找到合适的提取溶剂实现同步提取是困难的。此外,食品基质富含油脂、蛋白等,会对目标分析物的提取和检测造成干扰。目前的方法只能检测单一或少量危害物,难以实现多类多种危害物的同步提取和同步检测。据所知,目前还没有关于同步检测AA、HMF、HAs和AGEs的报道。
发明内容
针对当前热加工食品中多类危害物共存的问题,本发明提出一种基于两步提取法和超高效液相色谱-串联质谱(ultra performance liquid chromatography-tandem massspectrometry,UPLC-MS/MS)同时检测4类17种热加工危害物的检测方法,具体包括丙烯酰胺、5-羟甲基糠醛、13种杂环胺和2种晚期糖基化末端产物。
实现本发明上述目的的技术方案为:
一种同时检测4类17种热加工危害物的检测方法,所述4类17种热加工危害物包括:丙烯酰胺、5-羟甲基糠醛、13种杂环胺:2-氨基-3-甲基咪唑[4,5-f]喹啉(IQ)、2-氨基-3,8-二甲基咪唑[4,5-f]喹啉(MeIQx)、1-甲基-9H-吡啶[3,4-b]吲哚(Harman)、9H-吡啶[3,4-b]吲哚(Norharman)、2-氨基-9H-吡啶[2,3-b]吲哚(AαC)、2-氨基-3-甲基-9H-吡啶[2,3-b]吲哚(MeAαC)、3-氨基-1,4-二甲基-5H-吡啶[4,3-b]吲哚乙酸酯(Trp-P-1)、3-氨基-1-甲基-5H-吡啶[4,3-b]吲哚乙酸酯(Trp-P-2)、2-氨基-6-甲基二吡啶并[1,2-α:3'2'-d]咪唑氢氯酸盐(Glu-P-1)、2-氨基二吡啶并[1,2-α:3'2'-d]咪唑氢氯酸盐(Glu-P-2)、2-氨基-1-甲基-6-苯咪唑[4,5-b]吡啶(PhIP)、2-氨基-1,6-二甲基咪唑[4,5-b]吡啶(DMIP)、2-氨基-5-苯基吡啶(Phe-P-1)、2种晚期糖基化末端产物:羧甲基赖氨酸(CML)、羧乙基赖氨酸(CEL),所述方法包括步骤:
S1:在样品中加入正己烷,涡旋混匀后超声处理,离心,丢弃正己烷相以除去脂质;加入二甲基酮,用超声辅助提取丙烯酰胺、5-羟甲基糠醛和游离杂环胺,离心,收集二甲基酮层上清液,加入混合内部标准品溶液,净化干燥后,用初始流动相复溶;
S2:将步骤S1离心余下的沉淀物转移到另一管中,加入四氢硼酸钠还原;加入氢氯酸水解;水解产物中加入混合内部标准品溶液,通过固相萃取小柱(Oasis MCX),用氨化甲醇洗脱,干燥后,用初始流动相复溶;
S3:配制一系列不同浓度的混合标准工作溶液,建立17种物质的标准曲线;步骤S1、步骤S2复溶得到的产物分别或混合后采用UPLC-MS/MS分析,内部标准品法定量;
其中步骤S1复溶的产物用于检测丙烯酰胺、5-羟甲基糠醛、13种杂环胺(AA、HMF和13种游离HAs);步骤S2复溶的产物用于检测13种结合HAs和2种结合AGEs。
以下是本发明的优选技术方案。
步骤S1中,所述样品为待测的热加工食品样品,按每克样品加入10mL正己烷的比例,涡旋,超声5~15分钟,离心丢弃正己烷相,以除去脂质。
该步骤可重复三次。
步骤S1中,每克样品加入10mL二甲基酮并涡旋1~2分钟,采用超声辅助提取10~30分钟后,将混合物以5000~20000rpm连续离心5~20分钟。
步骤S1中,所述混合内部标准品溶液的终浓度为200~1000μg/L;加入的净化物质为无水硫酸镁(MgSO4)和N-丙基乙二胺(primary secondary amine,PSA)。
可选地,所述混合内部标准品溶液加入体积为每克样品20μL,内部标准品溶液浓度为50mg/L 13C3-AA、10mg/L 13C6-HMF和10mg/L 4,7,8-TriMeIQx。加入混合内部标准品溶液后用无水MgSO4和PSA净化。
加入AA、HMF和HAs对应的内部标准品13C3-AA、13C6-HMF和4,7,8-TriMeIQx,有两点作用:其一是消除基质效应的干扰,减少掩蔽或增强效应,造成含量低估或高估;其二是校正前处理过程造成的损失,避免含量低估。
其中,无水MgSO4的作用是吸附样品中的水分,促进目标分析物转移至有机相,提高萃取效率;PSA的作用是吸附部分杂质,净化提取液。每克样品二者的加入量可分别为4.0g和0.03g。
步骤S2中,将离心后余下的沉淀物转移到聚四氟乙烯管中用于结合态HAs和AGEs分析。本方法可以同步转化结合态HAs和AGEs,实现准确定量,减少前处理的时间成本和有机试剂的投入。
步骤S2中,还原操作为:用pH 9.2的硼酸盐缓冲液和1M四氢硼酸钠(0.1M氢氧化钠溶解)溶液处理10~15小时。还原溶液用氯仿-甲醇混合溶液沉淀蛋白。
该还原步骤旨在减少果糖赖氨酸,从而避免其在酸水解过程中形成新的AGEs,造成含量高估。
具体可以采用如下操作:称量1g样品,在4℃下使用10mL硼酸盐缓冲液(0.2M,pH9.2)和5mL四氢硼酸钠(1M,溶于0.1M氢氧化钠溶液)还原12小时。还原溶液优选用10mL氯仿-甲醇(2:1,v/v)混合溶液沉淀蛋白。
步骤S2中,水解操作为:在6M氢氯酸中水解20~28小时。冷却至室温后,过滤蛋白水解产物并用超纯水稀释。
更优选地,采用的水解条件是加入10mL 6M氢氯酸,在110℃水解24小时。
所述固相萃取小柱,可以是Oasis MCX,或本领域已有的可提取带有阳离子交换基团的化合物的吸附装置。用于洗脱的氨化甲醇浓度为1~10%(v/v)。
其中,步骤S2中,所述混合内部标准品溶液的终浓度为200~400μg/L。
更优选地,所述混合内部标准品溶液的加入体积为每克样品20μL,内部标准品溶液浓度分别为10mg/L 4,7,8-TriMeIQx,20mg/LD4-CML和20mg/L D4-CEL。
此处加入内部标准品的作用同上。
步骤S1和S2中,用于复溶的初始流动相为1%甲醇-甲酸水,v/v。
已有技术中,氮吹后一般采用水/乙腈(1:1,v/v)溶解,而本方法采用初始流动相复溶,相比于常规方式,可以减小进样时溶剂效应的影响。
步骤S3中,在UPLC-MS/MS分析前,优选的UPLC条件为:
色谱柱:BEH C18柱;流动相:0.1%甲酸水(v/v)(A)和甲醇(B);梯度洗脱:99%A,0–0.5分钟;99–75%A,0.5–1.5分钟;75–62%A,1.5–2.5分钟;62–55%A,2.5–3.5分钟;55–52%A,3.5–4.0分钟;52–46%A,4.0–5.0分钟;46–35%A,5.0–5.9分钟;35–99%A,5.9–7.0分钟。
可选地,UPLC运行时间为7分钟;流速:0.3mL/min;柱温:30℃;进样量:1μL。
优选的MS/MS条件为:
ESI:正离子模式;毛细管电压:0.5kV;离子源温度:100℃;脱溶剂温度:500℃;锥孔气流量:150L/h;脱溶剂气体流量:1000L/h;碰撞气体流速:0.12mL/min。
所述UPLC-MS/MS分析可采用Acquity UPLCTM I-Class***和Xevo TQ-S三重四极杆质谱仪联用或本领域已有的超高效液相色谱质谱联用设备。
本发明的有益效果在于:
1.本发明提出两步提取法,操作简单高效,能一次性提取实际样品中的4类17种热加工伴生危害物。普通方法只能实现单一或少量危害物的提取,本方法与现有方法相比,提取的危害物种类更多,耗费的前处理时间减少50%以上。
2.前处理过程容易造成危害物二次生成或损失,导致含量高估或低估。在本发明中,采取了相对应的校正措施,保证食品中热加工危害物的准确定量。
3.本发明用UPLC-MS/MS可以在7分钟内实现同步检测。用BEH C18柱,0.1%甲酸水和甲醇做流动相,可以使17种分析物和5种内标完全分离。方法的准确度为98.13~100.96%,检测限为0.01~0.89μg/L,定量限为0.02~2.96μg/L,几乎所有分析物在各自的线性范围内均表现出较高的相关系数(R2>0.999)。日内精密度和日间精密度分别为0.25~9.90%和0.17~12.43%。
4.该方法可在多种食品基质中应用,具体包括高淀粉、高蛋白、高脂和高糖食品。
附图说明
图1:优化条件下17种目标分析物和5种内部标准品的色谱图:1:CML和D4-CML;2:CEL和D4-CEL;3:AA和13C3-AA;4:IQ;5:DMIP;6:Glu-P-2;7:HMF和13C6-HMF;8:Glu-P-1;9:MeIQx;10:Norharman;11:Phe-P-1;12:Harman;13:4,7,8-TriMeIQx;14:PhIP;15:Trp-P-2;16:AαC;17:Trp-P-1;18:MeAαC。
图2:Acquity UPLC HSS T3色谱柱的分离色谱图。
图3:3.03mM乙酸铵的分离色谱图。
图4:甲醇和乙腈的分离色谱图。
图5:17种目标分析物和5种内部标准品的MRM色谱图。
图6:本检测方法的流程图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
如无特别说明,说明书中采用的技术手段均为本领域已知的技术手段。所用原料均可市购。
本测定方法预备阶段是配置各标准溶液:
S1:将AA、HMF和13种HA标准品溶解在甲醇中;两种AGE标准品溶解在超纯水中,分别获得1mg/mL的标准储备溶液;
用初始流动相逐步稀释标准储备溶液来制备一系列含有AA、HMF、HAs和AGEs的混合标准工作溶液,备用。
实施例1:质谱(MS/MS)条件的优化
为了使目标分析物的灵敏度和选择性最大化,直接将标准品注入质谱仪来监测和优化MS/MS参数。根据17种分析物的结构特征,选择ESI正离子模式,采用多反应监测模式(MRM)。通过优化毛细管电压和锥孔电压,提高分子离子的响应性。优化碰撞电压,在二次质谱扫描中,选择具有最高响应的碎片离子定量,第二高响应的碎片离子定性。优化后的毛细管电压:0.5kV;离子源温度:100℃;脱溶剂温度:500℃;锥孔气流量:150L/h;脱溶剂气体流量:1000L/h;碰撞气体流速:0.12mL/min。AA、HMF、AGEs和HAs的最佳MS/MS参数如表1所示。
表1 17种分析物和5种内部标准品物的保留时间和MS/MS参数
a:内部标准品
实施例2:液相(UPLC)条件的优化:
UPLC条件的优化对于基线分离和17种分析物的准确定量至关重要。
色谱柱优化:
对于这些目标分析物,常用的色谱柱是Acquity UPLC HSS T3柱(2.1mm×100mm,1.8μm)和Acquity UPLC BEH C18柱(2.1×100mm,1.7μm)。因此,本实施例测试并比较了这两种常用的色谱柱对目标分析物的分离效果。从结果来看,在HSS T3柱上,部分HAs的峰形较差,如MeAαC,Phe-P-1,Harman和Norharman(图2);而在BEH C18柱上,17种分析物的峰形对称且尖锐。因此,最终选择BEH C18色谱柱。
流动相的优化:
流动相的选择有助于提高分析物的电离和分离效率。通常在流动相中添加甲酸或铵盐,用来改善分析物的峰形、灵敏度和分离效率。因此,将流动相B固定为乙腈,比较了0.1%甲酸水和3.03mM乙酸铵(pH 4.0)这两种不同的流动相A的分离效果。当流动相A为3.03mM乙酸铵时,IQ、MeIQx、Aαc和MeAαC等HAs出现了拖尾峰(图3)。然而,当0.1%甲酸水为流动相A时,峰形尖锐且对称,无拖尾现象。因此,最终采用0.1%甲酸水作为流动相A。
进一步地,把流动相A固定为0.1%甲酸水时,比较了甲醇和乙腈两种不同的流动相B。结果表明,甲醇为所有分析物提供了更好的保留和分离效率(图4)。因此,采用甲醇作为流动相B。
在优化后的方法中,选择了BEH C18柱,流动相由0.1%甲酸水和甲醇组成。针对17种目标分析物的分离效果,进一步优化了梯度洗脱程序。为了延长高极性分析物AA和AGEs的保留时间,流动相A从99%开始。优化后的梯度洗脱程序:99%A,0–0.5分钟;99–75%A,0.5–1.5分钟;75–62%A,1.5–2.5分钟;62–55%A,2.5–3.5分钟;55–52%A,3.5–4.0分钟;52–46%A,4.0–5.0分钟;46–35%A,5.0–5.9分钟;35–99%A,5.9–7.0分钟。最终,使用7分钟梯度洗脱程序,将每个目标分析物及其内部标准品清晰地洗脱为单一峰,没有任何干扰峰,证明了所开发方法的高度特异性(图5)。优化条件下17种分析物的色谱图如图1所示。
实施例3:前处理过程的优化
提取溶剂的优化:
这些分析物的极性范围跨度很广,寻找合适的溶剂,实现同步提取极具挑战性。本实施例在空白猪肉样品中加入AA、HMF和13种HAs标准品,通过测试不同溶剂萃取后的回收率来评价提取效果。首先测试了超纯水、乙腈和甲醇的提取效果。其中,乙腈和甲醇是常用的有机提取溶剂。超纯水对AA和HMF的提取效果较好,但由于杂环胺属于中等极性化合物,提取效果并不理想。此外,超纯水容易提取食品中的水溶性物质,导致溶液粘稠,难以进行后续操作。甲醇提取的特异性不强,容易同时提取目标分析物和干扰物质,不利于氮吹后分析物的重建。乙腈对大部分分析物的提取效果好,但对AA的提取效果差。考察AA在其他有机溶剂中的溶解性后,又测试了二甲基酮的提取效果,对所有目标分析物均表现出良好的回收率,最终选择二甲基酮作为提取溶剂。
酸水解的优化:
结合态的HAs和AGEs在食品中占比很高,但不能直接被检测,必须先转换为游离态。酸水解可以通过水解蛋白质将结合态HAs和AGEs转化为游离态。通过对不含分析物的生猪肉样品进行酸水解测试,结果发现酸水解过程不会产生新的HAs,但会产生CML和CEL,造成AGEs含量高估。因此,在本方法中进行了改进:在酸水解前增加还原步骤,加入1M四氢硼酸钠,还原果糖赖氨酸等Amadori产物,防止酸水解过程形成新的AGEs。还原处理有效校正样品中AGEs含量的高估,且证实不会干扰结合态HAs的转化。随后,考查了这些分析物的酸稳定性,少量HAs具有酸不稳定性。加入对应的内标校正酸水解过程的损失,避免结合态HAs和AGEs含量的低估。优化后的方法能同时转化结合态HAs和AGEs,且不会造成含量高估或低估。
实施例4:方法的验证
根据SANTE/11813/2017指南中关于线性、检测限(limit of detection,LOD)、定量限(limit ofquantitation,LOQ)、准确度、回收率、精密度、基质效应和残留等参数的标准,验证了所提出的方法。
线性、LOD、LOQ和准确度验证:
通过标准曲线的相关系数(R2)评估线性度,其中校准浓度为自变量(x),各自的面积比(化合物面积/各自的内部标准品面积)为因变量(y)。LOD和LOQ分别定义为信噪比(S/N)为3和10时的稀释浓度。准确度评估为观察到的浓度与规定浓度之间的百分比。AA范围为31.25至2000μg/L(31.25、62.5、125、250、500、1000和2000μg/L),HMF范围为3.9065至500μg/L(3.9063、7.8125、15.625、31.25、625、125和500μg/L)、CEL、CML、AαC、MeAαC、Norharman、Glu-P-1、Glu-P-2、Trp-P-1和Trp-P-2范围为3.125至400μg/L(3.0625、6.125、12.25、25、50、100、200和400μg/L)。IQ、MeIQx、PhIP、DMIP、Phe-P-1和Harman的范围为3.125至200μg/L(3.0625、6.125、12.25、25、50、100和200μg/L)。每个水平加入20μL混合内部标准品溶液(50mg/L 13C3-AA、10mg/L 13C6-HMF、10mg/L4,7,8-TriMeIQx、20mg/L D4-CML和20mg/L D4-CEL)。使用加权最小二乘线性回归分析获得数据,加权因子为1/x。所有分析物在相应范围内显示出良好的线性,矩阵中几乎所有的测定系数(R2)均大于0.999,LODs为0.01~0.89μg/L,LOQs为0.02~2.96μg/L,准确度为98.13~100.96%。
表2:17种危害物的方法学评价
回收率、精密度、基质效应和残留情况验证:
在高淀粉食品(饼干)、高脂肪食品(炭烤坚果)、高糖食品(杏干果脯)和高蛋白食品(植物肉)这4类具有代表性的食品中,分别添加三个浓度水平(50、100和200%,m/m)的混合标准工作溶液,测试方法的回收率。精密度(日内和日间精密度)通过上述三个浓度水平的加标样品的重复分析(n=6,n=3)进行评估,并用相对标准偏差(RSD,%)表示。用稳定的内部标准品(13C3-AA、13C6-HMF、D4-CML、D4-CEL和4,7,8-TriMeIQx)校正分析物的基质效应。在测定校准范围内的最高标准浓度后,通过注射两个空白来评估方法残留。绝大部分分析物的回收率在70~130%的范围内,日内精密度和日间精密度分别为0.25~9.90%和0.17~12.43%,符合要求。注射的两个空白未观察到分析物的残留。
综上,该方法具有准确度高、检测限和定量限低、线性相关性好、精密度良好、特异性强的优点。
实施例5:方法的应用
本实施例提供一种同时检测4类17种热加工危害物的方法,检测的流程见图6。
选取4类具有代表性的食品基质,高淀粉食品(饼干和薯片)、高蛋白食品(猪肉饼和植物肉)、高脂食品(炭烤坚果)和高糖食品(杏干果脯),测定其中AA、HMF、HAs和AGEs的含量。
将粉碎的食品样品(1g)分别称重到50mL离心管中,并加入正己烷(10mL)。离心管通过涡旋混合器振荡1分钟,并超声处理10分钟,然后以10000rpm离心10分钟。丢弃己烷相以除去脂质。此步骤重复三次。然后,向离心管中加入二甲基酮(10mL)并涡旋1分钟。采用超声辅助提取,20分钟后,将混合物以10000rpm连续离心10分钟。
将二甲基酮层上清液收集在另一离心管中用于AA、HMF和13种游离HAs分析,加入20μL混合内部标准品溶液(50mg/L 13C3-AA、10mg/L 13C6-HMF和10mg/L 4,7,8-TriMeIQx)。随后加入无水MgSO4(4.0g)和PSA(0.03g)。将离心管密封并立即涡旋2分钟。在10000rpm离心10分钟后,将二甲基酮层转移到清洁的10mL离心管中,然后在温和的氮气流下干燥。残余物用初始流动相(1mL,1%甲醇-甲酸水,v/v)复溶。
将沉淀物转移到聚四氟乙烯管中用于结合HAs和AGEs分析。对于沉淀物,在4℃下使用10mL硼酸盐缓冲液(0.2M,pH 9.2)和5mL四氢硼酸钠(1M,溶于0.1M氢氧化钠溶液)还原12小时。然后加入10mL氯仿-甲醇(2:1,v/v)混合溶液沉淀蛋白。以10000rpm离心10分钟,随后在沉淀物中加入10mL 6M氢氯酸,在110℃下水解24小时。冷却至室温后,过滤蛋白水解产物并用超纯水稀释至50mL。接着,取1mL水解产物,加入20μL混合内部标准品溶液(10mg/L4,7,8-TriMeIQx,20mg/L D4-CML和20mg/L D-CEL)。将混合溶液装入用甲醇预活化的固相萃取小柱(Oasis MCX,3mL/60mg,Waters)中。然后,用水(3mL)和甲醇(3mL)洗涤,最后用5%氨化甲醇(6mL,v/v)洗脱。将洗脱液在氮气下干燥,然后用初始流动相(1mL,1%甲醇-甲酸水,v/v)进行复溶。
在UPLC-MS/MS分析之前,通过0.22μm注射器过滤器过滤所有样品溶液。本实施例中,上清液和沉淀物复溶溶液分别进样。
UPLC-MS/MS分析条件:采用实施例2优化后的流动相和梯度洗脱程序,BEH C18色谱柱。采用实施例1列出的最佳MS/MS参数。
检测结果见表3和表4。
表3:食品中AA、HMF和游离杂环胺含量
注:n.d,未检出;n.q,未定量.
表4:食品中结合杂环胺和晚期糖基化末端产物含量
注:n.d,未检出;n.q,未定量.
虽然,以上通过实施例对本发明进行了说明,但本领域技术人员应了解,在不偏离本发明精神和实质的前提下,对本发明所做的改进和变型,均应属于本发明的保护范围内。
Claims (10)
1.一种同时检测4类17种热加工危害物的方法,其特征在于,所述4类17种热加工危害物包括:丙烯酰胺、5-羟甲基糠醛、13种杂环胺:2-氨基-3-甲基咪唑[4,5-f]喹啉(IQ)、2-氨基-3,8-二甲基咪唑[4,5-f]喹啉(MeIQx)、1-甲基-9H-吡啶[3,4-b]吲哚(Harman)、9H-吡啶[3,4-b]吲哚(Norharman)、2-氨基-9H-吡啶[2,3-b]吲哚(AαC)、2-氨基-3-甲基-9H-吡啶[2,3-b]吲哚(MeAαC)、3-氨基-1,4-二甲基-5H-吡啶[4,3-b]吲哚乙酸酯(Trp-P-1)、3-氨基-1-甲基-5H-吡啶[4,3-b]吲哚乙酸酯(Trp-P-2)、2-氨基-6-甲基二吡啶并[1,2-α:3'2'-d]咪唑氢氯酸盐(Glu-P-1)、2-氨基二吡啶并[1,2-α:3'2'-d]咪唑氢氯酸盐(Glu-P-2)、2-氨基-1-甲基-6-苯咪唑[4,5-b]吡啶(PhIP)、2-氨基-1,6-二甲基咪唑[4,5-b]吡啶(DMIP)、2-氨基-5-苯基吡啶(Phe-P-1)、2种晚期糖基化末端产物:羧甲基赖氨酸(CML)、羧乙基赖氨酸(CEL);所述方法包括如下步骤:
S1:在样品中加入正己烷,涡旋混匀后超声处理,离心,丢弃正己烷相以除去脂质;加入二甲基酮,用超声辅助提取丙烯酰胺、5-羟甲基糠醛和游离杂环胺,离心,收集二甲基酮层上清液,加入混合内部标准品溶液,净化干燥后,用初始流动相复溶;
S2:将步骤S1离心后余下的沉淀物转移到另一管中,加入四氢硼酸钠还原;加入氢氯酸水解;水解产物中加入混合内部标准品溶液,通过固相萃取小柱,用氨化甲醇洗脱,干燥后,用初始流动相复溶;
S3:配制一系列不同浓度的混合标准工作溶液,建立17种物质的标准曲线;步骤S1、步骤S2复溶得到的产物分别或混合后采用超高效液相色谱-串联质谱检测,内标法定量;
色谱柱:BEH C18柱;采用多反应监测模式。
2.根据权利要求1所述同时检测4类17种热加工危害物的方法,其特征在于,步骤S1中,所述样品为待测的热加工食品,按每克样品加入10mL正己烷的比例提取以除去脂质;通过涡旋混合器振荡后超声处理5~15分钟,然后离心,丢弃正己烷相以除去脂质。
3.根据权利要求1所述同时检测4类17种热加工危害物的方法,其特征在于,步骤S1中,每克样品加入10mL二甲基酮并涡旋1~2分钟,超声辅助提取10~30分钟后,将混合物以5000~20000rpm连续离心5~20分钟。
4.根据权利要求1所述同时检测4类17种热加工危害物的方法,其特征在于,步骤S1中,所述混合内部标准品溶液为13C3-AA、13C6-HMF和4,7,8-TriMeIQx,终浓度为200~1000μg/L;加入的净化物质为无水硫酸镁和N-丙基乙二胺。
5.根据权利要求1所述同时检测4类17种热加工危害物的方法,其特征在于,步骤S1和S2中,用于复溶的初始流动相为1%甲醇-甲酸水,v/v。
6.根据权利要求1所述同时检测4类17种热加工危害物的方法,其特征在于,步骤S2中,还原操作为:用pH 9.2的硼酸盐缓冲液和1M四氢硼酸钠溶液处理10~15小时,还原溶液用氯仿-甲醇混合溶液沉淀蛋白。
7.根据权利要求1所述同时检测4类17种热加工危害物的方法,其特征在于,步骤S2中,水解操作为:在6M氢氯酸中水解20~28小时。
8.根据权利要求1所述同时检测4类17种热加工危害物的方法,其特征在于,步骤S2中,所述混合内部标准品溶液为D4-CEL、D4-CML和4,7,8-TriMeIQx,终浓度为200~400μg/L。
9.根据权利要求1所述同时检测4类17种热加工危害物的方法,其特征在于,步骤S3中液相条件为,流动相:0.1%甲酸水(v/v)(A)和甲醇(B);梯度洗脱:99%A,0–0.5分钟;99–75%A,0.5–1.5分钟;75–62%A,1.5–2.5分钟;62–55%A,2.5–3.5分钟;55–52%A,3.5–4.0分钟;52–46%A,4.0–5.0分钟;46–35%A,5.0–5.9分钟;35–99%A,5.9–7.0分钟。
10.根据权利要求1所述同时检测4类17种热加工危害物的方法,其特征在于,步骤S3中,质谱条件为,ESI:正离子模式;毛细管电压:0.5kV;离子源温度:100℃;脱溶剂温度:500℃;锥孔气流量:150L/h;脱溶剂气体流量:1000L/h;碰撞气体流速:0.12mL/min。
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