CN111948324A - 一种同时测定热反应香料中10种极性杂环胺的方法 - Google Patents
一种同时测定热反应香料中10种极性杂环胺的方法 Download PDFInfo
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Abstract
本发明涉及一种同时测定热反应香料中10种极性杂环胺的方法,本发明方法具有操作简单、省时高效、仪器配置简单、分析成本低等优点,适宜相关企业进行产品质量监控,也可作为相关行业标准建立的参考。
Description
技术领域
本发明涉及食品香料检测领域,具体涉及一种热反应香料中极性杂环胺的检测方法。
背景技术
杂环胺化合物(Heterocyclic amines,HAs)由碳、氢及氮原子组成,具有杂环芳香族结构的化合物。此类化合物经常发现于经热处理过的高蛋白食品中。杂环胺是烹调富含蛋白质的食物过程中由蛋白质或氨基酸热解产生的一类具有致癌、致突变作用的多环芳香族化合物,其致突变性相当于迄今检测到的最有突变活力的毒物的水平。也已证明,正常烹调食品中均含有不同量的杂环胺。
HAs的生成方式主要为两类,第一类是由四种前驱物,即葡萄糖、氨基酸、肌酸(creatine)和肌酐酸(creatinine)经热反应而成的IQ型HAs,如IQ、MeIQ、IQx、MeIQx、7,8-DiMeIQx、4,8-DiMeIQx、PhlP等,这类化合物一般在普通家庭烹调温度(100-225℃)时就可形成;第二类为直接由单一氨基酸和蛋白质裂解生成的carboline型HAs,如Glu-P-1、Glu-P-2、Trp-P-1、Trp-P-2、AaC、MeaAaC等,这类化合物一般在300℃以上的高温时形成。而食品中的HAs生成量,主要取决于食品种类、加工方式、加热温度及时间,其中又以加热温度和时间为主要的影响因子,加热温度越高,时间越长,生成的HAs越多。
热反应香料是一种由食品原料和(或)允许在食品或反应香精中添加的原料加热制备而成的产物。反应香精制备时的反应温度一般不能超过180℃,在180℃的条件下反应一定时间,因此热反应香料的加工过程可能生成IQ型的HAs,因此研究热处理反应香料中IQ型的HAs对于食品安全性就尤为重要。
2016年8月国家发布《食品安全国家标准高温烹调食品中杂环胺类物质的测定》国标方法,并于2017年3月正式实施,标准适用于烤鱼、烤肉及其制品中MeIQ、MeIQx、4,8-DiMeIQx、7,8-DiMeIQx、PhIP的测定,样品采用氢氧化钠-甲醇溶液提取,固相萃取柱净化,液相色谱-串联质谱检测。其中,固相萃取柱为一次性器具,不可重复使用,因此成本较高;同时实验过程较为繁琐、费时。液相色谱-串联质谱价格较高,应用具有一定局限,目标检测物极性杂环胺数量较少。
发明内容
本发明的目的是克服上述现有技术的不足,提供一种同时测定热反应香料中10种极性杂环胺的方法;本发明方法建立一整套回流提取-高效液相色谱同时测定热反应香料中10种IQ型杂环胺类物质含量的分析方法。本发明方法具有操作简单、省时高效、仪器配置简单、分析成本低等优点,适宜相关企业进行产品质量监控,也可作为相关行业标准建立的参考。
本发明采用的技术方案为:
一种同时测定热反应香料中10种极性杂环胺的方法,该方法包括如下步骤:
1)、热反应香料中10种极性杂环胺的提取净化方法
准确称取5g反应香料于250mL圆底烧瓶中,加入5g硅藻土,加入50mL盐酸溶液(2mol/L)、10mL乙醇加热回流1h;得到的溶液冷却后过滤,用饱和NaOH溶液调节pH≥9;移入250mL分液漏斗中,用25mL二氯甲烷(含5%甲苯)萃取,加入0.5g硅藻土,振摇10min,静置分层,过滤,重复萃取两次,合并二氯甲烷,6000r/min离心10min;在旋转蒸发仪上,挥干溶剂;用甲醇定容到1mL;用0.2μm的滤膜过滤,得到清澈透明的溶液,然后HPLC分析。
2)、高效液相色谱法同时测定10种极性杂环胺的方法
采用二元流动相体系,流动相组成:A:0.01mol/L醋酸-醋酸胺(pH4.0),B:乙腈-甲醇(体积比2:3);进样量:10μL;流速:1.0ml/min;柱温:20℃;双波长扫描:DMIP、TMIP和PhIP为325nm,其他为258nm;流动相梯度洗脱程序:0~2min(B:12~15%,体积百分比),2~12min(B:15~20%,体积百分比),12~20min(B:20~30%,体积百分比),20~25min(B:30~60%,体积百分比),25~27min(B:60~12%,体积百分比)。
本发明所具有的有益效果:
本发明方法建立一整套回流提取-高效液相色谱同时测定热反应香料中10种IQ型杂环胺类物质含量的分析方法。本发明方法具有操作简单、省时高效、仪器配置简单、分析成本低等优点,适宜相关企业进行产品质量监控,也可作为相关行业标准建立的参考。
附图说明
图1为10种杂环胺类化合物混标溶液高效液相色谱图(波长258nm)。
图2为10种杂环胺类化合物混标溶液高效液相色谱图(波长325nm)。
图中:
1:2-氨基-1,6-二甲基咪唑[4,5-b]吡啶(DMIP)
2:2-氨基-1,5,6-三甲基咪唑[4,5-b]吡啶(TMIP)
3:2-氨基-3-甲基咪唑:[4,5-f]喹喔啉(IQx)
4:2-氨基-3-甲基咪唑[4,5-f]喹啉(IQ)
5:2-氨基-3,8二甲基咪唑[4,5-f]喹喔啉(MeIQx)
6:2-氨基-3,4-二甲基咪唑[4,5-f]喹啉(MeIQ)
7:2-氨基-3,7,8-三甲基咪唑[4,5-f]喹喔啉(7,8-DiMeIQx)
8:2-氨基-3,4,8-三甲基咪唑[4,5-f]喹喔啉(4,8-DiMeIQx)
9:2-氨基:-3,4,7,8-四甲基咪唑[4,5-f]喹喔啉(4,7,8-TriMeIQx)
10:2-氨基-1-甲基:-6-苯基-咪唑[4,5-b]吡啶(PhIP)
具体实施方式
下面结合具体实施例对本发明作进一步说明,但不限定本发明的保护范围。
实施例1
一种同时测定热反应香料中10种极性杂环胺的方法,该方法包括如下步骤:
1)、热反应香料中10种极性杂环胺的提取净化方法
准确称取5g反应香料于250mL圆底烧瓶中,加入5g硅藻土,加入50mL盐酸溶液(2mol/L)、10mL乙醇加热回流1h;得到的溶液冷却后过滤,用饱和NaOH溶液调节pH≥9;移入250mL分液漏斗中,用25mL二氯甲烷(含5%甲苯)萃取,加入0.5g硅藻土,振摇10min,静置分层,过滤,重复萃取两次,合并二氯甲烷,6000r/min离心10min;在旋转蒸发仪上,挥干溶剂;用甲醇定容到1mL;用0.2μm的滤膜过滤,得到清澈透明的溶液,然后HPLC分析。
2)、高效液相色谱法同时测定10种极性杂环胺的方法
采用二元流动相体系,流动相组成:A:0.01mol/L醋酸-醋酸胺(pH4.0),B:乙腈-甲醇(体积比2:3);进样量:10μL;流速:1.0ml/min;柱温:20℃;双波长扫描:DMIP、TMIP和PhIP为325nm,其他为258nm;流动相梯度洗脱程序:0~2min(B:12~15%,体积百分比),2~12min(B:15~20%,体积百分比),12~20min(B:20~30%,体积百分比),20~25min(B:30~60%,体积百分比),25~27min(B:60~12%,体积百分比)。
分别测定0.5、1、2、5、10、20、50、80、100μg/L的10种混标工作液,以各组分峰面积为纵坐标,质量浓度(ug/L)为横坐标绘制标准曲线,结果表明,10种极性杂环胺在一定范围内线性良好(见表1),该方法的线性范围可基本满足常规样品的定量分析的需要(GB/T27404-2008)。
表1 10种杂环胺类化合物的线性范围、回归方程及相关系数
测定低、中、高三个浓度(10、40、80ug/L)水平杂环胺混标在热反应香料基质中的回收率和精密度,10种杂环胺的加标回收率在73.48~102.4%之间,相对标准偏差(RSD)为3.24~7.22%,回收率和精密度符合检测要求,该法具有较高的准确性和可靠性。目标物混标溶液进样后色谱图如图1、2所示。
对比试验:
2.1考察不同流动相如醋酸-醋酸胺、甲酸-醋酸胺、磷酸-三乙胺、甲酸-三乙胺、醋酸-三乙胺(pH=3~5)为水相,有机相为乙腈,进行实验,结果发现,三乙胺使系统基线漂移严重,峰形差,而甲酸体系无法对DMIP、TMIP和IQx进行分离,易形成包埋峰。为实现良好分离效果,使用了0.01mol/L醋酸-醋酸胺为水相〔A相﹞,弱极性的乙腈-甲醇(2:3,B相)为有机相进行梯度洗脱,结果发现基线稳定,峰形好,溶剂峰小,分离度良好,是理想的流动相。
2.2流动相A在不同pH条件下(3.0、3.5、3.8、4.0、4.2、4.5、5.0、5.5、6.0)对HAs分离效果的影响,pH值降低,目标物保留时间提前,峰形尖锐,由于保留时间变化存在差异会造成部分峰形重叠;而pH值升高,目标物保留时间延长,峰形变宽,也会引起部分峰形重叠。在pH为4.0时,10种目标HAs物质能实现较好分离。
2.3使用DAD对10种目标物混合溶液在200~400nm波长下进行光谱扫描,IQ等7种目标物在258nm附近均有较大吸收,PhIP等3种目标物在325nm附近有较大吸收,为实现最佳检测状态,采用双波长扫描,258nm(检测IQx、IQ、MeIQx、MeIQ、7,8-DiMeIQx、4,8-DiMeIQx、4,7,8-TriMeIQx)和325nm(检测DMIP、TMIP和PhIP)。
2.4考察20、30、40℃色谱柱柱温下对HAs分离效果的影响,结果发现随着柱温升高,相邻色谱峰分离度下降,故确定柱温为20℃。
2.5考察回流提取和固相萃取两种方法,将前处理方法Ⅰ(回流提取)和前处理方法II(固相萃取)对样品的净化效果进行对比,用两种方法同时处理100ng/g的加标样品,结果表明回流提取方法回收率要高于固相萃取(如表2所示),同时回流提取方法具有操作简便,耗时少,成本较低等优点,本发明回流提取方法效果突出。
表2
应用实施例1
一种热反应香料的制备方法:
牛肉水解液60g(牛肉经过蛋白酶水解处理)、水解植物蛋白20g、酵母提取物6g、牛脂4g、谷氨酸钠3g、食盐2g、木糖2g、葡萄糖1g、甘氨酸1g、半胱氨酸0.5g和I+G 0.5g,在130℃下反应1h,冷却后过滤备用。
采用实施例1的方法,做3组重复实验,分别进行杂环胺测定,均未检出。
Claims (1)
1.一种同时测定热反应香料中10种极性杂环胺的方法,该方法包括如下步骤:
1)、热反应香料中10种极性杂环胺的提取净化方法
准确称取5g反应香料于250mL圆底烧瓶中,加入5g硅藻土,加入50mL盐酸溶液(2mol/L)、10mL乙醇加热回流1h;得到的溶液冷却后过滤,用饱和NaOH溶液调节pH≥9;移入250mL分液漏斗中,用25mL二氯甲烷(含5%甲苯)萃取,加入0.5g硅藻土,振摇10min,静置分层,过滤,重复萃取两次,合并二氯甲烷,6000r/min离心10min;在旋转蒸发仪上,挥干溶剂;用甲醇定容到1mL;用0.2μm的滤膜过滤,得到清澈透明的溶液,然后HPLC分析;
2)、高效液相色谱法同时测定10种极性杂环胺的方法
采用二元流动相体系,流动相组成:A:0.01mol/L醋酸-醋酸胺(pH4.0),B:乙腈-甲醇(体积比2:3);进样量:10μL;流速:1.0ml/min;柱温:20℃;双波长扫描:DMIP、TMIP和PhIP为325nm,其他为258nm;流动相梯度洗脱程序:0~2min(B:12~15%),2~12min(B:15~20%),12~20min(B:20~30%),20~25min(B:30~60%),25~27min(B:60~12%)。
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