加工肉制品中未知添加物质筛查与确证技术研究
摘要
本研究以加工肉制品为样品,分别建立了着色剂、防腐剂、甜味剂、抗氧化剂的检测方法,以适用于不同的检测目的。本研究研究采用凝胶渗透色谱(GPC)和固相萃取(SPE)法相结合的方式达到净化的目的,样品提取液通过GPC净化后,即去除了大分子脂类干扰物质,同时也实现了将脂溶性添加剂和非脂溶性添加剂分离的目的。通过对各类添加剂与填料之间键合关系的调查研究,并对比了多种商品固相萃取柱和自制固相萃取小柱的净化效果和标准品损失程度,针对不同的添加剂标准品选择出了不同的固相萃取净化方法。
着色剂部分,分析了10种食品添加剂和33种工业染料,采用了分级提取的方法,通过使用正己烷均质提取脂肪和脂溶性色素,达到了将脂溶性和水溶性色素分离的目的,脂溶性色素部分利用凝胶渗透色谱净化,水溶性色素部分经过5%氨水提取,C18固相萃取小柱净化后,以乙酸铵缓冲溶液/乙腈梯度洗脱,在多组可变波长的条件下,高效液相色谱/二极管矩阵检测器检测完成分离和液相色谱定量检测；并建立了43种着色剂标准品的飞行时间质谱数据库,通过对100个肉类样品进行筛查,查出极个别的样品中含有苏丹红Ⅰ和苏丹红Ⅳ,应用液相色谱/飞行时间质谱对阳性样品进行定量确证,其中样品A中SudanⅠ含量约为2.23±0.8μg/kg,SudanⅣ含量约为2.03±0.9μg/kg；样品B中SudanⅠ含量约为3.06±0.5μg/kg.本方法不仅可以满足日常检测的需要,而且能够应对肉制品污染或非法添加工业染料等突发事件的发生。
防腐剂部分,选择中性氧化铝固相萃取小柱作为净化手段,在磷酸缓冲溶液/乙腈梯度洗脱下,完成分离和液相色谱定量检测；方法回收率在80.85%-109.26%之间,相对标准偏差在0.66%-5.63%之间。对香肠和火腿样品进行了测定,其中检出山梨酸在香肠中含量为0.704g/kg,在火腿中含量为0.027g/kg,均低于国家标准,复合要求。同时建立了23种防腐剂的液相色谱-飞行时间质谱数据库,为筛查出阳性样品后进行进一步确证奠定基础。
甜味剂部分,以甲醇：缓冲盐：丙酮的不同体积比作为两种流动相,在进行液相色谱-电喷雾电离源质谱的检测时,分析物质的离子化效率受到了样品基质中的强极性物质强力抑制,通过对流动相中甲醇的不同比例进行研究,克服了离子抑制效应,方法的灵敏度达到了0.01 mg/kg。
抗氧化剂部分,以乙酸水溶液/甲醇作为流动相,实验选用Agilent Zorbax C18分析色谱柱进行梯度洗脱,在波长280nm下检测。实验中样品的加标回收率在80.9%-118.2%之间,相对偏差均小于2%。本方法中丁基羟基茴香醚(BHA)、2,6-二叔丁基对甲基苯酚(BHT)、叔丁基对苯二酚(TBHQ)和没食子酸丙酯(PG)的检出限分别为0.173mg/L,0.232 mg/L,0.123 mg/L,0.079 mg/L。在对实际样品的检测中,分别在腊肠样品中查出BHA 0.068g/kg和BHTO.104g/kg;在火腿样品中查出BHTO.027g/kg,均在国家标准要求范围之内可以满足检测工作的要求,能够用于火腿、腊肠和培根肉等加工肉制品的日常检测使用。
本研究属国内外首次利用液相色谱-飞行时间质谱进添加剂的筛查和确认技术研究,建立了部分添加剂的液相色谱-飞行时间质谱的标准品数据库。这种筛查检测技术对于全未知肉制品样品中添加剂的筛查检测非常重要,其特点是利用飞行时间质谱的更宽的线性范围和更高的质量精度,来筛查及确认肉制品中添加剂的含量,为加工肉制品的质量监控和抽查提供有效的技术手段。
In this study, the sample processed meat products, were established coloring agents, preservatives, sweeteners, anti-oxidant test method to test for different purposes. In the purification, the study by gel permeation chromatography (GPC) and solid phase extraction (SPE) which combines the means of achieving the purpose of purification, the sample extract purified by GPC, the lipid molecules in addition to interfering substances, but also achieve the fat-soluble additives and water-soluble additives, the purpose of separation. Through various types of additives and fillers bonded relationship between research and compare a variety of goods made by solid phase extraction column and solid phase extraction column purification effect and extent of the damage standards, standards for different additives choose the Different solid phase extraction method.
Colorants part of the analysis of 10 kinds of food additives and 33 industrial dyes, using a sequential extraction method, using hexane extraction of fat and fat-soluble pigment homogeneous, reaching the separation of fat-soluble and water-soluble pigment purpose Some fat-soluble pigments purified by gel permeation chromatography, water-soluble pigments extracted some after 5% ammonia, C18 solid phase extraction column purification, followed by ammonium acetate buffer/acetonitrile gradient, in the conditions of multiple variable wavelength, The high performance liquid chromatography/photodiode array detector to complete separation and quantitative analysis of liquid chromatography; and established a standard 43 colorants time of flight mass spectrometry database, through the screening of 100 meat samples, found a very Individual samples contain Sudan I and SudanⅣ, application of liquid chromatography-time of flight mass spectrometry for quantitative confirmation of positive samples, including samples of A, Sudan I content of about 2.23±0.8μg/kg, Sudan IV content of about 2.03±0.9μg/kg; sample B in the Sudan I content of about 3.06±0.5μg/kg. This method not only to meet the needs of daily testing, and able to cope with added meat products contaminated or illegal industrial dye and other unexpected events.
Preservatives section, select neutral alumina as solid phase extraction column purification means, in phosphate buffer/acetonitrile gradient, the complete separation and quantitative analysis of liquid chromatography; recovery rates between 80.85%-109.26%, Relative standard deviation of 0.66%-5.63%, respectively. Sausage and ham samples were determined, which were detected in the sorbic acid content in the sausage 0.704g/kg, content in the ham 0.027g/kg, were lower than the national standard, complex requirements.23 kinds of preservatives also established liquid chromatography-time of flight mass spectrometry database for screening out the positive samples were further confirmed after the foundation.
Sweeteners part, use the methanol:buffer salt:the different volume ratio of acetone as the mobile phase two, during liquid chromatography-electrospray ionization mass spectrometry for detection, analysis of material by the ionization efficiency in the sample matrix Potent inhibitors of polar substances, through the mobile phase of methanol in different proportions to study, to overcome the ion suppression effect, the sensitivity reached 0.01 mg/kg.
Anti-oxidants Part, use acetic acid solution/methanol as mobile phase, experimental analysis of selected Agilent Zorbax C18 column with gradient elution, test at 280nm. Recoveryies of spiked samples between 80.9%-118.2%, relative deviations were less than 2%. The method of butylated hydroxyanisole (BHA),2,6-di-tert-butyl methyl phenol (BHT), tert-butyl hydroquinone (TBHQ) and propyl gallate (PG) The detection limit were 0.173mg/L, 0.232mg/L,0.123mg/L,0.079mg/L. In the detection of real samples, the samples were found in the sausage BHA 0.068g/kg and BHT 0.104g/kg; in ham samples found BHT0.027g/kg, were within the scope of national standards to meet the testing requirements Work requirements, can be used for ham, sausages and bacon and other processed meat daily testing use.
This study is the first time at home and abroad liquid chromatography-time of flight mass spectrometry screening and confirmation of additives into the technical studies, the establishment of some additives in liquid chromatography-time of flight mass spectrometry of the standard database. This detection technology for screening samples of all the unknown additives in meat products is very important screening tests, which is characterized by time of flight mass spectrometry using a wider linear range and higher quality of accuracy, to screen and Confirm the content of additives in meat products, processed meat products for the quality control and sampling to provide effective technical means.
着色剂部分,分析了10种食品添加剂和33种工业染料,采用了分级提取的方法,通过使用正己烷均质提取脂肪和脂溶性色素,达到了将脂溶性和水溶性色素分离的目的,脂溶性色素部分利用凝胶渗透色谱净化,水溶性色素部分经过5%氨水提取,C18固相萃取小柱净化后,以乙酸铵缓冲溶液/乙腈梯度洗脱,在多组可变波长的条件下,高效液相色谱/二极管矩阵检测器检测完成分离和液相色谱定量检测；并建立了43种着色剂标准品的飞行时间质谱数据库,通过对100个肉类样品进行筛查,查出极个别的样品中含有苏丹红Ⅰ和苏丹红Ⅳ,应用液相色谱/飞行时间质谱对阳性样品进行定量确证,其中样品A中SudanⅠ含量约为2.23±0.8μg/kg,SudanⅣ含量约为2.03±0.9μg/kg；样品B中SudanⅠ含量约为3.06±0.5μg/kg.本方法不仅可以满足日常检测的需要,而且能够应对肉制品污染或非法添加工业染料等突发事件的发生。
防腐剂部分,选择中性氧化铝固相萃取小柱作为净化手段,在磷酸缓冲溶液/乙腈梯度洗脱下,完成分离和液相色谱定量检测；方法回收率在80.85%-109.26%之间,相对标准偏差在0.66%-5.63%之间。对香肠和火腿样品进行了测定,其中检出山梨酸在香肠中含量为0.704g/kg,在火腿中含量为0.027g/kg,均低于国家标准,复合要求。同时建立了23种防腐剂的液相色谱-飞行时间质谱数据库,为筛查出阳性样品后进行进一步确证奠定基础。
甜味剂部分,以甲醇：缓冲盐：丙酮的不同体积比作为两种流动相,在进行液相色谱-电喷雾电离源质谱的检测时,分析物质的离子化效率受到了样品基质中的强极性物质强力抑制,通过对流动相中甲醇的不同比例进行研究,克服了离子抑制效应,方法的灵敏度达到了0.01 mg/kg。
抗氧化剂部分,以乙酸水溶液/甲醇作为流动相,实验选用Agilent Zorbax C18分析色谱柱进行梯度洗脱,在波长280nm下检测。实验中样品的加标回收率在80.9%-118.2%之间,相对偏差均小于2%。本方法中丁基羟基茴香醚(BHA)、2,6-二叔丁基对甲基苯酚(BHT)、叔丁基对苯二酚(TBHQ)和没食子酸丙酯(PG)的检出限分别为0.173mg/L,0.232 mg/L,0.123 mg/L,0.079 mg/L。在对实际样品的检测中,分别在腊肠样品中查出BHA 0.068g/kg和BHTO.104g/kg;在火腿样品中查出BHTO.027g/kg,均在国家标准要求范围之内可以满足检测工作的要求,能够用于火腿、腊肠和培根肉等加工肉制品的日常检测使用。
本研究属国内外首次利用液相色谱-飞行时间质谱进添加剂的筛查和确认技术研究,建立了部分添加剂的液相色谱-飞行时间质谱的标准品数据库。这种筛查检测技术对于全未知肉制品样品中添加剂的筛查检测非常重要,其特点是利用飞行时间质谱的更宽的线性范围和更高的质量精度,来筛查及确认肉制品中添加剂的含量,为加工肉制品的质量监控和抽查提供有效的技术手段。
In this study, the sample processed meat products, were established coloring agents, preservatives, sweeteners, anti-oxidant test method to test for different purposes. In the purification, the study by gel permeation chromatography (GPC) and solid phase extraction (SPE) which combines the means of achieving the purpose of purification, the sample extract purified by GPC, the lipid molecules in addition to interfering substances, but also achieve the fat-soluble additives and water-soluble additives, the purpose of separation. Through various types of additives and fillers bonded relationship between research and compare a variety of goods made by solid phase extraction column and solid phase extraction column purification effect and extent of the damage standards, standards for different additives choose the Different solid phase extraction method.
Colorants part of the analysis of 10 kinds of food additives and 33 industrial dyes, using a sequential extraction method, using hexane extraction of fat and fat-soluble pigment homogeneous, reaching the separation of fat-soluble and water-soluble pigment purpose Some fat-soluble pigments purified by gel permeation chromatography, water-soluble pigments extracted some after 5% ammonia, C18 solid phase extraction column purification, followed by ammonium acetate buffer/acetonitrile gradient, in the conditions of multiple variable wavelength, The high performance liquid chromatography/photodiode array detector to complete separation and quantitative analysis of liquid chromatography; and established a standard 43 colorants time of flight mass spectrometry database, through the screening of 100 meat samples, found a very Individual samples contain Sudan I and SudanⅣ, application of liquid chromatography-time of flight mass spectrometry for quantitative confirmation of positive samples, including samples of A, Sudan I content of about 2.23±0.8μg/kg, Sudan IV content of about 2.03±0.9μg/kg; sample B in the Sudan I content of about 3.06±0.5μg/kg. This method not only to meet the needs of daily testing, and able to cope with added meat products contaminated or illegal industrial dye and other unexpected events.
Preservatives section, select neutral alumina as solid phase extraction column purification means, in phosphate buffer/acetonitrile gradient, the complete separation and quantitative analysis of liquid chromatography; recovery rates between 80.85%-109.26%, Relative standard deviation of 0.66%-5.63%, respectively. Sausage and ham samples were determined, which were detected in the sorbic acid content in the sausage 0.704g/kg, content in the ham 0.027g/kg, were lower than the national standard, complex requirements.23 kinds of preservatives also established liquid chromatography-time of flight mass spectrometry database for screening out the positive samples were further confirmed after the foundation.
Sweeteners part, use the methanol:buffer salt:the different volume ratio of acetone as the mobile phase two, during liquid chromatography-electrospray ionization mass spectrometry for detection, analysis of material by the ionization efficiency in the sample matrix Potent inhibitors of polar substances, through the mobile phase of methanol in different proportions to study, to overcome the ion suppression effect, the sensitivity reached 0.01 mg/kg.
Anti-oxidants Part, use acetic acid solution/methanol as mobile phase, experimental analysis of selected Agilent Zorbax C18 column with gradient elution, test at 280nm. Recoveryies of spiked samples between 80.9%-118.2%, relative deviations were less than 2%. The method of butylated hydroxyanisole (BHA),2,6-di-tert-butyl methyl phenol (BHT), tert-butyl hydroquinone (TBHQ) and propyl gallate (PG) The detection limit were 0.173mg/L, 0.232mg/L,0.123mg/L,0.079mg/L. In the detection of real samples, the samples were found in the sausage BHA 0.068g/kg and BHT 0.104g/kg; in ham samples found BHT0.027g/kg, were within the scope of national standards to meet the testing requirements Work requirements, can be used for ham, sausages and bacon and other processed meat daily testing use.
This study is the first time at home and abroad liquid chromatography-time of flight mass spectrometry screening and confirmation of additives into the technical studies, the establishment of some additives in liquid chromatography-time of flight mass spectrometry of the standard database. This detection technology for screening samples of all the unknown additives in meat products is very important screening tests, which is characterized by time of flight mass spectrometry using a wider linear range and higher quality of accuracy, to screen and Confirm the content of additives in meat products, processed meat products for the quality control and sampling to provide effective technical means.
引文
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