三聚氰胺和亚硝酸盐卫生检验新方法研究
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摘要
三聚氰胺和亚硝酸盐都是含氮物质,是重要的卫生检测项目。三聚氰胺是广泛使用的化工原料,可诱发肾衰竭而导致死亡,禁止用于食品和动物饲料中。但由于三聚氰胺含氮量高达66%,高出氨基酸的3~4倍,近年来有人向奶粉中违规添加三聚氰胺,虚假提高蛋白质含量,对人体健康构成重大威胁,三聚氰胺事件向社会提出了卫生检验新任务。因此,研究、建立三聚氰胺新的卫生检测方法具有现实意义。亚硝酸盐广泛存在于人们赖以生存的环境当中,在体内亚硝酸根可转化形成强致癌物质N-亚硝胺。本文进行了三聚氰胺和亚硝酸盐的卫生检测新方法研究。
     第一章:分别概述了三聚氰胺和亚硝酸盐卫生检验技术的发展趋势和研究进展;提出了几种卫生检测新方法研究工作的新思维。
     第二章:应用荧光光谱、共振散射光谱方法研究了三聚氰胺–乙醇之间的相互作用。①应用荧光光谱法初步探讨了三聚氰胺对乙醇荧光的猝灭机理,根据不同温度下的热力学常数确定了三聚氰胺与乙醇之间的作用力类型;②首先应用共振散射光谱研究了三聚氰胺与乙醇的反应机理;在荧光猝灭理论分析基础上,首次应用共振散射光方法分析了荧光猝灭的作用力类型;两者对比研究结果一致。
     第三章:应用荧光光谱技术,建立了三聚氰胺的卫生检验新方法。①在pH3.3 BR缓冲溶液中,三聚氰胺与甲醛、安息香缔合,使体系的荧光强度增强,建立了一种荧光光谱法水相测定三聚氰胺的新方法。在8.0×10~(-8)~2.4×10~(-5) mol/L范围内,体系荧光强度改变值(ΔF)与三聚氰胺的浓度呈现良好的线性关系,线性回归方程为ΔF=68.9c - 0.381(r = 0.9998),检出限为2.4×10~(-8) mol/L。方法用于乳制品中三聚氰胺的测定,样品加标回收率为95.7%~110.0%,RSD=2.5%~4.1%,结果满意。②在水介质中,三聚氰胺与荧光素钠缔合,使体系的荧光强度增强;应用酸效应对型体分布的影响,研究了反应机理;建立了荧光光谱法水相测定三聚氰胺的新方法。在4.8×10~(-3)~90.0μg/mL、1.6×10~(-2)~280.0μg/mL范围内,体系荧光强度改变值(ΔF)与三聚氰胺的浓度分别呈现良好的线性关系,线性回归方程分别为ΔF=2660.3ρ+ 36.67(r = 0.9992)、ΔF=768.92ρ+ 160.66(r =0.9996),检出限分别为1.4、5.0 ng/mL。文中研究了反应产物的光谱特征和适宜的反应条件;方法简便、快速、准确实用。
     第四章:应用荧光光谱法研究了检测亚硝酸根新方法,首次研究了抗癌物质大蒜素(DADS)对其反应的抑制机理。在硫酸介质中,应用致癌物质NO_2~-与吖啶红(AR)发生亚硝化反应,导致体系荧光猝灭;加入大蒜素抑制亚硝化反应,降低了体系荧光猝灭。首次建立了亚硝酸根的同步荧光、三维荧光和共振荧光几种检测新方法;与直接荧光法相比,新方法具有灵敏度更高、检出限更低、线性范围更宽等优点。应用DADS阻抑AR-NO_2~-体系荧光猝灭结果,阐述了阻抑原理,为研究DADS的抗癌作用提供了新的研究方法和理论依据。
Melamine and nitrite are all the matter containing nitrogen element, and the important hygienic detection items at present. The former is used mainly as chemical industry material. It does not be permit to used as food additives and feedingstuff even now because its level of nitrogen (66% by mass, m:m) is higher more than the amino acids by 3-4 times, resulting in the failure of kidney and death. But melamine was illegally used in recent years due to that it can increase the protein content falsely, and threatening greatly to the health. Therefore, it is practical importance to study methods of hygienic detection of melamine. While the widely existed nitrite could be turned to N-nitrosamine in the human body, which is strong carcinogen. It was scientific researches on new methods of the hygienic detection of melamine and nitrite in this paper.
     Part 1, the headways trend and research statuses were summarized briefly about melamine and nitrite respectively. The novel ideas on the health laboratory technologies for melamine and nitrite in scientific researches were developed.
     Part 2, the interactions of melamine with ethanol were reviewed by the fluorescence spectroscopy method and one by Resonance Scattering Spectroscopy(RSS) in aqueous solution.①The fluorescence quenching mechanism of ethanol was investigated after adding melamine, and apparent binding constant K was measured by spectrofluorometric method. Thermodynamic parameters obtained from measured data at different temperatures showed the binding force of melamine and ethanol.②The binding between melamine and ethanol had been investigated by RSS. The effort types of fluorescence quenching mechanism were analyzed firstly by RSS on the basis of the fluorescence quenching mechanism theory. Results were consistent with each other.
     Part 3, novel spectrofluorometric methods were developed for the determination of melamine.①A novel spectrofluorometric method was proposed based on the formation of ion-association complex between melamine and formaldehyde and benzoinum in pH3.3 Britton-Robinson (BR) buffer solution, resulting in the enhancement of fluorescence intensity. It was found that the enhanced intensity was propotional to the concentration of melamine in the range of 8.0×10~(-8)~2.4×10~(-5) mol/L, linear regression equation wasΔF=68.9c - 0.381(r = 0.9998) with the detection limit of 2.4×10~(-8) mol/L. The average recoveries were 95.7%~110.0% with relative standard deviation(RSD) of 2.5%~4.1%. Satisfactory results were obtained for the determination of melamine in dairy sample.②The other one was proposed in the water phase based on fluorescence intensity enhancement after fluorescein sodium forms ion-association complex with melamine. The reaction mechanism studied applying the influence of the acid effect on the species distribution. It was found that the enhanced intensity was propotional to the concentration of melamine in the range of 4.8×10~(-3)~90.0μg/mL and 1.6×10~(-2)~280.0μg/mL respectively, linear regression equation wereΔF=2660.3ρ+ 36.67(r=0.9992),ΔF=768.92ρ+ 160.66(r =0.9996)with the detection limits of 1.4 ng/mL, 5.0 ng/mL respectively. The optimum conditions of spectrofluorometric methods were investigated. It showed that the method was fast, convenient and accurate.
     Part 4, two topics were studied on the determination of NO_2~- and inhibiting mechanism of diallyl disulfide(DADS) based on spectrofluorometric method. The nitrosation reaction of NO_2~- (carcinogen) and acridine red (AR) could make the system of fluorescence intensity decreased, while DADS could inhibit part of nitrosation reaction and quenching of the system of fluorescence in the sulphuric acid medium. Novel spectrofluorometric methods were constructed to assay NO_2~- by synchronous fluorescence, three-dimensional fluorescence, resonance fluorescence firstly in this paper. Compared with the direct fluorescent method, the new ones had higher sensitivity, lower detection limit and larger detection range and so on. Principal of the repression was expounded by applying with the results of fluorescence quenching of the system of AR-NO_2~-, then novel methods and theory evidence was supplied for studying the antitumous effect of DADS.
引文
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