重氮偶联分光光度法测定人体血液中亚硝酸盐含量的比较研究及亚硝酸盐在大鼠主要检材中的分布

发布时间：2018-01-03 19:04

本文关键词：重氮偶联分光光度法测定人体血液中亚硝酸盐含量的比较研究及亚硝酸盐在大鼠主要检材中的分布　出处：《昆明医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的选择六组具有代表性的重氮偶联分光光度法测定人体血液中亚硝酸盐含量的最佳偶合剂,探索各组重氮偶合剂的最适反应条件,建立六组人体血液中亚硝酸盐的分光光度计定量分析方法；用所建立的方法,分别测出六组偶联-分光光度法测定亚硝酸盐含量的线性范围、检出限、人体血液中亚硝酸盐的含量和各组方法的精密度及回收率,比较不同试剂重氮偶联测定血液中亚硝酸盐含量的优越性,最终确定最适宜分析生物检材中亚硝酸盐含量的重氮偶联分光光度法。建立急性亚硝酸盐中毒大鼠模型,选用最适宜分析生物检材中亚硝酸盐含量的重氮偶联分光光度法,即对氨基苯磺酸溶液与4-氨基-5-羟基萘-2,7-二磺酸单钠盐溶液分别为重氮试剂和偶联试剂测定亚硝酸盐中毒大鼠模型心血、肝脏、胃及内容物和肾脏中亚硝酸盐的含量,对实验数据进行分析,阐明急性亚硝酸盐中毒大鼠主要脏器中亚硝酸盐的分布状况。方法 1.通过查阅文献,选定六组具有代表性的偶联-分光光度法测定亚硝酸盐含量的试剂方法：(1)对氨基苯磺酰胺和盐酸萘乙二胺分别为重氮试剂和偶氮试剂；(2)对氨基苯磺酸和8-羟基喹啉分别为重氮试剂和偶氮试剂；(3)对氨基苯磺酸和4-氨基-5-羟基萘-2,7-二磺酸单钠盐分别为重氮试剂和偶氮试剂；(4)对氨基苯磺酸和1-苯基-3-甲基-5-吡唑啉酮分别为重氮试剂和偶氮试剂；(5)4-氨基-5-羟基萘-2,7-二磺酸单钠盐(H酸)既作为重氮试剂又作为偶氮试剂(偶联反应在pH7.5条件下进行)；(6)4-氨基-5-羟基萘-2,7-二磺酸单钠盐既作为重氮试剂又作为偶氮试剂(偶联反应在pH4.5条件下进行)。 2.随机抽取10个健康成年人静脉血各1mL混合(内含抗凝剂),抽取1mL混合血置于250mL容量瓶中,加入50mL超纯水和10mL氢氧化钠溶液(20.0g/L),再加入10mL硫酸锌溶液(0.42mol/L),混合均匀。置60℃水浴中加热10min,取出冷却至室温,定容至250mL,混合均匀。放置30min,使用定量滤纸过滤并弃去初滤液50mL,加入干燥的亚硝酸钠160μg,使之完全溶解。 3.每组试剂方法分别固定其它反应条件,改变一种反应条件,测其最适宜的反应参数,确定总反应最适宜条件；建立六组试剂方法,测定每组方法的相关参数,分别用六组方法测定血液中亚硝酸盐含量及回收率,并进行六组方法结果的比较研究。 4.将14只健康雄性Sprague-Dawley大鼠随机分为空白对照组(n=4)、生理盐水灌胃对照组(n=4)和亚硝酸钠灌胃实验组(n=6)。分别对每组动物进行编号。生理盐水对照组和实验组大鼠均灌胃给药。空白对照组采用断头法处死,生理盐水对照组于给药后60min采用断头法处死,实验组在给药后任其死亡。快速提取所有大鼠心血、肝脏、胃及内容物和肾脏,以超纯水浸泡萃取其中的亚硝酸盐。 5.以对氨基苯磺酸溶液与4-氨基-5-羟基萘-2,7-二磺酸单钠盐溶液分别为重氮试剂和偶联试剂,采用标准工作曲线法测定各组大鼠心血、肝脏、胃及内容物和肾脏中亚硝酸盐的含量,采用SPSS软件对定量分析结果进行统计学分析。结果 1.对氨基苯磺酰胺溶液与盐酸萘乙二胺溶液分别为重氮试剂和偶联试剂反应条件：室温下,在10mL比色管中加入4.0g/L pH1盐酸对氨基苯磺酰胺溶液1mL及梯度浓度亚硝酸钠溶液1mL,静置3min进行重氮反应,后加入1.0g/L盐酸萘乙二胺溶液1mL,加水至刻度,混匀,静置1min进行偶合反应,以未加亚硝酸钠的混合溶液为参比进行吸光度测定。此反应生成紫红色的偶联产物,在最大吸收波长540nm处,亚硝酸盐的浓度在0.1-1.51μg/mL范围内符合Beer定律,相关系数丫为0.99975,检出限为3.86ng/mL; 2.对氨基苯磺酸溶液与8-羟基喹啉溶液分别为重氮试剂和偶联试剂反应条件：室温下,在10mL比色管中加入4.0g/L pH1盐酸对氨基苯磺酸溶液1mL及系列梯度浓度亚硝酸钠溶液1mL,静置1min进行重氮反应,后加入0.6g/L8-羟基喹啉溶液1mL及pH12.5氨水1mL,加水至刻度,混匀,静置1mmin进行偶合反应,以未加亚硝酸钠的混合溶液为参比进行吸光度测定。此反应生成桔黄色偶联产物,在最大吸收波长500nm处,亚硝酸盐的浓度在0.2-4.6μg/mL范围内符合Beer定律,相关系数γ为0.99995,检出限为26.13ng/mL; 3.对氨基苯磺酸溶液与4-氨基-5-羟基萘-2,7-二磺酸单钠盐溶液分别为重氮试剂和偶联试剂反应条件：室温下,在10mL比色管中加入1.0g/L pH1盐酸对氨基苯磺酸溶液1mL及系列梯度浓度亚硝酸钠溶液1mL,静置3mmin进行重氮反应,后加入1.0g/L4-氨基-5-羟基萘-2,7-二磺酸单钠盐溶液1mL及pH4.5醋酸-醋酸钠缓冲液1mL,加水至刻度,混匀,静置30min进行偶合反应,以未加亚硝酸钠的混合溶液为参比进行吸光度测定。此反应生成粉红色偶联产物,在最大吸收波长520nm处,亚硝酸盐的浓度在0.2-5.25μg/mL范围内符合Beer定律,相关系数γ为0.99970,检出限为61.58ng/mL； 4.对氨基苯磺酸溶液与1-苯基-3-甲基-5-吡唑啉酮溶液分别为重氮试剂和偶联试剂反应条件：室温下,在10mL比色管中加入8g/L pH0.45盐酸对氨基苯磺酸溶液1mL及系列梯度浓度亚硝酸钠溶液1mL,静置1min进行重氮反应,后加入pH7.5磷酸盐缓冲溶液1mL及1.0g/L1-苯基-3-甲基-5-吡唑啉酮溶液1mL,加水至刻度,混匀,静置20min进行偶合反应,以未加亚硝酸钠的混合溶液为参比进行吸光度测定。此反应生成金黄色偶联产物,在最大吸收波长390nm处,亚硝酸盐的浓度在0.2-6.0μg/mL范围内符合Beer定律,相关系数γ为0.99998,检出限为15.13ng/mL; 5.4-氨基-5-羟基萘-2,7-二磺酸单钠盐在pH7.5磷酸盐缓冲液条件下与亚硝酸盐反应反应条件：室温下,在10mL比色管中加入8g/L pH1盐酸4-氨基-5-羟基萘-2,7-二磺酸单钠盐溶液1mL及系列梯度浓度亚硝酸钠溶液1mL,静置3min进行重氮反应,后加入pH7.5磷酸盐缓冲溶液(磷酸二氢钠-磷酸氢二钠缓冲溶液)1mL,加水至刻度,混匀,静置6min进行偶合反应,以未加亚硝酸钠的混合溶液为参比进行吸光度测定。此反应生成紫红色偶联产物,在最大吸收波长550nm处,亚硝酸盐的浓度在0.8-7.25μg/mL范围内符合Beer定律,相关系数γ为0.99081,检出限为632.89ng/mL; 6.4-氨基-5-羟基萘-2,7-二磺酸单钠盐在pH4.5醋酸盐缓冲液(醋酸-醋酸钠缓冲溶液)条件下与亚硝酸盐反应反应条件：55℃水浴条件下,在10mL比色管中加入6.0g/LpH1盐酸4-氨基-5-羟基萘-2,7-二磺酸单钠盐溶液1mL及系列梯度浓度亚硝酸钠溶液1mL静置3min进行重氮反应,后加入pH4.5醋酸-醋酸钠缓冲液1mL,加水至刻度,混匀,静置6min进行偶合反应,以未加亚硝酸钠的混合溶液为参比进行吸光度测定。此反应生成紫红色偶联产物,在最大吸收波长550nm处,亚硝酸盐的浓度在0.4-5.25μg/mL范围内符合Beer定律,相关系数γ为0.99930,检出限为193.99ng/mL。 7.除4-氨基-5-羟基萘-2,7-二磺酸单钠盐(偶氮反应在pH7.5条件下完成)方法,其他五种方法灵敏度高且线性好。对氨基苯磺酸和4-氨基-5-羟基萘-2,7-二磺酸单钠盐分别为重氮组分和偶氮组分、对氨基苯磺酸和1-苯基-3-甲基-5-吡唑啉酮分别为重氮组分和偶氮组分、4-氨基-5-羟基萘-2,7-二磺酸单钠盐(偶氮反应在pH7.5条件下完成)和H酸(偶氮反应在pH4.5条件下完成)四种方法线性范围上限高,可测较大浓度亚硝酸盐含量；对氨基苯磺酰胺和盐酸萘乙二胺分别为重氮组分和偶氮组分方法线性范围下限低,可测痕量亚硝酸盐的含量。对氨基苯磺酸和8-羟基喹啉分别为重氮组分和偶氮组分、对氨基苯磺酸和4-氨基-5-羟基萘-2,7-二磺酸单钠盐分别为重氮组分和偶氮组分、对氨基苯磺酸和1-苯基-3-甲基-5-吡唑啉酮分别为重氮组分和偶氮组分和4-氨基-5-羟基萘-2,7-二磺酸单钠盐(偶氮反应在pH4.5条件下完成)方法稳定性较好。对氨基苯磺酸和1-苯基-3-甲基-5-吡唑啉酮分别为重氮组分和偶氮组分方法测得的血液亚硝酸盐回收率最高,对氨基苯磺酸和4-氨基-5-羟基萘-2,7-二磺酸单钠盐分别为重氮组分和偶氮组分方法的回收率次之,但测定的人体血液中亚硝酸盐含量最接近加入量,准确度最好。 8.对照组大鼠未见任何不良反应,实验组大鼠在濒死期均出现皮肤黏膜发绀和呼吸困难等症状,且均在90min内死亡。经对氨基苯磺酸和4-氨基-5-羟基萘-2,7-二磺酸单钠盐分别为重氮组分和偶氮组分方法检测,空白对照组和生理盐水组大鼠检材中均未测出亚硝酸盐。亚硝酸钠给药实验组测得胃及内容物中亚硝酸钠含量最多,血液中含量次之,肝脏中亚硝酸钠含量最少。结论对氨基苯磺酸溶液与4-氨基-5-羟基萘-2,7-二磺酸单钠盐溶液分别为重氮试剂和偶联试剂方法,稳定性较好,线性范围广,测量人体血液亚硝酸盐含量实验中,其准确度及回收率较高,最适合测定生物检材中亚硝酸盐的含量。检测急性亚硝酸钠中毒案例最好的生物检材为胃及内容物和心血,在今后司法检测鉴定亚硝酸盐中毒的案例中可采用对氨基苯磺酸溶液与4-氨基-5-羟基萘-2,7-二磺酸单钠盐溶液分别为重氮试剂和偶联试剂方法。
[Abstract]:objective
Six was the best coupling agent Determination of nitrite content in human blood diazo coupling representative spectrophotometry, the optimum reaction conditions were explored diazo coupling agent, spectrophotometry to establish six groups of nitrite in human blood count quantitative analysis method; with the establishment of the six groups were analyzed. Coupled spectrophotometric determination of nitrite content in the linear range, detection limit, precision and recovery of each method and content of nitrite in human blood rate, comparative superiority of determination of nitrite content in the blood of the diazo coupling reagent, and ultimately determine the most suitable diazo coupling analysis of nitrite content in biological samples by spectrophotometry spectrophotometric method.
Establishment of a rat model of acute nitrite poisoning, choose the most suitable diazo coupling analysis of nitrite content in biological samples by spectrophotometry, namely p-aminobenzenesulfonic acid solution and 4- amino -5- -2,7- two hydroxy naphthalene sulfonic acid monosodium salt solution respectively as diazo reagent and coupling reagent determination of nitrite poisoning rat model of blood, liver, content the nitrite contents of stomach and kidney and the analysis of experimental data, to clarify the distribution of nitrite in major organs of rats with acute nitrite poisoning.
Method
1. through the literature, selected six groups of reagent method determination of nitrite content of coupling - representative spectrophotometric method: (1) Sulfanilamide and hydrochloric Naphthylethylenediamine respectively diazo and azo reagents; (2) sulfanilate and 8- hydroxyquinoline were diazo and azo reagents (3); p-aminobenzenesulfonic acid and 4- amino -5- hydroxy naphthalene sulfonic acid monosodium salt -2,7- two respectively, diazo and azo reagents; (4) sulfanilic acid and 1- phenyl -3- methyl -5- pyrazolone respectively diazo and azo reagents; (5) 4- -5- -2,7- amino hydroxy naphthalene two sulfonic acid monosodium salt (H) as diazo and azo reagents (as coupling reaction under the condition of pH7.5); (6) 4- -5- -2,7- two amino hydroxy naphthalene sulfonic acid monosodium salt as diazo and azo reagents (as coupling reaction under the condition of pH4.5).
2. randomly selected 10 healthy adults 1mL mixed venous blood (containing anticoagulants), mixed blood from 1mL to a 250mL volumetric flask, add 50mL ultrapure water and 10mL sodium hydroxide solution (20.0g/L), adding 10mL solution of zinc sulfate (0.42mol/L), mixed evenly. The heating 10min 60 C water bath, remove the cooling to room temperature and set the volume at 250mL, mixed evenly. Placed 30min, using quantitative filter and discard primary filtrate 50mL, adding dry sodium nitrite 160 G, which is completely dissolved.
3. each reagent method respectively fixed other reaction conditions, a change in the reaction conditions, the optimum reaction parameters, determine the total reaction the most suitable conditions; establish six group reagent method, related parameters determination method in each group, respectively with six sets of methods for determination of nitrite content and recovery rate of blood, comparative study and six the results of the group method.
4. a total of 14 healthy male Sprague-Dawley rats were randomly divided into control group (n=4), saline control group (n=4) and sodium nitrite perfused experimental group (n=6) respectively. The number of each animal. The saline control group and experimental group rats were intragastric administration of blank. The control group were killed by decapitation, the saline control group after administration by 60min were killed by decapitation, the experimental group in the administration after its death. The rapid extraction of all rats blood, liver, stomach and kidney and the contents of nitrite, ultra pure water for extraction of them.
5. with p-aminobenzenesulfonic acid solution and 4- amino -5- -2,7- two hydroxy naphthalene sulfonic acid monosodium salt solution respectively as diazo reagent and coupling reagent, determination of blood, the rats liver by standard working curve method, and the contents of nitrite content in the stomach and kidney, the quantitative analysis results were statistically analyzed by SPSS software.
Result
1. sulfanilamide solution and hydrochloric acid naphthalene ethylenediamine respectively diazo reagent and coupling reagent reaction conditions: room temperature, 10mL colorimetric tube and add 4.0g/L pH1 hydrochloride on 1mL and concentration gradient BENZENESULFONAMIDES solution of sodium nitrite solution 1mL, static 3min diazo reaction, after adding 1.0g/L hydrochloric acid naphthalene ethylenediamine 1mL, add water to the scale, mixing, static 1min coupling reaction, the reference absorbance was determined by mixed solution without nitrite. This reaction produces coupling product purple, the maximum absorption wavelength of 540nm, the concentration of nitrite in the range of 0.1-1.51 in the range of g/mL accords with Beer law. The correlation coefficient y is 0.99975, the detection limit is 3.86ng/mL;
2. sulfanilic acid solution and 8- hydroxyquinoline solution respectively diazo reagent and coupling reagent reaction conditions at room temperature in the 10mL colorimetric tube into 4.0g/L pH1 hydrochloric acid p-aminobenzenesulfonic acid solution 1mL and series of concentrations of sodium nitrite solution 1mL, static 1min diazo reaction, after adding 0.6g/L8- hydroxyquinoline 1mL and pH12.5 1mL ammonia solution, add water to the scale, mixing, static 1mmin coupling reaction, the reference absorbance was determined by mixed solution without nitrite. This reaction produces orange coupling product, the maximum absorption wavelength of 500nm, nitrite concentration in the range of g/mL 0.2-4.6 with Beer related law the coefficient is 0.99995, the detection limit is 26.13ng/mL;
3. sulfanilic acid solution with 4- -5- -2,7- two amino hydroxy naphthalene sulfonic acid monosodium salt solution respectively as diazo reagent and coupling reagent reaction conditions at room temperature in the 10mL colorimetric tube into 1.0g/L pH1 hydrochloric acid p-aminobenzenesulfonic acid solution 1mL and series of concentrations of sodium nitrite solution 1mL, static 3mmin diazo after the reaction, adding 1.0g/L4- amino -5- hydroxy naphthalene sulfonic acid sodium salt solution two -2,7- single 1mL and pH4.5 acetic acid sodium acetate buffer 1mL, add water to the scale, mixing, static 30min coupling reaction, the reference of absorbance was determined by mixed solution without nitrite. This reaction produces pink coupling products. The maximum absorption wavelength of 520nm, nitrite concentration in the range of g/mL 0.2-5.25 with Beer law, related coefficient is 0.99970, the detection limit is 61.58ng/mL;
4. sulfanilic acid solution with 1- phenyl -3- methyl -5- pyrazolone solution respectively for diazo reagent and coupling reagent reaction conditions at room temperature in the 10mL colorimetric tube into 8g/L pH0.45 hydrochloric acid p-aminobenzenesulfonic acid solution 1mL and series of concentrations of sodium nitrite solution 1mL, static 1min diazo reaction after joining pH7.5, 1mL phosphate buffer and 1.0g/L1- phenyl -3- methyl -5- pyrazolone solution 1mL, add water to the scale, mixing, static 20min coupling reaction, the reference absorbance was determined by mixed solution without nitrite. This reaction produces gold coupling products in the maximum absorption wavelength of 390nm 0.2-6.0, the nitrite concentration in the range of g/mL with the Beer law, related coefficient is 0.99998, the detection limit is 15.13ng/mL;
5.4- -5- -2,7- two amino hydroxy naphthalene sulfonic acid monosodium salt in pH7.5 phosphate buffer conditions and nitrite reaction conditions: room temperature, 10mL colorimetric tube and add 8g/L pH1 4- -5- -2,7- hydrochloride amino hydroxy naphthalene sulfonic acid sodium salt solution and two single 1mL series of concentrations of sodium nitrite solution 1mL, static 3min weight nitrogen reaction after adding pH7.5 phosphate buffer solution (sodium dihydrogen phosphate - two sodium hydrogen phosphate buffer solution) 1mL, add water to the scale, mixing, static 6min coupling reaction, the reference absorbance was determined by mixed solution without nitrite. This reaction produces purple coupling products in the maximum absorption wavelength of 550nm 0.8-7.25, the nitrite concentration in the range of g/mL with the Beer law, related coefficient is 0.99081, the detection limit is 632.89ng/mL;
6.4- -5- -2,7- two amino hydroxy naphthalene sulfonic acid monosodium salt in pH4.5 acetate buffer (sodium acetate buffer solution) under the condition of nitrite and reaction conditions: 55 DEG C water bath under the condition of 10mL in the cuvette adding 6.0g/LpH1 HCl 4- amino -5- hydroxy naphthalene sulfonic acid sodium salt solution two -2,7- single 1mL and a series of sub concentration gradient sodium nitrate solution 1mL static 3min diazo reaction, after adding pH4.5 acetic acid sodium acetate buffer 1mL, add water to the scale, mixing, static 6min coupling reaction, the reference absorbance was determined by mixed solution without nitrite. This reaction produces purple coupling products at the maximum absorption wavelength at 550nm, the nitrite concentration in the range of g/mL 0.4-5.25 with Beer law, related coefficient is 0.99930, the detection limit is 193.99ng/mL.
In addition to the 7. 4- -5- -2,7- two amino hydroxy naphthalene sulfonic acid monosodium salt (azo reaction under the condition of pH7.5), the other five methods with high sensitivity and good linearity. Sulfanilic acid and 4- amino -5- hydroxy naphthalene sulfonic acid monosodium salt -2,7- two respectively as diazo component and azo group, sulfanilic acid and 1- phenyl -3- methyl -5- pyrazolone respectively as diazo component and azo group, 4- -5- -2,7- two amino hydroxy naphthalene sulfonic acid monosodium salt (azo reaction under the condition of pH7.5) and H (azo reaction was completed under the condition of pH4.5) four methods of linear range limit, measurable greater concentration of nitrite content; Sulfanilamide and hydrochloric Naphthylethylenediamine respectively as diazo component and azo group method is linear in the range of lower limit, we can measure the contents of trace nitrite. P-aminobenzenesulfonic acid and 8- hydroxyquinoline respectively as diazo component and azo group, 4-aminobenzene 4- -5- amino acid and hydroxy naphthalene sulfonic acid monosodium salt -2,7- two respectively as diazo component and azo group, sulfanilic acid and 1- phenyl -3- methyl -5- pyrazolone respectively as diazo component and azo group and 4- -5- -2,7- two amino hydroxy naphthalene sulfonic acid monosodium salt (azo reaction in pH4.5 under the condition of stable method.) sulfanilic acid and 1- phenyl -3- methyl -5- pyrazolone respectively as diazo component and azo group method measured the blood nitrite recovery rate was highest, sulfanilic acid and 4- amino -5- hydroxy naphthalene sulfonic acid monosodium salt -2,7- two respectively as diazo component and azo composition for the recovery time, but the determination of nitrite content in human blood is most close to the amount of the best accuracy.
8. control rats without any adverse reactions, the experimental group was observed in rat skin mucous membrane cyanosis and dyspnea and other symptoms in the dying period, and all died within 90min. By p-aminobenzenesulfonic acid and 4- amino -5- hydroxy naphthalene sulfonic acid monosodium salt -2,7- two respectively as diazo component and azo component method, the control group and the saline group rats samples were not detected in nitrite. Sodium nitrite administered experimental group measured gastric contents and most nitrite content in the blood content of sodium nitrite content in the liver at least.
conclusion
P-aminobenzenesulfonic acid solution with 4- -5- -2,7- two amino hydroxy naphthalene sulfonic acid monosodium salt solution respectively as diazo reagent and coupling reagent method, good stability, wide linear range, measurement of human blood nitrite content in the experiment, the accuracy and recovery rate is high, the most suitable for the determination of nitrite content in biological samples.
Acute sodium nitrite poisoning cases detected the best biological samples for the stomach and the contents and efforts, in case of nitrite poisoning in judicial identification using p-aminobenzenesulfonic acid solution and 4- amino -5- -2,7- two hydroxy naphthalene sulfonic acid monosodium salt solution respectively as diazo reagent and coupling reagent method.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：D919.1

【参考文献】