| Objective:Select six representative best coupler of diazonium coupling spectrophotometry determination methods to test the nitrite content in the blood of human body. Establish six groups of spectrophotometer quantitative analysis methods for determination of nitrite in human blood after discovered the optimum reaction conditions. Use the established methods to determine the range, DL, nitrite content in human blood, precision and recycling rate of each method. Compare the different methods’superiority to make sure the optimum method for determination of nitrite in biological material.Establish nitrite poisoning rats model, and choose sulfanilic acid and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo reagent and coupling reagent respectively to determine the nitrite in heart blood, liver, stomach and contents, and kidneys of rats model. Analyze the experimental data and clarify the distribution of nitrite of in the main organs of rat model with acute nitrite poisoning.Methods:1. Look up literature reference to choose six represented groups of coupling-spectrophotometric methods for determination of nitrite in human blood:(1) sulfonamide and N-ethylenediamine as diazo reagent and coupling reagent respectively. (2)sulfanilic acid and8-hydroxyquinoline as diazo reagent and coupling reagent respectively.(3)sulfanilic acid and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo reagent and coupling reagent respectively.(4)sulfanilic acid and1-phenyl-3-methyl-5-pyrazolone as diazo reagent and coupling reagent respectively.(5)4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo reagent and coupling reagent (coupling reaction in pH7.5condition).(6)4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo reagent and coupling reagent (coupling reaction in pH4.5condition).2. Extract each of10healthy adult1mL venous blood randomly which containing anticoagulant, and mix the blood. Extract1mL mixed blood into250mL volumetric flask, add50mL ultrapure water and10mL (20.0g/L) sodium hydroxide solution and add10mL (0.42mol/L) zinc sulfate solution, mix them evenly. Then put it in60℃water, heating10minutes, take out and cool it to room temperature, dilute it to250mL, mixed evenly.30min at room temperature, use quantitative filter paper to filter and abandon the50mL primary filtrate, add160μg dry sodium nitrite, completely dissolve it.3. Fixed other conditions, change one reaction condition to make sure the most suitable conditions, measure the optimum reaction parameters. Establish six reagent methods and determine every group’s related parameters. Respectively use six groups of methods to determine the nitrite content and recovery rate in the blood. Compare the results of the six methods.4. Devide14healthy male Sprague-Dawley rats randomly into blank control group (n=4), normal saline lavage in the control group (n=4) and sodium nitrite lavage experimental group (n=6), number the rats in each groups. Normal saline control group and experimental group rats were given lavage administration. Use beheading method to put the rats of blank control group to death. Norma saline lavage in the control group,60min after dosing, put them to death by using beheading method. The experimental group uses dosing as its death method. Extract all the rats’ blood, liver, stomach and contents and kidney, use ultrapure water immersion to extract their nitrite.5. Take sulfanilic acid solution and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt solution as diazo reagent and coupling reagent respectively. Use standard working curve method to determine each group of rats’ heart blood, liver, stomach and kidneys’ content of nitrite.Results:1. The reaction condition of the sulfonamide solution and N-ethylenediamine solution as diazo reagent and coupling reagent respectively:at room temperature, in10mL colorimetric tube with4g/L pH1hydrochloric acid to aminobenzene sulfonamide solution1mL and gradient concentration of nitrite solution1mL, let stand for3min diazo reaction, add1g/L1mL of N-ethylenediamine solution, add water to the scale, blending, and let stand for1min, with no nitrite mixed solution as a reference for determination of absorbance. The reaction produces purple coupling product, the maximum absorption wavelength at540nm, the concentration of nitrite within the scope of0.1to1.5μg/mL which was in accordance with Beer’s law, the correlation coefficient γ:0.99975, the detection limit:3.86ng/mL.2. The sulfanilic acid solution and8-hydroxyquinoline solution as diazo reagent and coupling reagent respectively:at room temperature, add4in10mL colorimetric tube pH1g/L of sulfanilic acid solution1mL and1mL series gradient concentration of nitrite solutions, let stand for1min for diazo reaction, add0.6g/L8-hydroxyquinoline solution1mL and pH12.5ammonia1mL, adding water to the scale, blending, and let stand for1min, with no nitrite mixed solution as a reference for determination of absorbance. This reaction generates the orange coupling product, at the maximum absorption wavelength which is500nm, the concentration of nitrite is within the scope of0.2to4.6μg/mL as in accordance with the Beer’s law, the correlation coefficient of γ:0.99995, detection limit:26.13ng/mL.3. The reaction condition of sulfanilic acid and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo reagent and coupling reagent respectively:at room temperature, in10mL colorimetric tube with1g/L pH11mL and series of sulfanilic acid solution concentration gradient nitrite solution1mL, let stand for3min diazo reaction, add1g/L amino-4-5-hydroxy beta-2,7-disulfonic acid monosodium salt solution1mL and pH4.5-acetic acid sodium acetate buffer1mL, add water to the scale, blending, and let stand for30min, with no nitrite mixed solution as a reference for determination of absorbance. This reaction produces pink coupling reaction products, at the maximum absorption wavelength520nm, the concentration of nitrite is within the scope of0.2 to5.25μg/mL as in accordance with Beer’s law, the correlation coefficient of γ is0.99970, the detection limit:61.58ng/mL.4. The reaction condition of sulfanilic acid and1-phenyl-3-methyl-5-pyrazolone as diazo reagent and coupling reagent respectively:at room temperature, add8to10mL colorimetric tube pH0.45g/L of sulfonic acid hydrochloride solution1mL and the corresponding concentrations of nitrite solution1mL, let stand for1min for diazo reaction, after join the phosphate buffer solution pH7.51mL and1g/L1-phenyl-3-methyl-5pyrazolone solution1mL, add water to the scale, blender, let stand20min, with no nitrite mixed solution as a reference for determination of absorbance. This reaction generates golden coupling product, the maximum absorption wavelength at390nm, the concentration of nitrite within the scope of0.2to6.0μg/mL in accordance with Beer’s law, the correlation coefficient of γ0.99998, detection limit:15.13ng/mL.5.4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo reagent and coupling reagent (coupling reaction in pH7.5condition):at room temperature, add8to10mL colorimetric tube pH1g/L hydrochloric acid4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt solution1mL and the corresponding concentrations of nitrite solution1mL, let stand for3min diazo reaction, join after pH7.5PBS1mL, add water to the scale, blender, let stand6min, with no nitrite mixed solution as a reference for determination of absorbance. This reaction generates purple coupling products, maximum absorption wavelength at550nm, the concentration of nitrite within the scope of0.8to7.25μg/mL in accordance with Beer’s law, the correlation coefficient of γ is0.99081, the detection limit:632.89ng/mL.6.4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo reagent and coupling reagent (coupling reaction in pH4.5condition):55℃water bath condition, join in10mL colorimetric tube6g/L pH1hydrochloric acid naphthalene hydroxyl amino4-5-1-2,7-disulfonic acid monosodium salt solution1mL and the corresponding concentrations of nitrite solution1mL, let stand for3min diazo reaction, then add pH4.5acetic acid sodium acetate buffer1mL, add water to the scale, blender, let stand6min, with no nitrite mixed solution as the reagent reference to determine absorbance. This reaction generates purple coupling products, maximum absorption wavelength at550nm, the concentration of nitrite is within the scope of0.4to5.25μg/mL in accordance with Beer’s law, the correlation coefficient of γ is0.99930, the detection limit:193.99ng/mL.7. Except4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt method (azo reaction under the condition of pH7.5), the other five kinds of methods have high sensitivity and linear. Sulfanilic acid and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo components and azo compositions respectively, sulfanilic acid and1-phenyl-3-methyl-5-pyrazolone as diazo components and azo components respectively,4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt (azo reaction under the condition of pH7.5) and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt (azo reaction under the condition of pH4.5) four methods have higher limit of linear range, they can measure larger doses of nitrite content; Para sulfonamide and N-ethylenediamine as diazo and azo method have lower linear range limit, they can trace low nitrite content. Sulfanilic acid and8-hydroxyquinoline as diazo components and azo component, the amino benzene sulfonic acid and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo components and azo compositions respectively, sulfanilic acid and1-phenyl-3methyl-5-pyrazolone as diazo components and azo compositions and amino-4-5-hydroxy beta-2,7-disulfonic acid monosodium salt methods (azo reaction under the condition of pH4.5) have good stability. Sulfanilic acid and1-phenyl-3-methyl-5-pyrazolone as diazo components and azo component method respectively measure blood nitrite, highest in aminobenzene sulfonic acid and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt diazo component respectively and the recovery rate of azo composition method of times, but the measurement of content of nitrite in human blood becomes most accurate when the amount of nitrite content close to the addition.8. The control rats did not demonstrate any adverse reaction, the experimental group rats in near-death period grow cyanosis and breathing difficulties and other symptoms of skin mucous membrane, and all died within90min. Of aminobenzene sulfonic acid and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt diazo respectively part a and part azo testing blank control group and normal saline lavage group failed in the examination of the measure of nitrite. The nitrite lavage for medicine and experimental group measured that the stomach has the most nitrite content, the blood the second most, the liver minimum.Conclusion:The method that take sulfanilic acid and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo reagent and coupling reagent respectively has good stability, wide linear range, the accuracy and recovery rate is higher in measurement of human blood nitrite content in the experiments and it is the most suitable method for determining the content of nitrite in specimens.The best biomaterial for detection of acute nitrite poisoning case is gastric specimens and contents and heart blood. In the judicial test cases, it can be used for the identification of nitrite poisoning with sulfanilic acid and4-amino-5-hydroxynathalene-2,7-disulphonic acid monosodium salt as diazo reagent and coupling reagent respectively for the future. |
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