Study On The Detections Of Morphine And Catecholamine Transmitters By Ag(Ⅲ) Chemiluminescence

Capillary Electrophoresis（CE）is a new liquid phase separation technology which is based on the capillary as the separation channel and the high voltage direct current electric field as the driving force.This technique advented since 1980s has been widely used in many fields,such as medical testing,clinical testing,environmental testing and forensic toxicity testing.With the diversified separation patterns,it can satisfy the analysis requirement of most substances,showing numerous advantages such as fast separation speed,high separation efficiency,low analysis cost and low sample consumption.CE realizes the transformation of analytical separation technology from micro-level to nano-level,making analytical techniques for both single cell analysis and single cell molecular analysis possible,and has brought new opportunities for the separation and analysis of biological macromolecules such as proteins.Chemiluminescence（CL）is a type of radiation phenomenon which is accompanied by chemical reaction.Base on the linear relationship between the concentration of the substance and signal strength under certain circumstances,chemiluminescence can be used to analyze substances that can directly produce chemiluminescence or can affect chemiluminescence signals.Chemiluminescence is a highly sensitive and simple trace analysis method without excitation light and external light source,which makes it avoid the noise from Rayleigh scattering and Raman scattering.The method that combinated capillary electrophoresis and chemiluminescence can combine the advantages of these both techniques.In recent years,the combination of the two technologies has been widely used in medical testing,clinical testing,environmental testing,forensic toxicity testing and many other fields.Ag（Ⅲ）complex[Ag（HIO₆）₂]^5--Luminol is a new chemiluminescence system.In alkaline medium,Ag（Ⅲ）complex which has strong oxidation ability can oxidize luminol to produce chemiluminescence.Based on the fact that some substances can enhance or inhibit the chemiluminescence signal,and within a certain concentration range,the enhancement or inhibition of the chemiluminescence signal has a strong linear relationship with the concentration of the substance,a new method called capillary electrophoresis chemiluminescence was established to detect morphine in urine and blood,and simultaneously detect five neurotransmitters including epinephrine（E）,norepinephrine（NE）,3-methoxy-4-hydroxy-phenylethylene glycol（MHPG）,3-methoxy-4-hydroxy-amygdalate（VMA）and 3-methoxy-4-hydroxy acid（HVA）in urine.The new method has the advantages of simple operation,good separation effect,short analysis time,less reagent consumption and environmentally friendly.Part 1 Detection of morphine in urine and blood by capillary electrophoresis-chemiluminescenceObjective:To establish a new determination of morphine in urine and blood by capillary electrophoresis chemiluminescence and successfully apply it to the qualitative and quantitative analysis of morphine in urine and blood.Method:The optimum experimental conditions were obtained:Ag（Ⅲ）complex concentration was 7.0×10^-5 mol/L,in which NaOH concentration was 0.04 mol/L,Na₂CO₃ concentration was0.008 mol/L,Luminol concentration was 2 mmol/L,borax concentration was 5 mmol/L,separation voltage was 15 kV and the injection time was 12 s.HLB solid phase extraction column was used to purify and concentrate urine and blood samples,and the disposed samples were injected under optimized conditions after nitrogen was dried and resolved.Morphine was separated from other substances in biological samples by capillary channel and high voltage DC electric field.When it reached the detection window,it interacts with Ag（Ⅲ）complex-luminol system,which can affect the redox reaction of the system and the chemiluminescence signal,and in alkaline medium,morphine has an inhibitory effect on the chemiluminescence signal of this reaction,and within a certain concentration range,the degree of inhibition has a strong linear relationship with the concentration of morphine.Based on this principle,a standard curve was established and the concentration of the morphine can be calculated.Results:Under the optimum detection conditions,the detection limit of morphine was 0.75μg/mL.The linear equation wasΔI=3.8265c+14.14（R²=0.9998）and the linear range was 2³0μg/mL.The relative standard deviation（RSD）was 2.01%（n=7）.The average recoveries of urine and blood samples were 109.03%and 101.28%,respectively.Conclusion:A new method for the determination of morphine in urine sample and blood sample by capillary electrophoresis with chemilu-minescence was developed,and was successfully applied in detecting morphine in urine sample and blood sample.The results of separation and analysis were satisfying.he benefits of this method include easy operation,low cost,less sample consumption and environmentally friendly.Part Ⅱ Simultaneous determination of adrenaline,noradrenaline and metabolitesinurinebycapillaryelectrophoresis-chemiluminescenceObjective:To establish a new method for simultaneous determination of epinephrine（E）,norepinephrine（NE）,3-methoxy-4-hydroxy-phenylethylene glycol（MHPG）,3-methoxy-4-hydroxy-picrylic acid（VMA）and 3-methoxy-4-hydroxyacetic acid（HVA）in urine by capillary electrophoresis-Ag（Ⅲ）complex-luminol chemiluminescence,and to apply it successfully to the simultaneous detection of these five sustances in urine.Method:Urine samples were treated by strong acid cation exchange resin and ultra-high-speed low-temperature centrifugation,then the processed samples were injected to be separated and analyzed under optimized conditions after then filtered.Under the optimized experimental conditions,the Ag（Ⅲ）complex concentration was 2 mmol/L including0.05 mol/L NaOH and 0.04 mol/L Na₂CO₃,luminol concentration was 3 mmol/L,borax concentration was 5 mmol/L,sodium cholate concentration was 3 mmol/L,separation voltage was 15 kV,and the injection time was 15 s.These target substances were separated by capillary electrophoresis and had a reaction with Ag（Ⅲ）complex-luminol chemiluminescence system at the detection window.In alkaline medium,epinephrine has a enhancement effect on the chemilu-minescence intensity of Ag（Ⅲ）complex with luminol,and norepinephrine,3-methoxy-4-hydroxy-phenylethylene glycol,vanilla mandelic acid and pervanillic acid all can inhibit the singal of this chemiluminescence system.Within a certain range of substance concentration,the degree of enhancement or inhibition has a good linear relationship with substance concentration.According to this,the standard curve of each substance was established and the contents of sample can be measured respectively.Results:Under the optimized detection conditions,the linear equations of E,NE,MHPG,VMA,HVA were respectivelyΔI=5.8429c+10.833,R²=0.9983,ΔI=5.8534c+52.882,R²=0.9984,ΔI=13.234c+84.315,R²=0.9986,ΔI=2.3409c+19.318,R²=0.9999,ΔI=9.9333c+9.906,R²=0.9994.The linear ranges were 2¹2μg/mL,2³0μg/mL,2²0μg/mL,2⁶0μg/mL and 2²0μg/mL,respectively.The relative standard deviations were4.50%,4.97%,3.23%,3.36%and 3.07%respectively（n=7）.The detection limits of epinephrine,norepinephrine,homovanillic acid were 0.50μg/mL,3-methoxy-4 hydroxy-phenylethylene glycol was 0.10μg/mL and vanilla mandelic acid was 0.75μg/mL.The average recoveries of these five substances were 103.96%,101.76%,100.65%,106.48%,and102.87%,respectively.Conclusion:A new method for simultaneous determination of epinephrine,norepinephrine,3-methoxy-4-hydroxy-phenylethylene glycol,vanilla mandelic acid and homovanillic acid in urine by capillary electrophoresis-Ag（Ⅲ）complex-luminol chemiluminescence method has been established.The method is easy operation,sensitive,low cost,environmentally friendly and has less sample consumption,the effect was satisfying.