| Objective:To establish the derivatization method for analysis of propofol,which was used to improve the sensitivity when use UV-visible and fluorescence spectrophotometric method and avoid signal interference from endogenous substances in plasma.Then a automated method was developed for detection of propofol,basing on derivatization of propofol combining with micro sequential injection- lab-on-valve(μSI-LOV) and fiber optic sensing technology.The μSI-LOV was used to carry out the derivatization reaction of propofol and the fiber optic sensing was used to detect the derivative products.Finally, the analysis for propofol realized the automation and integration.Method:1.The derivatization of propofol and analysis: To study azo-coupling derivatization fo propofol,after the derivatization reaction of the diazonium salt with propofol, the coupling product was obtained and measured at the maximum absorption wavelength(λmax),using a single factor variable method to investigate reaction conditions of azo-coupling for propofol; To study fluorescence derivatization for propofol, after the reaction of the DNS-Cl with propofol, the derivative products were obtained which were extracted by a pophilicity of organic solvent and measured at detection wavelength by fluorescence spectrophotometry,the reaction conditions of fluorescence derivatization for propofol were investigated by single factors variable method.To establish the method for the determination of propofol in plasma which based on diazo-couple derivatization combining Uv- vis spectrophotometry,the method was applied to analyse propofol in simulated biological samples; 2.To develop the automated method for analysis of propofol which based on μSI-LOV carrying out the diazo coupling reaction,the derivatization products were detected by the optical fiber sensing- visible spectrophotometry,the best optimum experimental parameters were studied by using the single factor variable method; The method was developed for the detection of propofol in plasma which based on the automated method.The method was applied to analyse propofol in simulated biological samples;To Build the method which based on the automated method for analysis of propofol in vivo rats’ plasma.The jugular vein catheterization was carried out in rats’ right jugular vein.Blood samples were collected from the jugular at different time points after drug administration.Samples were immediately centrifuged and plasma was separated and analyzed. Results: 1.The best reaction condition of azo-coupling for propofol:The sodium nitrite which was dissolved in distilled water and reacted with sulfanilic acid which was dissolved in hydrochloric acid(0.06mol/L),after 5min,the diazonium salt(5.780×10-4mol/L) was obtained;Then propofol reacted with the diazonium salt,adding 1ml sodium hydroxide solution(0.7mol/L) at room temperature for 10 min.The orange coupling product was obtained and remained stable within 20 min,whose λmax at 483nm; The excitation and emission wavelengths(λex and λem) of fluorescence derivatizative product of propofol at 345,500 nm respectively.The optimum derivatization conditions as follows:Acetonitrile as the solvent for DNS-Cl and propofol,the derivatives is stable within 10 min which can be obtained by the labeling reaction of DNS-Cl with propofol(DNS-Cl:propofol=8:1,mole ratio) in the presence of sodium hydroxide(0.1mol/L) at 60℃ for 5 min in the dark,then using cyclohexane to extract for 1min.In the method of diazo-coupling derivatization combing Uv- vis spectrophotometry for determination of propofol in plasma,the linear range of propofol was 6~21μg/ml which showed a good linear relationship(r=0.9960); The methodological recovery of three le vels of concentration of high,medium and low were between 102.9%and 104.5%, the precision(RSD) were 4.1~5.5%;Repeatability met the requirements; 2.Optimum experiment parameters of azo-coupling combining with the optical fiber sensing–μSI-LOV: the aspiration of 50μL diazonium salt(1.156×10-3mol/L), 50μL propofol solutions(or the blank), 40μL NaOH solution(0.2mol/L) at the same flow rate of 50μL/s and the solutions were mixed in the holding coil(there was no stop flow),then the products was reversely prope lled to Z- flow cell at a flow rate of 15μl/s.;In the method of azo-coupling combing the optical fiber sensing-μSI-LOV for determination of propofol in plasma, the linear range of propofol was 3~18μg/ml which showed a good linear relationship(r=0.9990);The methodological recovery of three levels of concentration of high, medium and low were between 89.8% and 99.6%, the the precision( RSD) were 4.3~5.4%; Repeatability met the requirements;In the method of azo-coupling combing the optical fiber sensing-μSI-LOV for determination of propofol in vivo rats’ plasma,the linear range of propofol was 3~24μg/mL which showed a good linear relationship(r=0.9960);The methodological recovery of three levels of concentration of high, medium and low were between 92.7% and 105.5%, the precision(RSD) were 5.3~7.5%. The method can measure the propofol concentration in vivo rats’ plasma within 10 min after drug administration. Conclusions:1. The absorption wavelength performed red shift and the absorbance had an increase after the azo-coupling reaction for propofol, the derivatization condition was eas y to control and reaction rate was fast. After derivatization of propofol by using DNS-Cl,The λex and λem performted red shift,but he fluorescence intensity of derivatizative product less than the underivatized propofol. The fluorescence derivatization reaction was not applied to analysis propofol in plasma,offering the investigation results for providing a reference for further study.Diazo-coupling derivatization combined with Uv-vis spectrophotometry can be used to analyze propofol in plasma,which can avoid the interference from endogenous substances in the plasma. The method was simple and fast 2.The combination of diazo coupling reaction of propofol and fiber optic sensing-μSI-LOV which made the analysis rate has been greatly improved. The analysis for propofol realized the automation and integration.The automated method can be used to analyze propofol in plasma, which was faster and more sensitive compared with the method of azo-couping derivatization combining UV-vis spectrophotometry. The sensitivity was not very ideal when the automated method was used to analysis propofol in vivo rats’ plasma.It is necessary that the automated method is further improved to enhance sensitivity,so that the method can detect propofol at the lower concentration. |
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