| Chromatography is a method of separation and analysis of the mixture.For the method is good at separation,combined with modern chromatographic detectors with high sensitivity,chromatography has become an important method for the analysis of complex mixtures.Among them,the most widely used high-performance liquid chromatography(HPLC),since 1960 s,after decades of development,both in theory and practice are maturing,it has been widely used in medicine,food,environment,agriculture and life sciences as well as other fields.When compared with traditional liquid chromatography,high performance liquid chromatography has high efficiency,high speed,high sensitivity and high automation;compared with gas chromatography,it also has wide application range,high selectivity,low temperature as well as the advantages of easy collection and preparation.Mycotoxins are a kind of toxic metabolites produced by toxigenic filamentous fungi,which can survive in a variety of crops,especially in and after crop harvest.Mycotoxins can enter into animals and human body through feed or food,which can cause multiple organ damages,and do harm to human's and animal's safety.Therefore,for the detection of mycotoxins has become very important.However,due to the presence of trace amounts mycotoxins in environmental samples,and the survival of the complex matrixs,which will undoubtedly bring great difficulties to the detection.The detection of aflatoxin,which is familiar to people,there are mature detection methods;but for fumonisins,which is also common and threaten to the health of human beings,has been the lack of an effective and reproducible detection method.To solve this problem,I conducted a method research in the first part of the thesis,and the results are satisfactory.In the second part,combined with the application of high performance liquid chromatography technology,and online cleaning device,as well as considering the sample detection limit and linear range at the same time,ultraviolet(UV)and fluorescence detector(FLD)were combined applied,on the study of anesthetic lidocaine and propofol.Lidocaine and propofol are widely used in clinical anesthesia,in pediatric surgery,artificial abortion analgesia and so on,but there is no joint detection research literature.Based on such reality,in this study I established a method for simultaneous detection of lidocaine and propofol,in order to guide the clinical medication,and achieved satisfactory results.PART 1 A new method of high performance liquid chromatography procedure for determination of fumonisins B1 and B2 coupled with online pre-column derivatizationObjective: To establish a new method for the determination of fumonisins B1 and B2 with online pre-column derivatization coupled with high performance liquid chromatography and fluorescence detection.Method: With the application of high performance liquid chromatography combined with fluorescence detector of high sensitivity,fumonisins B1 and B2 in maize samples were detected.The collected samples were extracted twice by 80% methanol solution after being battered,then centrifugated,the supernatant was filtered through the membrane,and then diluted with PBS buffer solution,stand-by.Then,the sample solution was put through immunoaffinity column(IAC)for purification and concentration,the final elution was analyzed after being filtered.Before the detection,the sample should under derivatization procedure,with derivative agents of o-phthalaldehyde(OPA)and 2-mercaptoethanol(2-ME),and then transfered to C18 column,finally,deteced by fluorescence detector(FLD),with external standard method quantified.Results: Under the optimized conditions,the linear range of fumonisins B1 and B2 were measured 0.1~5.0 ?g/mL(FB1)and 0.06~2.5 ?g/mL(FB2),the linear regression equations were Y = 7832.79+116714.74X(FB1),Y = 4499.88+53501.98X(FB2),linear correlation coefficients were 0.9999 and 0.9992,respectively.The lowest detection limits were 0.10 and 0.26 mg/kg,respectively.Seven parallel determinations for FB1 of 1.0 mg/L and FB2 of 0.5 mg/L every day,and seven days of continuous measurement,the relative standard deviation of FB1 was measured 4.38%(intra-day)and 6.90%(interday);the relative standard deviation of FB2 was 3.15%(intra-day)and 8.50%(inter-day).The recoveries of the standard addition of maize samples were 85.6%~119.2%.Conclusion: The method is simple,rapid and highly automated,which reduces the error in the process of artificial derivation,and the results are accurate and reliable.PART 2 Simultaneous determination of lidocaine and propofol by online solid phase extraction(SPE)high performance liquid chromatography with double detectorsObjective: To establish a new method for the simultaneous determination of lidocaine and propofol in plasma by high performance liquid chromatography(HPLC)and dual detectors,coupled with online purification technology.Method: Based on the high performance liquid chromatography,combined with dual detectors and online purification technology,which makes the combined detection of lidocaine and propofol come true.In the study,chromatographic conditions and online purification conditions were optimized.Finally,blood samples were centrifuged by 12000 r/min for 10 minutes,collected the supernatant,protein was precipitated with methanol,which followed by direct injection.Through the purification by online solid phase extraction column,gradient elution by reversed-phase C18 column and detected by ultraviolet detector series fluorescence detector,and quantified by external standard method at last.Then,the wistar rats were injected to test drugs and the blood were detected.Results: Under the optimized conditions,the linear range of lidocaine was measured for 0.08~80 mg/L,the linear regression equation was Y =-0.2263+0.8281 X,the correlation coefficient was r = 0.9999,the lowest detection limit was 24 ?g/L.Six consecutive parallel determinations for lidocaine at the concentrations of 0.2,2.0 and 20 mg/L,respectively,the relative standard deviations were 1.2~5.0%.For the detection of lidocaine in blood,the recoveries were in the range of 95.4%~108.9%.Under the optimized conditions,the linear range was measured with propofol for 0.0025~20 mg/L,the linear regression equation was UV: Y =-0.4053+0.8262 X,r = 0.9999,FLD: Y =-731.3385+1999852.5522 X,r = 0.9999,the lowest detection limit was 0.75 ?g/L.Six consecutive parallel determinations for propofol at the concentrations of 0.05,0.5 and 5.0 mg/L,respectively,obtained the relative standard deviations were 1.9~3.5%.Similarly,the purification and detection of propofol in blood were also conducted,the recoveries were between 96.9%~98.6%.Conclusion: The method is simple,rapid and easy to realize automation.It can be used to monitor the concentration of lidocaine and propofol in blood. |
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