| High performance liquid chromatography(HPLC) is the most widely used in the modern instrumental analysis. It has the merits of high separation efficiency and high selective. In the system of the HPLC, the detector plays an important role. The most common detectors are the fluorescence detector, ultraviolet detector and electrochemical detector and so on.Resonance Rayleigh scattering(RRS) as a new spectral analysis method was developed since the 1990 s. It has obtained the broad attention of the research workers because of its simply operate process, quickly analyze speed and the high sensitive. But the selectivity of RRS is bad, it is vulnerable to the interference of the complex matrix and difficult to effectively detect the substance which has the similar structure. Thus, HPLC combined with RRS can use the advantage of high separation efficiency of HPLC to make up the shortage of the selective of RRS. At the same time, it can perform the advantages of high sensitivity of RRS. On the other hand, it also can promote the further development of high performance liquid chromatography. The main research content of this article were as follows: 1. High performance liquid chromatography combined with resonance Rayleigh scattering for the detection of six psychotropic drugs in human urineIn this article, a simple, reliable, high selectivity and sensitivity analysis method of HPLC-RRS for the determination of six psychotropic drugs was established. The six psychotropic drugs were Doxepin hydrochloride(DH), Promethazine hydrochloride(PMZ), Imipraine hydrochloride(IMP), Amitriptyline hydrochloride(AH), Chlorpromazine hydrochloride(CPZ) and Clomipramine hydrochloride(CHL), respectively. In this study, the mobile phase was acetonitrile: 0.1% triethylamine solution( adjusted the p H to 3.1 by using phosphoric acid) = 40 : 60(V/V) with the flow rate of 0.4 m L · min-1. Column temperature was 40 ℃ and sample quantity was 20 μL. After separated by HPLC, the six drugs were mixed with BR buffer solution(BR) one by one in the first tee and protonation, then the mixture was mixed with erythrosine(Ery) in the second tee. Finally the association reaction was completed in the heat tube. This method was based on the weak intensity of the RRS of six drugs and its significantly enhancement after combined with Erythrosine(Ery) in p H 4.0~4.7 BR buffer solution. In this paper, we have optimized a series of the experimental conditions including the detection wavelength, flow rate, the reaction temperature, the reaction tube length and so on. The quantum chemistry calculation, the technology of scanning electron microscope(SEM) and the UV absorption spectrum and so on were used for the discussion of the reaction mechanism. In the optimization experimental conditions, the range of the limit of detection(LOD, S/N=3) were 0.42~21.05 ng · m L-1 and the linear correlation coefficient all above 0.9916. This method was used for the detection of the adding standard recovery of six psychotropic drugs in human urine and obtained the satisfactory results. In the end, The method was successfully applied to the detection of the IMP in the patient human urine. 2. High performance liquid chromatography combined with resonance Rayleigh scattering for the detection of antihistamines drugs in human urineAfter separated by HPLC, three kinds of antihistamines drugs, including Pheniramine(PH), chlortrimeton(CHL), diphenhydramine(DIP) will enter into a post-column derivatization device one by one. Then the three antihistamines drugs will combine with Acid Red 94(AR) to form 1:1 ionic associate and the signal of RRS enhanced signficantly. Based on this principle, a new method of HPLC-RRS for the detection of above three antihistamines drugs was established. This study used a phenomenex Luna C8 column for the separation and the separation flow rate was 0.4 m L · min-1, Column temperature was 35 ℃ and the sample quantity was 20 u L. A series of the post-column experiment conditions including BR buffered solution flow rate, AR solution flow rate, the detection wavelength, the reaction acidity, the reaction temperature and so on were studied. The methods of molar ratio and iso-molar continuous variation were adopt to determine the composition ratio of DIP and AR. The reason of the RRS enhanced also was studied. It was found that there was a good linear relationship between the peak area with the concentration of the kinds of drugs in a certain range. The limit of the detection(LOD) of PH, CHL, DIP were 29.71, 8.49, 36.75 ng · m L-1, respectively. Which based on the S/N = 3. In the end, this method was successfully applied to the detection of the three antihistamines drugs in human urine. 3. Study on the interaction between mitoxantrone and erythrosine by resonance Rayleigh scattering spectra and fluorescenceMitoxantrone(MTX) is an amino-anthraquinone anticancer drug, It has outstanding efficacy and lower cardiac toxicity and it has been approved by the U.S Food and Drug Administration(FDA) since 2000. The RRS signal of Ery is very weak, but its fluorescence intensity is very strong. When it combined with MTX, its RRS signal will significantly enhance and its fluorescence intensity will quench. Based on this principle, two method of RRS and fluorescence quenching for the detection of MTX was established. Compared with the method of fluorescence quenching, the sensitivity of RRS method was more highly. Therefore, RRS method was more suitable for the detection of MTX in the concentration of microscale or trace amount.In this study, a series of reaction conditions such as acidity, the concentration of Ery, the reaction time and so on were optimized. A series of possible interfering substance was researched. The binding site and the combination mode of Ery and MTX were studied. The results indicated that Ery would combined with MTX by electrostatic attraction and hydrophobic force. Under the optimization conditions, the range of linearity of RRS and fluorescence were 0.03~3.0 μg · m L-1 and 0.1~1.5 μg · m L-1, respectively. The correlation coefficient(R) were 0.9991 and 0.9989, respectively. The detection limits(LOD) were 2.1 and 15.0 ng · m L-1, respectively. In the end, this method was applied to the detection of MTX in human urine and obtained the satisfactory results. |
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