| High performance liquid chromatography(HPLC) is a kind of very important separation and analysis techniques. Liquid is used as mobile phase, and buffer, a single solvent or mixed solvent were pumped into the chromatographic column by the high pressure pump. Then mobile phase components are separated in the chromatographic column, flow into the detector for testing, to analyze of the sample. Resonance Rayleigh scattering(RRS), as a well-developed analytical technique, has drawn much more attention in recent years owing to its high sensitivity and simplicity. Furthermore, it has been increasingly applied to detect proteins, nucleic acids, anti-cancer drugs, pesticides, metal ions, vitamins, and surfactants. High performance liquid chromatography(HPLC) was combined with resonance Rayleigh scattering, a high sensitivity and high selectivity of high performance liquid chromatography-resonance Rayleigh scattering detection technique(HPLC-RRS) was established. And it has been applied to detect amino acids, protein, local anesthetics, antidepressants, α1-adrenoceptor antagonists, indole alkaloids and flavonoids drugs. High performance liquid chromatography-resonance Rayleigh scattering has very prominent advantages:(1) the sensitivity of high performance liquid chromatography- resonance Rayleigh scattering is higher than that of UV-detector, and higher than or close to the level of fluorescence detector and chemiluminescence detector;(2) high performance liquid chromatography-resonance Rayleigh scattering can be applied to both fluorescence and UV matters, and also can be applied to not fluorescence and UV matters;(3) high performance liquid chromatography-resonance Rayleigh scattering overcomes the drawback of RRS poor selectivity and poor repeatability, expands the resonance Rayleigh scattering spectra research in the new field, and further expands the application range of high performance liquid chromatography(HPLC).This paper mainly studies high performance liquid chromatography-resonance Rayleigh scattering determination of local anesthetics and flavonoids drugs in human urine. Experiments on a series of experimental parameters are optimized, such as high performance liquid chromatographic separation conditions and resonance Rayleigh scattering testing conditions, and the reaction mechanism of the resonance Rayleigh scattering has been discussed fully. This article is mainly composed of the introduction and text. The introduction briefly summarized the high performance liquid chromatography and resonance Rayleigh scattering, and introduced the high performance liquid chromatography-resonance Rayleigh scattering detection principle, application status and development prospects. The text part includes the following three experimental system.1. High performance liquid chromatography and resonance Rayleigh scattering detection method for determination of three local anesthetics in urine.A highly selective and sensitive method of reversed phase high performance liquid chromatography(RP-HPLC) coupled with resonance Rayleigh scattering(RRS) was developed for determination of procaine, bupivacaine and tetracaine. Separation of three local anaesthetics was achieved at 35 oC on a C18 column. The mobile phase was 30:70(v/v) acetonitrile/ triethylamine-phosphoric acid buffer(pH=2.9) at flow rate of 0.3 mL/min. The RRS detection was conducted by taking the advantage of the strong RRS enhancement of local anaesthetics with erythrosine reaction in acidic medium. Under optimum conditions, the limit of detection(S/N=3) values were in the range of 2.4~11.2 ng/m L. Recoveries from spiked human urine samples were 95.8%~104.5%. The proposed method applied to the determination of local anaesthetics in human urine has achieved satisfactory results. In addition, the mechanism of reaction was fully discussed.2. High performance liquid chromatography and resonance Rayleigh scattering detection methods and mechanism research of three flavonoids.High performance liquid chromatography and resonance Rayleigh scattering method is set up to determine of three flavonoids. This experiment is mainly based on the three flavonoids with ethyl violet can form ion association and the resonance Rayleigh scattering signal significantly enhanced. High performance liquid chromatography realized determination of three flavonoids at the same time. RRS signal detection wavelength is λex = λem = 325 nm. Quantum chemistry calculation, SEM and UV absorption spectroscopy were used to discuss the reaction mechanism. The limit of detection(S/N=3) values were in the range of 5.4 ~ 10.3 ng/m L. Linear range was 0.08~20 μg/m L and the correlation coefficients were all above 0.9974. The results showed that the high performance liquid chromatography-resonance Rayleigh scattering method has a good linear relationship.3. High performance liquid chromatography and resonance Rayleigh scattering detection method for determination of quercetin and genistein in human urine.This experiment was based on quercetin and genistein with crystal violet to form ion association, leading to a resonance Rayleigh scattering signal significant enhancement, thus a good selective and highly sensitive high performance liquid chromatography-resonance Rayleigh scattering was estabished, it was successfully applied to determine quercetin and genistein in urine. High performance liquid chromatography separation parameters and resonance Rayleigh scattering detection conditions are optimized, in order to get high sensitivity and accuracy. Quercetin and genistein detection limit(S/N = 3)were 6.3 ng/m L and 8.6 ng/m L, respectively. Standard addition method was used for determination of quercetin and genistein in human urine, recoveries were 97.1%~103.1%. Therefore, the method could very well determine quercetin and genistein in human urine. |
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