| Resonance Rayleigh scattering technique is a rapid, simple and sensitive analysis tool that is developed since 1993. The next 20 years, the resonance Rayleigh scattering technology is gradually used in inorganic element analysis, pharmaceutical analysis, biochemical analysis, nano materials, etc. High performance liquid chromatography (HPLC) is with high selectivity and high efficiency, fast separation characteristics and can be used in the physical examination and content determination at the same time, so it has become a powerful tool for drug research. In recent years, since the resonance Rayleigh scattering and the advantages of high performance liquid chromatograph coupled techniques was found as a new kind of technology, it has been successfully applied to protein, amino acid and drug analysis. This paper established a no post-column derivative HPLC-RRS detection reagent, and it use a stable ternary system of HPLC-RRS combined detection technology, this not only improves the stability of the method, but further expand the application range of RRS. In addition, in this paper, to further improved HPLC-RRS combination device on the basis of predecessors’, we use a matching flow pool, controlling reaction length, reaction temperature which is of great significance for improving the sensitivity of the method.This article mainly research application of high performance liquid chromatography (HPLC) and resonance Rayleigh scattering spectrometry determination of alpha-1 receptor blockers, anticancer drugs of mercaptopurine and cephalosporin antibiotics. We use standard addition method to validated the practical application in human urine samples of this three kinds of drugs which has obtained satisfactory results. This paper include the introduction and the research content, the introduction part briefly outlines the resonance Rayleigh scattering technology, high performance liquid chromatography (HPLC) technique, and high performance liquid chromatography (HPLC) coupled with resonance Rayleigh scattering detection technique and its combination device improvement. The research content generally cover the following three aspects:Chapter 1:High performance liquid chromatograph coupled with Resonance Rayleigh scattering detection method for the determination of three α1-adrenoceptor antagonistsHerein, a resonance Rayleigh scattering (RRS) detection method combined with high performance liquid chromatograph (HPLC) without any post-column probe was developed for the separation and determination of three α1-adrenoceptor antagonists. The quantitative analysis is benefiting from RRS signal enhancement upon addition of methanol which induced molecular aggregation to form an hydrophobic interface between aggregates and water that produce a sort of superficial enhanced scattering effect. A good chromatographic separation among the compounds was achieved using a Gemini 5u C18 reversed phase column (250 mm × 4.6 mm; 4 μm) with a mobile phase consisting of methanol and ammonium acetate-formic acid buffer solution (25 mM; pH=3.0) at the flow rate of 0.7 mL min-1. The RRS signal was monitored atλ ex= λem = 354 nm. A limit of detection (LOD) of 0.065-0.70 μg L-1 was reached and a linear range was found between peak height and concentration in the range of 0.75-15 μg L-1 for doxazosin mesyllate (DOX),0.075-3.0 μg L-1 for prazosin hydrochloride (PRH), and 0.25-5 μg L-1 for terazosin hydrochloride (TEH), with linear regression coefficients all above 0.999. Recoveries from spiked urine samples were 88.4-99.0% which is within acceptable limits. The proposed method is convenient, reliable and sensitive which has been used successfully in human urine samples.Chapter 2:A resonance Rayleigh scattering coupled with high performance liquid chromatography method for the estimation of 6-mercaptopurine and mechanism studyA simple, sensitive, and selective resonance Rayleigh scattering hyphenated chromatographic method was established for the estimation of 6-mercaptopurine based on the enhancement of RRS signal which is originate from formation of coordination complex between 6-mercaptopurine and metal palladium (Ⅱ). The separation and detection conditions were optimized. The chromatographic separation was achieved at 30℃ on a reverse phase column of Synergi Hydro-RP (250 mm×4.6mm; 4μm), with (25:75, v/v) methanol-water as the mobile phase and a flow rate of 0.3 mL min-1. The RRS signal was detected by fluorescence detector at λex= λem= 315 nm. The analytical parameters were provided by the hyphenated system, the linear of 6-mercaptopurine response from 0.0615 to 2.40 μg L-1 and the limit of detection (S/N=3) was 0.019 μg L-1.The presented method has been applied to determine 6-mercaptopurine in human urine samples which obtained satisfactory results.The mechanism and binding mode were discussed by infrared spectra, quantum calculation, Scanning electron microscopy and absorption spectroscopy.Chapter 3:Resonance Rayleigh scattering detection coupled with high performance liquid chromatography method for the determination of cephalosporinsA facile, sensitive and selective resonance Rayleigh scattering (RRS) coupled with high performance liquid chromatography (HPLC) method based on the ternary ion-association complex was established in human urine samples for the simultaneously separation and determination of cephalosporins. Owing to poor thermodynamic stability which led to the unstable RRS signal, a more stable ternary system with palladium(II) and 4,5-dibromofluorescein (DBF) as post-column probes was presented in this study. Four analytes of Ceftazidime(CZD), ceftriaxone(CTRX), cefoperazone(CPZ) and cefotaxime (CFTM) were separated by isocratic elution with the mobile phase of 30 mM phosphate buffer(pH= 3.5) and MeOH. The RRS signals were detected by a common fluorescence detector at λex= λem= 295 nm. With this new strategy, continuous mobile analysis of the compounds was successfully performed. Good linearities (r2> 0.9990) for all calibration curves were obtained and the detection limits at a signal-to-noise ratio of 3 for CZD, CTRX, CPZ and CFTM were 0.694,0.685,2.81 and 0.699 ng mL-1, respectively. Recoveries from spiked urine and plasma samples were between 97.4% and 101.3%. The applicability of the proposed method has been validated in human urine and plasma samples with satisfactory results. |
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