| Resonance Rayleigh scattering (RRS) is a newly-developed analytical method. Nowadays, due to its sensitivity, simplicity and speediness, most of RRS techniques have been applied to the research of biologic molecules such as protein, nucleic acid and the determination of trace amounts of inorganic ions. Recently, RRS has been successfully incorporated with flow injection analysis (FIA) and capillary electrophoresis (CE) method. The incorporation methods exert RRS advantages of high sensitivity and high selectivity while avoiding its bad reproducibility and poor stability.High performance liquid chromatography (HPLC) is the most commonly used method for the quantitative analysis. RRS can be also coupled with HPLC as a new detection technique, and which will play an important role in the development of HPLC for the improvement of sensitivity in the determination of trace components. However, the advantages of HPLC coupled with RRS (HPLC-RRS) have not been attached enough importance to, and which will be unfavorable to expanding the application of HPLC-RRS. In this assay, influencing factors about the spectra and HPLC chromatograms are discussed in details and particularly researching process is also presented. There are three parts in this assay as follows:1. Development of a non-derivatization quantitative method by coupling a reversed-phase high performance liquid chromatography with resonance Rayleigh scattering for the determination of Amikacin in different pharmaceutical formulations. The detection principle is based on the enhancement of RRS intensity of ion-association complex formed from AMK and PSB used as a molecular recognition probe. The HPLC coupled with RRS detection scheme was implemented post-column by probe solution with the column eluent prior to detection. The RRS signal was detected by fluorescence detector atλex=λem=362nm. In this assay, a good separation was achieved on an Agilent Zorbox SB-C18 column (250mm×4.6mm i.d., 5μm) with a guard column (Security Guard C18, 4×2mm, Phenomenex) with the mobile phase consisting of acetonitrile-water (7:93, v/v) at 0.50mL·min-1. The assay quantifies over a linear range of 1.0μg·mL?1 to 15.0μg·mL?1 (R2=0.9993) with a minimum detectable limit of 0.5μg·mL-1 (S/N=3). The recovery of extraction was 96.5%~101.0%, and the intra-assay RSD (same-day) and the inter-assay RSD (different day) were lower than 2.44% and 4.58%, respectively. The method has been applied successfully to the determination of AMK in different pharmaceutical formulations with satisfactory results.2. Development of a non-derivatization quantitative method by coupling a reversed-phase high performance liquid chromatography with resonance Rayleigh scattering for simultaneous determination of three aminoglycoside antibiotics in mixture samples. Three aminoglycoside antibiotics (AGs) as targets in mixture samples, including tobramycin, etimicin and netilmicin, are be simultaneously separated and determined by HPLC-RRS, which can greatly enhance the selectivity of classic RRS assays. In this assay, a good separation was achieved on an Agilent Zorbox SB-C18 column (250mm×4.6mm i.d., 5μm) with a guard column (Security Guard C18, 4×2mm, Phenomenex). The selected optimized mobile phase was methanol-water (5:95, v/v), containing 0.2% trifluoroacetic acid with the flow rate of 0.5mL·min-1. The probe solution was PSB with the concentration of 2.0×10?5mol·L?1 diluted by BR buffer solution at the flow rate of 0.2mL·min-1. The effluents were monitored atλex=λem=362nm by fluorescence detector. In mixture samples of three AGs sulfate pharmaceutical raw materials, the simultaneous separation and determination of tobramycin, etimicin and netilmicin was feasible. The assay quantifies over a linear range of 2.0μg·mL?1 to 20.0μg·mL?1 of AGs, and the mean recoveries of AGs from spiked samples ranged from 97.7% to 108.4% (RSD%≤4.81%, n=5). Further, the developed method was applied successfully to the determination of tobramycin, etimicin and netilmicin in pharmaceutical formulations and the mixture analyte with satisfactory separation and high repeatability.3. A reversed-phase high performance liquid chromatography coupled with resonance Rayleigh scattering detection for the determination of Mitoxantrone in healthy human plasma and urine. The detection principle is based on the enhancement of RRS intensity of ion-association complex formed from MXT and ammonium molybdate used as a molecular recognition probe. The RRS signal was detected by fluorescence detector atλex=λem=365nm. In this assay, a good separation was achieved on an Agilent Zorbox SB-C18 column (250mm×4.6mm i.d., 5μm) with a guard column (Security Guard C18, 4×2mm, Phenomenex) with the mobile phase consisting of acetonitrile-water (22:78, v/v) at 0.50mL·min-1. The limit of detection (S/N=3) for MXT was 0.6μg·mL-1. A calibration curve ranged from 1.0μg·mL?1 to 15.0μg·mL?1 was showed to be linear. The presented method was applied for the determination of MXT spiked in blank human plasma and urine samples. |
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