| In the thesis, CE was combined with on-line sweeping and hollow fiber-basedliquid-phase microextraction technique. Efficient and sensitive methods were developedfor the determination of Strychnos alkaloids, carbamazepine in Chinese herbal medicinesand biological samples. The interactions of Strychnos alkaloids, fangchinoline,tetrandrine and carbamazepine with serum albumin were studied by fluorescencequenching spectra, synchronous fluorescence spectra, three-dimensional fluorescencespectra and ultra-violet spectra. This thesis is mainly concerned with the followingaspects:(1) The investigation of an on-line sweeping preconcentration method in micellarelectrokinetic chromatography (MEKC) for the determination of Strychnos alkaloids,namely strychnine and brucine.After experimental optimizations, the best separation was achieved in 50 mmol L-1H3PO4 (pH 2.0) containing 100 mmol L-1 SDS and acetonitrile in a ratio of 4:1 (v/v),with an applied voltage of -20 kV at 20℃. The sample matrix consisted of 100 mmol L-1H3PO4 (pH 2.0), and sample introduction was performed at 0.5 psi for 270 s, withphotodiode array detection at 203 nm. Compared with the conventional MEKC injectionmethod, up to 100-fold improvement in concentration sensitivity was achieved in termsof peak height by using this sweeping injection technique. In the method, the compoundberberine was used as the internal standard for the improvement of the experimentalreproducibility. The calibration curve was linear over a range of 0.5-15μg mL-1 for bothstrychnine and brucine, with a correlation coefficient of 0.998 and 0.997, respectively.The detection limits (S/N=3:1) for strychnine and brucine were 0.05μg mL-1 and 0.07μg mL-1, respectively. The Sweeping-MEKC method has been successfully applied to theanalysis of strychnine and brucine in Strychnos nux-vomica L and its Chinese medicinalpreparations.(2) The establishment of a new method for the enrichment of Strychnos alkaloids inbiological samples via liquid-phase mieroextraction (LPME) based on porous polypropylene hollow fibers combined with on-line sweeping in micellar electrokineticchromatography (MEKC).Strychnos alkaloids were first extracted from urine sample, in which the concentrationof NaOH was 0.5 mmol L-1. The unionized analytes were subsequently extracted into1-octanol impregnated in the pores of hollow fibers, and then into 100 mmol L-1 H3PO4inside the hollow fiber. The extract was analyzed directly by on-line sweeping in micellarelectrokinetic chromatography (MEKC). In the method, the compound berberine wasused as the internal standard for the improvement of the experimental reproducibility.The calibration curve was linear over a range of 20-200 ng mL-1 for both strychnine andbrucine in human urine sample, with a correlation coefficient of 0.996 and 0.997,respectively. The detection limits (S/N=3:1) for strychnine and brucine were 1 ng ml-1and 2 ng mL-1, respectively. The LPME-sweeping method has been successfully appliedto the analysis of strychnine and brucine in real urine sample, indicating thatLPME-sweeping-MEKC is a promising combination for the analysis of basic drugspresent at low levels in some biological matrices.(3) The development of a sensitive and simple micellar electrokinetic chromatography(MEKC) method for the determination of antiepileptic drug, Carbamazepine(CBZ), usingsweeping on-line concentration method with photodiode array detection.The effect of pH, concentration of the running buffer solution, organic modifier,voltage and injection time on the concentration efficiency was investigated. An untreatedfused-silica capillary was used (50 cm; effective length, 40 cm, 75μm i.d.) for theanalysis. The background solution (BGS) was 50 mmol L-1 NaH2PO4 (pH 3.0) and 100mmol L-1 SDS containing 20％acetonitrile with an applied voltage of -20 kV at 25℃.Sample introduction was performed at 0.5 psi for 90 s with diode array detection at 214nm. In the method validation, the calibration curve was linear over a range of 0.5-40μgmL-1 for CBZ with a correlation coefficient of 0.998. The detection limit (S/N=3:1) ofCBZ was 0.10μg mL-1. About 100-fold improvement in concentration sensitivity wasachieved in terms of peak height by the sweeping method compared to conventionalinjection method. The Sweeping-MEKC method has been successfully applied to theanalysis of CBZ in tablet and human serum.(4) The development of a simple capillary zone electrophoresis method for thedetermination of levodopa and methyldopa in human serum.The influence of pH, concentration of the running buffer solution, voltage andinjection time was investigated. The best separation was obtained with a fused-silicacapillary column(50 cm×75μm I. D.) in a running buffer of 40 mmol L-1 sodiumtetraborate (pH=9.5), with an applied voltage of 22 kV at 25℃. Sample introductionwas performed at 0.5 psi for 7 s combined with diode array detection at 200 nm. Thelinear range of methyldopa and levodopa were 0.001～0.064 mg mL-1 (r=0.9998) and 0.001～0.071 mg/mL (r=0.9994) respectively, the detection limit (S/N=3:1) ofmethyldopa and levodopa were 0.6μg mL-1 and 0.8μg mL-1.(5) The study on the interaction between brucine and bovine serum albumin (BSA)using fluorescence spectroscopy (FS) and ultraviolet spectroscopy (UV).The experimental results showed that brucine could quench the intrinsic fluorescenceof BSA. It was found that static quenching and non-radiation energy transfer were themain reasons leading to the fluorescence quenching. The apparent binding constants (KA)between brucine and BSA were 6.3×103 (27℃) and 7.7×103 (37℃) and the bindingsites (n) were 0.94 (27℃) and 0.97 (37℃). According to the F(?)rster theory ofnon-radiation energy transfer, the binding distances (r) were also obtained. The processof binding was a spontaneous molecular interaction in which entropy increased andGibbs free energy decreased, which indicated that the interaction between brucine andBSA was driven mainly by hydrophobic force.(6) The study on the interaction of fangchinoline and tetrandrine with bovine serumalbumin and human serum albumin (HSA) under different temperatures by fluorescencequenching spectra, synchronous fluorescence spectra, three-dimensional fluorescencespectra and ultra-violet spectra.It was shown that fangchinoline and tetrandrine had a quite strong ability to quenchthe intrinsic fluorescence of BSA or HSA. The Stem-Volmer curve on the fluorescence ofBSA or HSA quenched by fangchinoline and tetrandrine indicated the quenchingmechanism between fangchinoline or tetrandrine and BSA or HSA was a staticquenching. According to the F(?)rster theory of non-radiation energy transfer, the bindingdistances (r), the binding constants (KA) and the binding sites (n) were obtained. Forfangchinoline and BSA: KA=1.05×105, n=1.4, r=2.53 (t=27℃); KA=3.31×105,n=1.3, r=2.70(t=37℃); KA=7.24×105, n=1.2, r=2.92 (t=47℃)。Forfangchinoline and HSA: KA=5.04×105, n=1.2, r=3.52 (t=27℃); KA=5.38×105,n=1.3, r=3.81 (t=37℃); KA=5.67×105, n=1.4, r=4.38 (t=47℃)。For tetrandrineandBSA:KA=1.52×105, n=1.2, r=2.34(t=27℃); KA=2.03×105, n=1.3, r=2.48 (t=37℃); KA=2.89×105, n=1.4, r=2.71 (t=47℃)。For tetrandrine and HSA:KA=4.02×105, n=1.0, r=3.00(t=27℃); KA=4.76×105, n=1.2, r=3.14(t=37℃); KA=4.98×105, n=1.3, r=3.39(t=47℃)。The thermodynamic parametersshowed that the interaction between fangchinoline or tetrandrine and BSA or HSA wasdriven mainly by hydrophobic force. Synchronous spectrum and three- dimensionalfluorescence spectrum were used to investigate the structural changes of BSA and HSA.(7) The investigation of interaction between quercetin and bovine serum albumin usingfluorescence spectroscopy and ultraviolet spectroscopy.The apparent binding constants (KA) between quercetin and BSA were 2.8×108 (26℃)and 3.1×108 (36℃) and the binding sites (n) were 1.7±0.02. According to the F(?)rster theory of non- radiation energy transfer, the binding distances (r) were also obtained. Theexperimental results showed that the quercetin could quenching the intrinsic fluorescenceof BSA by forming the quercetin-BSA complex. It was found that both static quenchingand non-radiation energy transfer were the main reasons leading to the fluorescencequenching. The process of binding was a spontaneous molecular interaction in whichentropy increased while Gibbs free energy decreased, which indicated that the interactionbetween quercetin and BSA was driven mainly by hydrophobic force.(8) The investigation of the interaction between bovine serum albumin and CBZusing fluorescence spectroscopy and ultraviolet spectroscopy.The experimental results showed that the CBZ could insert into the BSA molecular,quenching the intrinsic fluorescence of BSA by forming the CBZ-BSA complex. It wasfound that both static quenching and non-radiation energy transfer were the main reasonsleading to the fluorescence quenching. The apparent binding constants (KA) betweenCBZ and BSA were determined to 1.8×104 (27℃) and 2.8×104 (37℃). The bindingsites values (n) were 0.97 (27℃) and 1.01 (37℃), respectively. According to the F(?)rstertheory of non-radiation energy transfer, the binding distances (r) between BSA and CBZwere 3.6 nm (27℃) and 3.4 nm (37℃) respectively. The process of the binding was amolecular interaction in which entropy and Gibbs free energy both decreased, whichindicated that the interaction between CBZ and BSA was mainly driven by hydrogenbond and Vander Waals force. |
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