| The coupling of high performance liquid chromatography (HPLC) with electrochemical detection (ECD) is an interesting research area in the analysis of complex sample. HPLC-ECD integrates both merits of high efficiency in HPLC and high sensitivity in ECD. In this thesis, a few new methods were developed for the analysis of several kinds of traditional Chinese medicines and biological samples by HPLC-ECD and HPLC with mass spectrometry (MS) detection. This work has demonstrated that HPLC-ECD can be well used in the analysis of biological and Chinese medicinal samples with excellent separation efficiency and high detection sensitivity and selectivity. This work includes following parts.1. An effective ultrasonic extraction method was used to extract two alkaloids of tetrandrine and fangchinoline from Stephania tetrandra S. Moore. The LC-ECD method has been developed for simultaneous determination of two alkaloids. Because of the good accuracy and selectivity, the developed LC-ECD method is suitable for quality control of commercial medicines containing TEN and FAN. The LC-ECD method could be used as an alternative technique to LC-UV in the simultaneous determination of TEN and FAN. The linear responses to TEN and FAN concentrations in the ranging of1.3×10-6～1.07×10-4mol/L and1.4×106～1.09×10-4mol/L were observed with good correlation coefficient. The detection limits were2.6×10-7mol/L and2.7×10-7mol/L, respectively, which were one order of magnitude lower than LC-UV method. The method has been applied to the analysis of Stephania tetrandra S with the average contents of TEN and FAN0.78%and0.40%, respectively. The recoveries of this LC-ECD method were distributed from95%to105%. The specificity and accuracy of the proposed LC-ECD method has been confirmed with LC-ESI-MS.2. A stable and sensitive LC-ECD method has been developed for simultaneous determination of four isoquinoline alkaloids. Traditional Chinese medicine Rhizoma Coptidis has been studied by this LC-ECD method. The average concentrations of jatrorrhizine, coptisine, palmatine, and berberine in Rhizoma Coptidis were0.74%,1.5%,0.51%and4.2%, respectively. Meanwhile, the proposed LC-ECD method has been applied to the analysis of rat plasma after oral administration of Rhizoma Coptidis extracts. The detection limits of four isoquinoline alkaloids were1.0×10-8mol/L and3.0×10-8mol/L, which were80times lower than LC-UV method. The flow injection analysis was used to evaluate the accuracy of of electrochemical detector and the RSDs of the peak area of berberine was tested as1.68%. There is no significant difference between this electrochemical detector and UV detector.3. A novel method was developed for the simultaneous determination of kynurenine (Kyn) and tryptophan (Trp) by HPLC-ECD with a multi-wall carbon nanotube (MWCNT)-modified glassy carbon electrode. The linear responses over Kyn and Trp concentrations in the ranging of1.0×10-6～8.0×10-5mol/L and8.0×10-7～1.6×10-4mol/L were observed with good correlation coefficient. The detection limits were5.0×10-7mol/L and4.0×10-7mol/L, respectively, which were3-5times lower than using the bare electrode detection. The Kyn and Trp concentrations in human plasma samples were determined as1.4×10-6mol/L and4.78×10-5mol/L, respectively. The analytical method for human plasma samples was validated and comfirmed by LC-UV and LC-MS. The recoveries were in the range of84.8-110%, and the precision was lower than5.9%.4. A high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method was developed for the separation of small molecular peptides with same amino acid composition but different sequences. Two tripeptides of Gly-Ser-Phe and Gly-Phe-Ser were used as a model sample. The separation behavior has been investigated and the separation conditions have been optimized. Under the optimum conditions, good repeatability was achieved. The developed method could provide a helpful reference for the separation of other peptides with same amino acid composition but different sequences in the study of proteomics and peptidomics. Meanwhile, this method could provide a powerful tool for the purity assay and quality monitoring of chemical synthesis of peptides.
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