| High performance liquid chromatography and capillary electrophoresis are two effectiveseparation and determination techniques, and the fluorescence detector is one of the mostcommonly used chromatographic detector. Chromatography in conjunction with fluorescencedetection, with its utmost separation efficiency and detetion sensitivity, has been documented asa powerful method for the determination of a variety of medicine compounds, especially forpharmaceutical and bio-analytical applications. The γ-aminobutyric acid analogue, such asbaclofen, gabapentin and GABA, has been widely used for the antiepileptic or symptomaticrelief of muscular spasm. The γ-aminobutyric acid needs to be derivatized because an aqueoussolution of the γ-aminobutyric acid does not show obvious absorption or fluorescence. For thisreason, chemical derivatization becomes a necessary procedure to transform the analytes intoderivatives that can be sensitively detected.In this graduation thesis,6-oxy-(N-succinimidylacetate)-9-(2'-methoxycarbonyl)fluorescein and5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein were chosen as derivatizationreagents, and several original studies were carried out as follows:(1) Two methods for the determination of baclofen(BAL) in urine and plasma based onhigh-performance liquid chromatography (HPLC) with UV-Vis and fluorescent detection,respectively, were developed for using a new synthesized fluorescent label,6-oxy-(N-succinimidylacetate)-9-(2'-methoxycarbonyl) fluorescein(SAMF). The samplewere detected after deriving in borate buffer (pH8.0) for15min at room temperature (25°C). The detection limits are1.065×10-3mg mL-1and1.065×10-2mg mL-1withHPLC-UV-Vis and HPLC-FD, respectively. The proposed method has been successfullyapplied to the analysis of BAL in human urine and plasma samples.(2) Capillary electrophoresis with laser-induced fluorescence detection has been used for theanalysis of baclofen. The fluorescent reagent5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein was used to label the BAL. Based on these established CE-LIF andSPE-CE-LIF method for the determination of baclofen(BAL) in plasma. At40°C, thederivatization reaction was completed within10min in borate buffer (pH9.0). The separation in CE-LIF method was performed with20mmol L–1sodium borate (pH9.5),and GABA as internal label. The separation in SPE-CE-LIF method was performed with40mmol L–1sodium borate (pH9.5), and dichlorofluorescein as internal label. Thedetection limits for BAL were1×10-11mol L–1and5×10-10respectively, and the methodwas successfully applied to monitoring of these BAL in human sample. Recovery of theBAL ranged from91.2to101.3%.(3) Fluorescein isothiocyanate (FITC),5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein(DTAF) and6-oxy-(N-succinimidylacetate)-9-(2'-methoxycarbonyl)fluorescein (SAMF)were evaluated for the ananlysis of GABA analog by micellar electrokineticchromatography (MEKC) with LIF detection. Several derivatization conditions of thethree labeling reagent are discussed. It was founded that the labelling reaction withSAMF was mild and rapid, so SAMF was adopted as the derivatization reagent in thisexperiment. A new SPE-CE-LIF method was established and at λem=473nm, the lowestdetectable analyte concentration ranged from0.4to4nmol L-1and LOD between2×10-11-3×10-11mol L-1. The method was successfully applied to monitoring of thesecompounds in human sample.(4) DTAF was used as pre-column label reagent for the sensitive analysis of chiral baclofenin serum by CE-LIF successfully. DTAF reacted with chiral baclofen rapidly at45°C inborate buffer (pH=9.0) for40min. The derivatives were baseline separated in40mmolL–1borate buffer (pH=9.6) consisted of2mmol L(-1)HP-β-CD within20min. The LODwas6×10-11mol L(-1). The method was successfully applied to monitoring of these chiralbaclofen in human sample and tablet. |
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