| Two parts, including literature review and research report, were described in this paper.The first part was literature review.In the review, the fluorescence derivative method was addressed from three aspects. Firstly, the main derivative methods, such as chemical reaction method, electrochemical reaction method and photochemical reaction method, were introduced. The mechanism and characteristic of each method was described with some real examples. Secondly, the latest literatures were referenced to summarize the applications of fluorescence derivative methods, included the applications in the analysis of metal ions and the applications in the analysis of drugs and organic compounds. In the last part, the status quo and the tendency of the fluorescence derivative method was discussed.The second part was research reports. It included four aspects.In the first section, a novel fluorimetric model of on-line electrochemical oxidation for the determination of methotrexate is described in this paper. The method was based on the oxidation of methotrexate into highly fluorescence product 2, 4-diaminopteridine -6-carboxylic acid by constant potential in the electrochemical oxidation flow cell. The stopped-flow techniques were employed. Under the optimal conditions, the fluorescence intensity of 2, 4-diaminopteridine-6-carboxylic acid was measured at excitation and emission wavelengths of 380 and 465nm, respectively. The calibration graph was linear over the concentration of methotrexate in the range of 2.0×10-7～1.0×10-5g/mL with a detection limit of 5.2×10-8g/mL(3σ). The method has been successfully applied to the determination of methotrexate in human urine samples and showed a％recovery ranging from 94.3 to 102.5％.In the second section, the fluorimetric method of on-line electrochemical oxidation was employed for the determination of folic acid. The method was based on the oxidation of folic acid into highly fluorescence product pteridine-6-carboxylic acid by constant potential in the electrochemical oxidation flow cell. Under the optimal conditions, the fluorescence intensity of pteridine-6-carboxylic acid was measured at excitation and emission wavelengths of 370 and 460nm, respectively. The calibration graph was linear over the concentration of folic aicd in the range of 1.0×10-7～1.0×10-5g/mL with a detection limit of 4.2×10-8g/mL(3σ). The method has been successfully applied to the determination of folic acid.A molecularly imprinted polymers solid phase extraction combined with flow injection fluorimetry for the determination of the fourth generation quinolone antimicrobial agent pazufloxacin mesilate was reported in the third section. Pazufloxacin mesilate was on-line enriched in the MISPE microcolumn and eluted to the flow cell for the fluorimetry determination by the CH3CN-HAc(95:5, v/v). The conditions of enrichment, elution and determination were carefully studied. Under the selected experimental conditions, the calibration graph of the fluorescence intensity versus pazufloxacin concentration was linear from 5×10-8g/mL to 4×10-6g/mL and the detection limit was 8.6×10-9g/mL(3σ). The relative standard deviation was 3.72％(n=7) for 1×10-7g/mL pazufloxacin. The experiments showed that the selectivity and sensitivity of fluorimetry could be greatly improved by the proposed method. It has been successfully applied to the determination of pazufloxacin mesilate in human serum and urine.The last section described the synthesis and evaluation of molecularly imprinted polymers as selective solid-phase extraction sorbent, coupled to electrochemical oxidation fluorimetry detection, for the efficient preconcentration and determination of methotrexate in serum and urine. Methotrexate was preconcentrated in the molecularly imprinted solid phase extraction microcolumn packed with molecularly imprinted polymers, and then eluted. The eluate was detected by fluorescence spectrophotometer after electrochemical oxidation. The conditions of preconcentration, elution, electrochemical oxidation and determination were carefully studied. Under the selected experimental conditions, the calibration graph of the fluorescence intensity versus methotrexate concentration was linear from 4×10-9g/mL to 5×10-7g/mL, and the detection limit was 8.2×10-10g/mL(3σ). The relative standard deviation was 3.92％(n=7) for 1×10-7g/mL methotrexate. The experiments showed that the selectivity and sensitivity of fluorimetry could be greatly improved by the proposed method.
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