in vivo analytical study on the endogenous agonist for cannabinoid receptor by using a microdialysis technique

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This research project was carried out in Graduate School of Pharmaceutical Sciences, University of Tokyo and School of Pharmaceutical Sciences, University of Shizuoka.In 2002, this research was performed in Graduate School of Pharmaceutical Sciences, University of Tokyo. The aim of this research is to determine anandamide (AN) in rat brain microdialysis (MD) sample by using a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection using a fluorescence reagent, DBD-COCl, which can react with primary alcohol of AN. In order to increase recovery of AN from MD probe, β-cyclodextrin derivatives were added in the perfusate of MD. As a result, the addition of 2,3,6-O-trimethyl-β-CD (TriMeCD) afforded to increase the recovery of AN.In 2003-4, this research was performed in School of Pharmaceutical Sciences, University of Shizuoka. Next, extraction conditions for AN determination from rat brain MD sample were investigated. Among them, a mixed solvent of ethyl acetate and toluene (50/50) was effective for the extraction of AN, and further, the addition of TriMeCD gave a larger AN peak. This is resulted form the prevention of undesired AN adsorption to inner surface of the experimental bottle. By using a fluorescence spectrophotometer, the fluorescence intensity of DBD-CO-AN augmented with the increase of CH_3CN percentage in the solvent. Thus, when using a tandem series of an ODS column (250 x 4.6 mm i.d., 3 μm) with a mobile phase containing 95% CH_3CN, the detection sensitivity was approximately 4.5-fold greater than that in the previous HPLC method (detection limit: approximately 6 nM). In the proposed HPLC method, AN in rat brain MD sample was successfully detected after administration of AN to rats. Using the HPLC method developed in this term, the research on the brain AN will be progressed.