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

本研究项目由东京大学药学院和静冈大学药学院共同完成。本研究于2002年在东京大学药学研究生院进行。本研究采用柱切换高效液相色谱法（HPLC）测定大鼠脑微透析（MD）样品中阿南达胺（AN）的含量，荧光试剂DBD-COCl可与阿南达胺的伯醇反应。为了提高MD探针中AN的回收率，在MD的灌注液中加入β-环糊精衍生物，结果表明，2,3,6- o -三甲基-β-CD （TriMeCD）的加入可以提高AN的回收率。本研究于2003- 04年在静冈大学药学院进行。其次，对大鼠脑MD样品中AN的提取条件进行了研究。其中乙酸乙酯与甲苯（50/50）的混合溶剂对AN的提取效果最好，另外，TriMeCD的加入使AN的提取峰更大。这是由于防止了不希望的AN吸附到实验瓶的内表面。用荧光分光光度计测定了DBD-CO-AN的荧光强度随溶剂中CH_3CN含量的增加而增强。因此，当采用ODS柱（250 × 4.6 mm, 3 μm）串联，流动相为95% CH_3CN时，检测灵敏度比之前的HPLC方法（检出限约为6 nM）提高了约4.5倍。采用高效液相色谱法，在给药后成功检测了大鼠脑MD样品中的AN。利用本学期开发的高效液相色谱（HPLC）方法，将进一步推进脑AN的研究。

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.