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

这项研究是在东京大学药学研究科和静冈大学药学研究室进行的。2002年，这项研究在东京大学药学研究科进行。本研究的目的是用柱切换高效液相色谱结合荧光试剂DBD-CoCl2对大鼠脑微透析(MD)样品中的AN进行检测。为了提高MD探针中AN的回收率，在MD的灌流液中加入β-环糊精衍生物。因此，添加2，3，6-O-三甲基-β-CD(TriMeCD)可以提高AN的回收率。2003-2004年，本研究在静冈大学药学院进行。其次，对从大鼠脑MD样品中提取测定的条件进行了研究。其中，乙酸乙酯/甲苯(50/50)混合溶剂对硝酸铵的萃取效果较好，加入三乙醇胺后，硝酸铵的萃取峰较大。这是由于防止了不需要的吸附到实验瓶的内表面造成的。用荧光分光光度计测得DBD-CO-AN的荧光强度随溶剂中CH3CN含量的增加而增强。因此，当使用串联的ODS柱(250×4.6 mm内径，3μm)，流动相中含有95%的CH_3CN时，检测灵敏度大约是以前的高效液相色谱方法的4.5倍(检测下限：大约6 nm)。在所建立的高效液相色谱方法中，大鼠给药后，成功地检测到了大鼠脑MD样本。利用本学期开发的高效液相色谱方法，将对脑AN的研究取得进展。

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.