Development of automatic system for the determination of biogenic amines in brain by microdialysis-HPLC with fluorescence detection

生物胺，如5-羟色胺和去甲肾上腺素，被认为是控制和调节许多大脑功能的神经递质，并与一些病理情况密切相关，如攻击性和捕食性行为、偏头痛、抑郁症和类癌综合征。此外，各种药物，如抗降压药，作用于中枢5-羟色胺能系统。测定5-羟色胺和/或去甲肾上腺素水平随时间的变化对于研究胺水平与各种药理活性之间的关系是非常重要的。脑微透析取样技术已成功地引入到神经递质的活体研究中。微透析法可用于连续测定自由活动动物的神经递质。本研究建立了一种基于苯甲胺柱前荧光衍生化的微孔高效液相色谱测定大鼠脑微透析液中胺类物质的方法。该方法基于六氰基铁酸钾(III)存在下胺与苯甲胺反应的荧光衍生化反应。将微透析探头植入大鼠脑内，以2.0微升/分钟的速度持续灌流林格液。每隔5min收集一次微透析液，加入由CAPS缓冲液(pH 12.0)、苯甲胺、六氰基铁酸钾(III)和甲醇组成的试剂溶液。在室温下放置5min即可完成衍生化反应。用等梯度洗脱的反相微孔柱可在15min内分离这些胺类的苄胺类化合物。5微升进样的检出限为30-100毫摩尔。考察了林格液中钾离子浓度的增加和一次注射甲基苯丙胺对微透析液中胺水平的影响。

Biogenic amines such as serotonin and norepinephrine are well known as neurotransmitters in the control and regulation of many brain functions, and have been strongly implicated in several pathological conditions such as aggressive and predatory behavior, migraine, depression and carcinoid syndrome. Moreover, various drugs such as antigepressants act on the central serotonergic syustem. It is very importnt to measure time-dependent changes of serotonin and/or norepinephrine levels for the investigations of the relationships between the amine levels and various pharmacentical activities. The brain microdialysis sampling technique has been successfully introduced into in vivo studies of the neurotransmitters. Microdialysis can be applied to the continuous measurement of the neurotransmitters of a freely moving animals.In this study, we have developed a microbore high-performance liquid chromatographic method based on precolumn fluorescence derivatization with benzylamine for the determination of the amines in microdialysate from rat brain. The method is based on the fluorescence derivatization of the amines by reaction with benzylamine in the presence of potassium hexacyanoferrate (III). A microdialysis probe was implanted in the rat brain, and continuously perfused at 2.0mul/min with Ringer solution. The microdialysate, collected every 5 min, was added with a reagent solution composed of CAPS buffer (pH 12.0), benzylamine, potassium hexacyanoferrate (III) and methanol. The derivatization reaction was completed by standing at room temperature for 5 min. The benzylamine derivatives of the amines could be separated within 15 minon a reverserd-phase microbore column with isocratic elution. The detection limits of the amines are 30-100 amol for a 5mul injection. The effects of increased potassium ion concentration in the Ringer solution and a single intraperitoneal injection of methanphetamine on the amine levels in microdialysate were examined.