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是众所周知的控制和调节许多脑功能的神经递质，并且与一些病理状况如攻击性和掠食行为、偏头痛、抑郁症和类癌综合征密切相关。此外，各种药物如抗抑郁药作用于中枢血清素能系统。测量血清素和/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物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应用于神经递质的体内研究。微透析可以应用于自由运动动物神经递质的连续测量。在本研究中，我们建立了一种基于柱前荧光衍生化的高效液相色谱方法，用于测定大鼠脑微透析液中的胺。该方法是基于在六氰高铁酸钾（III）存在下与苄胺反应的荧光衍生化。将微透析探针植入大鼠脑内，以2.0mul/min的速度连续灌注林格氏液。微透析液每5 min收集一次，加入由CAPS缓冲液（pH 12.0）、苄胺、六氰高铁酸钾（III）和甲醇组成的试剂溶液。在室温下静置5min完成衍生化反应。用反相微孔柱等密度洗脱，可在15 min内分离出胺类化合物的苯胺衍生物。5毫升注射剂的检出限为30-100 amol。研究了林格氏溶液中钾离子浓度升高和单次腹腔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.