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

1997 Fiscal Year Final Research Report Summary

• Project/Area Number: 08672493
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: Fukuoka University
• Principal Investigator: YAMAGUCHI Masatoshi Fukuoka University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (50117280)
• Project Period (FY): 1996 – 1997
• Keywords: bioactive amines / serotonin / norepinephrine / brain / microdialysis / liquid chromatography / benzylamine / fluorescence detection

Research Abstract

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.