Serotonin monitoring in microdialysate from rat brain by microbore-liquid chromatography with fluorescence detection
Introduction
Serotonin is well known as a neurotransmitter in the control and regulation of many brain functions, and has been strongly implicated in several pathological conditions such as aggressive and predatory behavior [1], migraine [2], depression [3]and carcinoid syndrome [4]. Moreover, various drugs such as antidepressants act on the central serotonergic system. It is very important to measure time-dependent changes of serotonin levels for the investigations of the relationships between serotonin levels and various pharmaceutical activities. The brain microdialysis sampling technique has been successfully introduced into in vivo studies of the neurotransmitters 5, 6, 7. Microdialysis can be applied to the continuous measurement of the neurotransmitters of a freely moving animal.
Liquid chromatography (LC) with electrochemical (EC) 8, 9, 10or fluorimetric 11, 12detection is usually used for the determination of serotonin in a microdialysate. Neither EC detection based on the oxidation reaction of a phenolic group nor fluorescence detection based on native fluorescence are sensitive for serotonin and related compounds, and microdialysates collected for 20–30 min were needed for the measurement of serotonin 8, 9, 10, 11, 12. Furthermore, those methods are not selective for these compounds. A sensitive and selective detection method which can monitor serotonin level changes in a short-time is required for microdialysis.
In previous work 13, 14, we have found that aromatic methylamines such as benzylamine react highly selectively and sensitively with 5-hydroxyindoles and catecholamines in weakly alkaline media in the presence of potassium hexacyanoferrate(III) to produce highly fluorescent benzoxazole derivatives. We have reported a sensitive and selective fluorimetric LC method with pre- [14]and post-column [15]derivatization using benzylamine for the determination of serotonin and related compounds in plasma and urine with simple pre-treatment. In this study, we have developed a microbore LC method based on pre-column fluorescence derivatization with benzylamine for the determination of serotonin in dialysates collected in a short time (2–5 min). The method was applied to serotonin monitoring using high potassium Ringer solution and after intraperitoneal injection of methamphetamine.
Section snippets
Chemicals and solutions
De-ionized and distilled water, purified with a Milli-Q II (Millipore, Milford, MA) system, was used for all aqueous solutions. Serotonin and its related compounds [5-hydroxytryptophan (5HTP) and 5-hydroxyindole-3-acetic acid (5HIAA)] were purchased from Sigma (St. Louis, MO). Benzylamine hydrochloride was purchased from Tokyo Kasei Kogyo (Tokyo) and was used after purification by recrystallization from absolute ethanol. Methamphetamine was obtained from Dainippon Seiyaku (Tokyo). N
LC conditions
The concentrations of neurotransmitters such as serotonin in the brain change in a short period by the effect of various stimulae and pharmaceuticals. Therefore, it is important to shorten the separation time in LC for measurement of serotonin in dialysates. Fig. 1(a) and (b) show typical chromatograms obtained with a standard solution of serotonin using mobile phases A and B, respectively. Other 5-hydroxyindoles (5HIAA and 5HTP) and catecholamines (dopamine, epinephrine and norepinephrine)
Conclusions
The proposed microbore LC method coupled with pre-column fluorescence derivatization with benzylamine permits the highly sensitive, selective and quick determination of serotonin and can be applied to the measurement of serotonin in microdialysates without prior sample purification. The method requires an extremely small portion of microdialysate (4–10 μl), and, therefore, should be useful for biological investigation of the brain.
Acknowledgements
The authors are grateful to Dr. M. Nakamura (Faculty of Pharmaceutical Sciences, Fukuoka University) for helpful suggestions. The financial support of the Grant-in-Aid for Scientific Research (No. 08672493) from the Ministry of Education, Science and Culture of Japan is greatly acknowledged.
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