Serotonin monitoring in microdialysate from rat brain by microbore-liquid chromatography with fluorescence detection

doi:10.1016/S0003-2670(97)00616-8

Analytica Chimica Acta

Volume 365, Issues 1–3, 5 June 1998, Pages 227-232

https://doi.org/10.1016/S0003-2670(97)00616-8 Get rights and content

Abstract

A sensitive fluorimetric microbore-liquid chromatographic method for the determination of serotonin in microdialysate from rat brain was developed. The method is based on the fluorescence derivatization of serotonin by reaction with benzylamine in the presence of potassium hexacyanoferrate(III). A microdialysis probe was implanted in the rat brain, and continuously perfused at 2.0 μl min⁻¹ with Ringer solution. The microdialysate, collected every 5 min, was added with a reagent solution composed of 0.3 M CAPS buffer (pH 12.0), 0.2 M benzylamine, 0.1 M potassium hexacyanoferrate(III) and methanol (1:1:5:10, v/v). The derivatization reaction was completed by standing at room temperature for 2 min. The benzylamine derivative of serotonin could be separated within 10 min on a reversed-phase microbore column (100×1.0 mm i.d., 5 μm TSK gel ODS-80TM) with isocratic elution. The detection limit (signal-to-noise ratio=3) of serotonin is 80 amol for a 5 μl injection. The effects of increased potassium ion concentration in the Ringer solution and a single intraperitoneal injection of methamphetamine on the serotonin level in microdialysate were examined.

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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Serotonin monitoring in microdialysate from rat brain by microbore-liquid chromatography with fluorescence detection

Abstract

Introduction

Section snippets

Chemicals and solutions

LC conditions

Conclusions

Acknowledgements

J. Chromatogr.

J. Chromatogr.

Life Sci.

Brain Res.

J. Chromatogr.

Eur. J. Pharmacol.

J. Chromatogr.

J. Chromatogr.

Biomed. Chromatogr.