engleski

Development of HPLC method for serotonin, dopamine and noradrenaline determination in rat brain tissue samples

Research of neurotransmitter levels in animal models for brain disorders, such as rat, is important for better understanding of different pathological processes. Serotonin, dopamine and noradrenaline are one of the most frequently analyzed neurotransmitters. Affordable analytical method which can be used for this purpose is high-performance liquid chromatography (HPLC). Aim of this study was to develop and validate a rapid HPLC method with UV detection for serotonin, dopamine and noradrenaline determination in rat brain tissue. Ten samples of prefrontal cortex of 22 days old Wistar rats were collected and homogenized in deproteinization solution, centrifuged and diluted for a run trough HPLC machine. Serotonin, dopamine and noradrenaline standards were initially used for spiking for identification of peaks in brain samples and later for making daily calibration curves. Reverse phase HPLC method was used, with C18 column as stationary phase and mix of formic acid and acetonitrile as a mobile phase. Gradient elution was optimized for flow (0.5-0.8 ml/min), duration (5-25 min), injection volume (10-50 µL) and organic phase percentage (0.5-20 %). Peaks were identified by UV detector at 280 nm wavelength. Best results were achieved with a flow of 0.7 ml/min, duration of 21 min, injection volume of 20 µL and gradient elution of the organic phase with highest peak of 12.5 % in 10-15th minute of elution. The method showed good selectivity for all three neurotransmitters. Linearity and repeatability were good for serotonin, while they were not completely satisfactory for dopamine and noradrenaline, pressumably due to their oxidative degradation. For the further development of the method, it is necessary to examine the quality of the sample preparation and the influence of the pH of the mobile phase on the separation of these compounds.

UV-HPLC, epinephrine, 5HT, catecholamine, neurotransmitters

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