[Determination of 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline, 5-hydroxytryptamine and 5-hydroxyindole acetic acid in the neonatal rat brains using high performance iquid chromatography-electrochemical detection]

Abstract

A simple method was developed for the analysis of 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline (6-OH-MTHbetaC), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat brain by high performance liquid chromatography with electrochemical detection (HPLC-ECD). The separation of the sample was performed on a Discovery HS F5 column (250 mm x 4.6 mm, 5 microm) with a mobile phase of the buffer (40 mmol/L citric acid + 20 mmol/L Na2HPO4 + 0.3 mmol/L EDTA2Na, pH 4.0)-methanol (78:22, v/v) at a flow rate of 1.0 mL/min. The electrochemical detector was Coularray Detector-4. This method showed good linearity (r > 0.9992) for the quantification of 6-OH-MTHbetaC, 5-HT and 5-HIAA in the concentration range of 1.0-500.0 microg/L. The limits of detection were 0.56, 0.26 and 0.53 microg/L, respectively. The recoveries of 6-OH-MTHbetaC, 5-HT and 5-HIAA spiked in rat brain samples were 87.1%-98.2%, 87.0%-95.3%, 90.1%-97.7%, respectively, and the relative standard deviations of intra-day and inter-day determinations were both less than 6.1%. In comparison with the control, the analysis of alcohol-exposed neonatal rat brain samples revealed a significant difference in the level of 6-OH-MTHbetaC (P < 0.05). The method was proved to be simple, highly sensitive, and could be applied in the analysis of 6-OH-MTHbetaC, 5-HT and 5-HIAA in the rat brain.