A Simple and Sensitive High-Performance Liquid Chromatography–Electrochemical Detection Assay for the Quantitative Determination of Monoamines and Respective Metabolites in Six Discrete Brain Regions of Mice
A rapid, sensitive, and reproducible assay is described for the quantitative determination of the monoamine neurotransmitters dopamine, norepinephrine and serotonin, their metabolites, and the internal standard 3,4-dihydroxybenzlyamine hydro-bromide in mouse brain homogenate using high-performance liquid chromatography with electrochemical detection. The method was validated in the following brain areas: frontal cortex, striatum, nucleus accumbens, hippocampus, substantia nigra pars compacta and ventral tegmental area. Biogenic amines and relevant metabolites were extracted from discrete brain regions using a simple protein precipitation procedure, and the chromatography was achieved using a C18 column. The method was accurate over the linear range of 0.300–30 ng/mL (r = 0.999) for dopamine and 0.300–15 ng/mL (r = 0.999) for norepinephrine, 3,4-dihydroxybenzlyamine hydro-bromide, homovanillic acid and 5-hydroxyindolacetic acid, with detection limits of ~0.125 ng/mL (5 pg on column) for each of these analytes. Accuracy and linearity for serotonin were observed throughout the concentration range of 0.625–30 ng/mL (r = 0.998) with an analytical detection limit of ~0.300 ng/mL (12 pg on column). Relative recoveries for all analytes were approximately ≥90% and the analytical run time was <10 min. The described method utilized minimal sample preparation procedures and was optimized to provide the sensitivity limits required for simultaneous monoamine and metabolite analysis in small, discrete brain tissue samples.
Allen, Serena A.; Rednour, Stephanie; Shepard, Samantha; and Pond, Brooks Barnes. 2017. A Simple and Sensitive High-Performance Liquid Chromatography–Electrochemical Detection Assay for the Quantitative Determination of Monoamines and Respective Metabolites in Six Discrete Brain Regions of Mice. Biomedical Chromatography. Vol.31(11). https://doi.org/10.1002/bmc.3998 PMID: 28474759 ISSN: 0269-3879