Simultaneous determination of multiple neurotransmitters and their metabolites in rat brain homogenates and microdialysates by LC-MS/MS

Abstract

Since neurotransmitters (NTs) and their metabolites are involved in some major neurological disorders, there is a demand for suitable analytical techniques allowing highly sensitive determination of NTs in biological samples. We developed and validated a derivatization method for the simultaneous determination of NTs and their metabolites in rat brain homogenates and microdialysates of the prefrontal cortex and hippocampus using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The NTs and their metabolites were derivatized with dansyl chloride under optimal conditions and separated by an Ultimate XB-C8 column. The method was used for the quantification of monoamine NTs like dopamine (DA), norepinephrine (NE) and serotonin (5-HT), and their metabolites including 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), vanilmandelic acid (VMA), 4-hydroxy-3-methoxyphenylglycol (MHPG), and 5-hydroxyindole-3-acetic acid (5-HIAA). Amino acid neurotransmitters like tryptophan (TRP), kynurenine (KYN), kynurenic acid (KA) and glutamate (GLU), glutamine (GLN) and γ-aminobutyric acid (GABA) were also taken into account. This method exhibited good linearity for all of the analytes with regression coefficients higher than 0.99. The intra-day and inter-day precision were no greater than 16%. The lower limit of quantification (LLOQ) values for NTs ranged from 0.4 to 9.7 pmol mL⁻¹. The mean recovery ranged from 90.3% to 115.0%. The described LC-MS/MS method was successfully applied for quantification of the NTs and their metabolites in the brain homogenates and microdialysates of rats with good precision and accuracy.