Development and validation of an ultra-high performance liquid chromatography–tandem mass-spectrometry (UHPLC–MS/MS) method for the simultaneous determination of neurotransmitters in rat brain samples

A simple method for the simultaneous determination of glutamate, γ-aminobutyric acid (GABA), choline, acetylcholine, dopamine, 5-hydroxyindole-3-acetic (5-HIAA), serotonin, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was developed by using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). These compounds are analysed in a single chromatographic run in less than 8 min, adding heptafluorobutyric acid (HFBA) in the mobile phase to improve the separation of the selected neurotransmitters. The analytes were detected using electrospray ionization (ESI)-MS/MS in positive mode with multiple reaction monitoring (MRM). Good linearity was obtained (R2 > 0.98) and the intra and inter-day precision of the method (expressed as relative standard deviation) were lower than 26%. Limits of quantification were lower than 2.440 μg/g of brain in all the cases, allowing the sensitive determination of these compounds in rat brain extracts. Therefore, the method was successfully applied for the quantitative determination of neurotransmitters in several rat brain regions (prefrontal cortex, striatum, nucleus accumbens and amygdala), detecting glutamate, GABA and choline at concentrations higher than 1000 μg/g, 30 μg/g and 100 μg/g respectively, whereas the other compounds were found at lower concentrations.

► Simultaneous determination of neurotransmitters in rat brain.
► Development of a fast, simple and reliable chromatographic method.
► Application in routine analysis or in the diagnosis of neural diseases.