Analysis of Glutamate, GABA, Noradrenaline, Dopamine, Serotonin, and Metabolites Using Microbore UHPLC with Electrochemical Detection

Abstract

The applicability of microbore ultrahigh performance liquid chromatography (UHPLC) with electrochemical detection for offline analysis of a number of well-known neurotransmitters in less than 10 μL microdialysis fractions is described. Two methods are presented for the analysis of monoamine or amino acid neurotransmitters, using the same UHPLC instrument. Speed of analysis of noradrenaline (NA), dopamine (DA), serotonin (5-HT), and the metabolites homovanillic acid (HVA), 5-hydroxyindole aceticacid (5-HIAA), and 3,4-dihydroxyphenylacetic acid (DOPAC) was predominated by the retention behavior of NA, the nonideal behavior of matrix components, and the loss in signal of 5-HT. This method was optimized to meet the requirements for detection sensitivity and minimizing the size of collected fractions, which determines temporal resolution in microdialysis. The amino acid neurotransmitters glutamate (Glu) and γ-aminobutyric acid (GABA) were analyzed after an automated derivatization procedure. Under optimized conditions, Glu was resolved from a number of early eluting system peaks, while the total runtime was decreased to 15 min by a 4-fold increase of the flow rate under UHPLC conditions. The detection limit for Glu and GABA was 10 nmol/L (15 fmol in 1.5 μL); the monoamine neurotransmitters had a detection limit between 32 and 83 pmol/L (0.16-0.42 fmol in 5 μL) in standard solutions. Using UHPLC, the analysis times varied from 15 min to less than 2 min depending on the complexity of the samples and the substances to be analyzed.

Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain

“…A frequently used in vivo method is the microdialysis technique, where extracellular fluid is collected continuously through a microdialysis probe inserted into the brain and the resulting fractions are subsequently analysed [4]. Ex vivo procedures involve post-mortem dissection of the animal's brain into specific sub-regions followed by analysis of the homogenized brain tissue [5].…”

“…For detection of the monoamines and their biogenic metabolites, three different detection principles are mostly applied: electrochemistry [4,[7][8][9][10][11], fluorescence [12,13] or mass spectrometry [14,15]. An advantage with electrochemical detection is that no derivatization of the analytes is required, since they are electrochemically active [10].…”

“…Only a few methods exist, where MHPG has been simultaneously quantified with the parent monoamine neurotransmitters despite the important role of MHPG in evaluating neurochemical imbalances of the noradrenergic system [7,9,16]. Some of the recently described methods for determination of monoamine neurotransmitters involve the use of instrumentation or cells specifically designed for monoamine analysis [4,9,10]. In addition, quantification of neurotransmitters from primary neuronal cell cultures has only been scarcely described in the literature [17][18][19].…”

Reassessment of the antioxidative mixture for the challenging electrochemical determination of dopamine, noradrenaline and serotonin in microdialysis samples

“…To quantify changes in the extracellular release of the monoamines in a specific brain area in response to a pharmacological stimulus, the in vivo microdialysis sampling technique is typically applied [9,10], combined with ion-pair (ultra-) high performance liquid chromatography ((U)HPLC) coupled to electrochemical detection (ECD) [9,[11][12][13][14][15][16]. Thanks to the latest advances in miniaturized and high throughput techniques, researchers can start exploring more challenging, small brain areas which may contain particularly low extracellular monoamine levels (pM range).…”

Aptamer biosensor for dopamine based on a gold electrode modified with carbon nanoparticles and thionine labeled gold nanoparticles as probe

“…Methods for dopamine detection include liquid chromatography [9], chemiluminescence [10], capillary electrophoresis [11], fluorescence [12], colorimetry [13,14] and electrochemical detection [15]. However, almost all these methods require sophisticated equipment and/or time-consuming procedures, which bring significant limitations on its application.…”