Bioanalytical research in neuroscience


Neurochemical and neuropharmacological research is strongly dependent on the availability of sensitive analysis methods for neurotransmitters, neuromodulators and drugs in brain microdialysis samples of rats and mice. The development of new, faster and sensitive analysis methods remains challenging for the bioanalyst due to the very low concentrations of these compounds in small volumes of dialysate, coupled to a low microdialysis recovery, in addition to the need for higher throughput.
The research group has ample experience in the development of miniaturized LC methods, coupled to fluorescence, electrochemical or tandem mass spectrometric (MS/MS) detection, for the quantification of neurotransmitters and neuromodulators in microdialysates. In addition, we have a large experience in the development and validation of analytical methods for the determination of drugs in drug products and biological samples with capillary electrophoresis, LC-UV, LC-fluorescence and LC-MS/MS.


Quantification of neuropeptides in microdialysates with nano LC-MS/MS

PIs: Ann Van Eeckhaut, Ilse Smolders, Yvette Michotte
PhD students: Katrien Maes, Yannick Van Wanseele
Technical support: Carina De Rijck, Gino De Smet

Neuropeptides seem to play an important role when the central nervous system is challenged. In order to obtain better insights into the central peptidergic effects, it is essential to monitor their concentration in the brain. Quantification of neuropeptides in dialysates is challenging due to their low extracellular concentrations (low pM range), their low microdialysis efficiencies, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to glass and polymeric materials. The quantification of neuropeptides in dialysates therefore necessitates the use of very sensitive nano LC–MS/MS methods. A number of LC–MS/MS and microdialysis parameters need to be optimized to achieve maximal sensitivity. This project focuses on the peptides of the neuromedin group.


HPLC with electrochemical detection for the simultaneous quantification of glutathione (GSH), glutathione disulfide (GSSG), cysteine and cystine in brain homogenates

PIs: Ann Van Eeckhaut, Ann Massie
PhD student: Katrien Maes
Technical support: Gino De Smet

Glutathione (GSH) is an important thiol tripeptide antioxidant in cells. It exists in two forms, namely reduced as GSH and oxidized as glutathione disulfide or GSSG. GSH is significantly favored over GSSG under healthy physiological conditions. However, pathological conditions causing oxidative stress have been found to result in a decreased GSH/GSSG ratio.
The aim of this project is to develop and validate a HPLC method with electrochemical detection for the simultaneous quantification of both redox couples GSH/GSSG and cysteine/cysteine in brain homogenates. This will provide us valuable information about the total redox state of the cell and will be applied to samples of models for neurological disorders before and after drug treatment, transgenic animals, etc.


UHPLC with electrochemical detection for quantification of monoamines in microdialysates

PI: Ann Van Eeckhaut
PhD student: Jolien Van Schoors
Technical support: Ria Berckmans

In this project, the coupling of an ultra-high pressure liquid chromatography (UHPLC) system with an electrochemical detector (ECD) for the analysis of the low concentrated monoamines in microdialysates will be investigated. The monoamines dopamine, noradrenaline and serotonin are neurotransmitters implicated in several neurological disorders. Monitoring of changes in these neurotransmitter concentrations in the brain using in vivo microdialysis sampling is an important tool in neuropharmacological research in the quest for new drug candidates.
Analytical challenges exist both at the level of the chromatography and the ECD. More sensitive methods are needed to allow detection of these monoamines in small brain areas, in smaller laboratory animals or in situations where catecholamine levels are decreased. Furthermore, there is a high demand for faster analysis to increase sample throughput.
For this purpose, the chromatographic parameters (stationary phase, mobile phase, flow rate and separation temperature) will be optimized. In addition, also the detection temperature, filtering of the signal, electrochemical cell type, electrode material and optimal working potential, will be studied. New developments in ECD such as new cell types and electrode material will be investigated in collaboration with Antec (the Netherlands), the manufacturer of amperometric ECD.


Enantioselective LC-method with fluorescence detection for quantification of glutamate and D-serine in biological samples.

PI: Ann Van Eeckhaut, Ilse Smolders
PhD student: Anissa El Arfani, Laura Walrave
Technical support: Carina De Rijck, Ria Berckmans
Scientific collaboration with Mia Lindskog (Karolinska Institutet, Sweden), Emmanuel Hermans (UCL)

We have a large background in chiral separations, especially with capillary electrophoresis. In this project, we adapted our existing LC-fluorescence method for the analysis of amino acids (Van Hemelrijck et al., 2005) to allow chiral separation of D-serine. D-serine is a physiological co-agonist of the N-methyl D-aspartate (NMDA) type of glutamate receptor which is a key excitatory neurotransmitter receptor in the brain. It binds with high affinity to a co-agonist site at the NMDA receptors and, along with glutamate, mediates several important physiological and pathological processes, including NMDA receptor transmission, synaptic plasticity and neurotoxicity (Wolosker et al., FEBS, 2008). To further elucidate its role, it is important to develop a sensitive analytical method which can detect and quantify D-serine in biological samples. For this purpose, the achiral thiol mercaptoethanolused in the derivatization procedure was replaced by N-isobutyryl-L-cysteine, allowing the formation of diastereomeric compounds. After optimization of the mobile phase composition and the gradient profile, the method was validated for the quantification of glutamate and D-serine in brain homogenates. The enantioselective analysis method was applied to analyse D-serine in brain homogenates of Flinders Sensitive Line rats, a rat model for depression (Gomez-Galan et al. 2012). The method is further optimized for quantification of glutamate and D-serine in other biological samples, such as microdialysates, CSF, plasma and cell cultures.

 

 

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