Ann Van Eeckhaut - Speaker

Yannick Van Wanseele - Contributor

Katrien Maes - Contributor

Ilse Smolders - Contributor

In order to investigate the potential role of neuropeptides in neurological disorders, their endogenous concentrations are monitored in the rodent brain using in vivo microdialysis and nano UHPLC-ESI-MS/MS. The low extracellular concentrations (low picomolar range) of these peptides, their tendency to stick to several surfaces, the low microdialysis efficiencies, the need for acceptable temporal resolution, the small sample volumes and the complexity of the matrix make the quantification of these neuropeptides in microdialysis samples challenging [1]. To achieve maximal sensitivity, the UHPLC, the MS/MS and microdialysis parameters need to be optimized. This study focuses on four peptides of the neuromedin family, i.e. neurotensin (NT), neuromedin N (NMN), neuromedin B (NMB) and neuromedin U (NMU).
To start, a strategy to improve method sensitivity by intervening on all aspects of standard preparation, i.e. from dissolution of the powder until the injection of the sample, was developed [2]. Furthermore, the sample composition in the UHPLC vial was optimized by using design of experiments. Reduction of peptide adsorption and optimization of the injection solvent resulted in a clear and quantifiable signal for the peptides under investigation. In addition, a shallow gradient slope at 300 nL/min on the analytical column maintained at 35°C, followed by two saw-tooth column wash cycles, resulted in the highest sensitivity and the lowest carry-over [3]. As MS sensitivity is hampered by the formation of multiple precursor ions during the ionization of peptides, the use of mixtures of superchargers (m-nitrobenzyl alcohol, sulfolane and dimethyl sulfoxide) to minimize the charge state distribution of NMU was investigated. It was possible to minimize the charge state distribution of NMU in favor of the +4 charge state, but the practical implications of the addition of these superchargers on sensitivity and method robustness need to be further investigated. Experiments are ongoing to compare the ionization of the peptides via ESI or the new Unispray source from Waters, including the effect of the different supercharging additives on the peptide charge state distribution.
The optimized assay allows for the accurate and precise quantification of 0.5 pM NT and NMN (2.5 amol on column) and 3.0 pM NMB (15.0 amol on column) in in vivo microdialysates. Hereby, the quantification limits needed for the simultaneous analysis of the three peptides in in vivo microdialysates is achieved, with only 5 µL of sample consumed. However, further improvements are still necessary. Indeed, also the low microdialysis efficiency for peptides hampers detectability. In vitro recoveries between 4 and 8% were obtained for these neuromedin peptides when using microdialysis probes with a 3 mm membrane and a cut-off value of 15 or 20 kDa and perfused at 2 µL/min [4]. Moreover, adsorption in microdialysis syringes and tubings are observed [5]. Strategies to reduce adsorption to the microdialysis set-up and to increase the recovery of the probes should be further investigated, especially when methods are developed for sampling in mouse models.
9 Sep 201812 Sep 2018

Event (Conference)

Title11th International Symposium on Drug Analysis and the 29th International Symposium on Pharmaceutical and Biomedical Analysis

ID: 39492805