Profiling metabolites in rat brain microdialysates by capillary electrophoresis-mass spectrometry using direct sample injection.
Marlien van Mever, LACDR, Leiden University, Leiden, The Netherlands (m.van.mever@lacdr.leidenuniv.nl)
Analysis of low-molecular-weight biomolecules in brain extracellular fluid plays an important role in the prediction, progression, and treatment outcome for various neurological diseases. Microdialysis is a powerful technique for in vivo sampling of extracellular fluid in the brain, thereby enabling the continuous measurement of neurotransmitters in freely-moving animals. However, metabolic analysis of microdialysis samples can be considered a huge analytical challenge due to limited sample amounts and low basal (nanomolar-range) concentrations of many metabolites. Capillary electrophoresis-mass spectrometry (CE-MS) emerged as a strong analytical tool for the profiling of polar and charged metabolites in volume-restricted biological samples. In this study, we have developed a CE-MS method for the profiling of amino acids and related compounds in rat brain microdialysates using direct sample injection.
CE was coupled to time-of-flight mass spectrometry (TOF-MS) employing a co-axial sheath-liquid interface and a bare fused-silica capillary. Amino acids were analyzed at low-pH separation conditions using 10% acetic acid (pH 2.2) as separation buffer. To increase the concentration sensitivity of CE-MS, an in-capillary preconcentration procedure was applied, which allowed large volume sample injections. A three-level Box-Behnken design (BBD) was used to optimize relevant CE-MS parameters for amino acids profiling in rat microdialysates.
The developed CE-MS system allowed the direct analysis of amino acids in rat brain microdialysis samples after only applying a centrifugation step. Very stable CE-MS patterns of amino acids and related compounds in microdialysis samples were obtained with a good migration-time repeatability. The optimized in-capillary preconcentration step allowed a sample injection of 105 nL (corresponding to circa 6% of the capillary volume), which resulted in detection limits down to the low nanomolar range for the amino acids. The CE-MS method provided a linear detector response for amino acids spiked into microdialysis samples over a wide concentration range, i.e. from 50 nM to 20000 nM. The influence of matrix effects on the quantification of amino acids was also investigated, and showed to be minimal for microdialysis samples. Overall, we propose here the first CE-MS method for the direct profiling of amino acids and related compounds in rat brain microdialysate samples. The the utility of this approach will be tested by analyzing a large set of rat brain microdialysate samples in a pharmacokinetic and biomarker discovery context using metabolomics.
Abstract Reference & Short Personal Biography of Presenting Author
Marlien van Mever is a PhD student at Leiden University within the group Biomedical Microscale Analytics, which is a part of the Department of the Leiden Academic Centre for Drug Research. Her project focuses mainly on the development of microscale analytical approaches for profiling metabolites in limited amounts of biological material. She currently focuses on brain metabolomics research using capillary electrophoresis – mass spectrometry.Organized & Produced by:
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