In-Depth Chemical Analysis of the Brain Extracellular Space Using <i>In Vivo</i> Microdialysis with Liquid Chromatography-Tandem Mass Spectrometry

Metabolomic analysis of samples acquired <i>in vivo</i> from the brain extracellular space by microdialysis sampling can provide insights into chemical underpinnings of a given brain state and how it changes over time. Small sample volumes and low physiological concentrations have limited the identification of compounds from this compartment, so at present, we have scant knowledge of its composition. As a result, most <i>in vivo</i> measurements have limited depth of analysis. Here, we describe an approach to (1) identify hundreds of compounds in brain dialysate and (2) routinely detect many of these compounds in 5 μL microdialysis samples to enable deep monitoring of brain chemistry in time-resolved studies. Dialysate samples collected over 12 h were concentrated 10-fold and then analyzed using liquid chromatography with iterative tandem mass spectrometry (LC-MS/MS). Using this approach on dialysate from the rat striatum with both reversed-phase and hydrophilic interaction liquid chromatography yielded 479 unique compound identifications. 60% of the identified compounds could be detected in 5 μL of dialysate without further concentration using a single 20 min LC-MS analysis, showing that once identified, most compounds can be detected using small sample volumes and shorter analysis times compatible with routine <i>in vivo</i> monitoring. To detect more neurochemicals, LC-MS analysis of dialysate derivatized with light and isotopically labeled benzoyl chloride was employed. 872 nondegenerate benzoylated features were detected with this approach, including most small-molecule neurotransmitters and several metabolites involved in dopamine metabolism. This strategy allows deeper annotation of the brain extracellular space than previously possible and provides a launching point for defining the chemistry underlying brain states.