In Vivo Neurochemical Monitoring Using Benzoyl Chloride Derivatization and Liquid Chromatography–Mass Spectrometry

Abstract

In vivo neurochemical monitoring using microdialysis sampling is important in neuroscience because it allows correlation of neurotransmission with behavior, disease state, and drug concentrations in the intact brain. A significant limitation of current practice is that different assays are utilized for measuring each class of neurotransmitter. We present a high performance liquid chromatography (HPLC) - tandem mass spectrometry method that utilizes benzoyl chloride for determination of the most common low molecular weight neurotransmitters and metabolites. In this method, 17 analytes were separated in 8 minutes. The limit of detection was 0.03–0.2 nM for monoamine neurotransmitters, 0.05–11 nM for monoamine metabolites, 2–250 nM for amino acids, 0.5 nM for acetylcholine, 2 nM for histamine, and 25 nM for adenosine at sample volume of 5 µL. Relative standard deviation for repeated analysis at concentrations expected in vivo averaged 7% (n = 3). Commercially available 13C benzoyl chloride was used to generate isotope-labeled internal standards for improved quantification. To demonstrate utility of the method for study of small brain regions, the GABAA receptor antagonist bicuculline (50 µM) was infused into rat ventral tegmental area while recording neurotransmitter concentration locally and in nucleus accumbens, revealing complex GABAergic control over mesolimbic processes. To demonstrate high temporal resolution monitoring, samples were collected every 60 s while neostigmine, an acetylcholine esterase inhibitor, was infused into the medial prefrontal cortex. This experiment revealed selective positive control of acetylcholine over cortical glutamate.

Asphyxia-activated corticocardiac signaling accelerates onset of cardiac arrest

“…To probe the neurochemical basis of the heightened cortical activities, we performed minute-by-minute microdialysis in the frontal and occipital lobes of unanesthetized rats (n = 7) before and during asphyxiation and analyzed cortical dialysates using liquid chromatography-mass spectrometry (LC-MS) (21). As expected, levels of brain glucose dropped rapidly in both frontal and occipital areas (Fig.…”

“…Dialysates collected in 1-min bins were derivatized for further analysis (SI Text). To capture an array of neurochemicals, we applied a variation of LC-MS analysis (21). Additional details about analysis can be found in SI Text.…”

Chemical Gradients within Brain Extracellular Space Measured using Low Flow Push–Pull Perfusion Sampling in Vivo

“…The 13 C-internal standard reagent contained 1% (vol) of a 13 Cbenzoyl chloride derivatized stock solution (described in detail elsewhere 24 ), 97% dimethyl sulfoxide, and 2% acetic acid. Calibration curves were prepared for each analyte in aCSF with the following concentrations: 0.5, 5, 10, 50, and 100 nM for DA, NM, NE, 5-HT, and ACh; 5, 10, 50, 100, and 1000 nM for DOPAC, HVA, 5-HIAA, Glu, GABA, and Asp; and 50, 100, 500, 1000, and 10 000 nM for Gly.…”

“…19,21−23 Fractions collected from probes in this work are analyzed using a recently developed liquid chromatography−mass spectrometry (LC-MS) method to assay 13 neurotransmitters and metabolites. 24 The method is used to measure extracellular chemical gradients across a few hundred micrometers in three brain regions where DA distributions are expected. We focus on DA because of interest in this neurotransmitter for its role in reward, addiction, and certain diseases.…”

Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure

“…Quantification of neurotransmitters in dialysate samples was performed by LC-MS/MS using established methodology (41,42). Briefly, 1 μL dialysate was added to 6.5 μL borate (100 mM), derivatized with benzoyl chloride [2% (vol/vol), acetonitrile (ACN)], and supplemented with an internal standard.…”