Simultaneous analysis of endogenous neurotransmitters and neuropeptides in brain tissue using capillary electrophoresis--microelectrospray-tandem mass spectrometry.

Abstract

Capillary electrophoresis was combined with highly sensitive microelectrospray-tandem mass spectrometry to simultaneously detect classical small molecule neurotransmitters as well as neuropeptides from discrete regions of the marmoset brain. A mixture of four classical neurotransmitters (glutamate, gamma-aminobutyric acid, acetylcholine, dopamine) and four neuropeptides (neurotensin, methionine-enkephalin, leucine-enkephalin and substance P 1-7) was studied to optimize the capillary electrophoresis conditions for separation, injection volume, and analysis time. Gamma-aminopropyltriethoxysilane-coated capillaries and acetic acid electrolytes were used to avoid interactions between the sample and the capillary surface and to obtain a high anodic electroosmotic flow, which resulted in a short analysis time. Detection was performed using tandem mass spectrometry in the selected reaction monitoring mode using a triple quadrupole mass spectrometer. Samples were dissolved in ammonium acetate to achieve a transient-isotachophoretic concentration step at the beginning of the separation and to make it possible to inject larger sample volumes, up to 140 nL. Small amounts of tissue from specific regions of the marmoset monkey brain were pretreated using solid-phase extraction as a clean-up and concentrating step. In the striatum we could detect endogenous glutamate, gamma-aminobutyric acid (GABA), acetylcholine and dopamine, as well as the neuropeptides methionine-enkephalin and substance P 1-7 in the same analysis, using only 58 mm3 of brain tissue.