Capillary hydrophilic interaction chromatography/mass spectrometry for simultaneous determination of multiple neurotransmitters in primate cerebral cortex

Abstract

A diverse array of neurotransmitters and neuromodulators control and affect brain function. A profound understanding of the signaling pathways and the neural circuits underlying behavior is therefore likely to require the tracking of concentration changes of active neurochemicals. In the present study, we demonstrate the feasibility of a method allowing the simultaneous determination of the concentrations of six neurotransmitters: acetylcholine, serotonin, dopamine, g-aminobutyric acid (GABA), glutamate and aspartate, in the extracellular brain fluid (EBF). We used hydrophilic interaction chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS) to analyze the EBF from the monkey brain. A push-pull sampling method was used to collect EBF from the prefrontal cortex (PFC) of conscious monkeys at flow rates in the range of low nL/min. The detection limits of acetylcholine, serotonin, dopamine, GABA, glutamate and aspartate were 0.015, 0.15, 0.3, 1.2, 6 and 15 femtomoles, respectively, allowing us to quantitatively determine the concentrations of these six neurotransmitters simultaneously from 500 nL in vivo samples. We conclude that HILIC/ MS/MS combined with the push-pull sampling method represents a sensitive technique for simultaneous monitoring of neurotransmitters from EBF samples.

Quantification of acetylcholine, an essential neurotransmitter, in brain microdialysis samples by liquid chromatography mass spectrometry

“…The highly volatile organic mobile phase used in HILIC facilitates the desolvation and compound ionization in the electrospray process and provides increased ESI-MS sensitivity (Kebarle, 2000). The separation of ACh based on HILIC has been conducted with polar stationary phases such as silica (Grumbach et al, 2004), aminopropyl, amide (Guo, 2005), polyhydroxyethyl aspartamide (Wang et al, 2008;Zhang et al, 2007b) or diol (Uutela et al, 2005). Uutela et al (2005) developed a HILIC method in which a diol column was used, not only for the separation of ACh, choline and the IS, but also for the separation of the analytes from inorganic salts and endogeneous compounds within a chromatographic run time of 10 min.…”

Analysis of multiple quaternary ammonium compounds in the brain using tandem capillary column separation and high resolution mass spectrometric detection

“…The striatum sample is prepared using 16 L solution per 1 mg of tissue. Each QAC was detected with signal to noise ratio 10. used for analysis of acylcarnitines but not free carnitine, acetylcholine or choline [30], while HILIC-MS allowed polar QACs such as acetylcholine and choline to be separated and detected [13,14]. Although a recent study has shown that acetylcarnitine and palmitoylcarnitine could be analyzed using HILIC-MS [31], the elution times of the two QACs were short and very close, reflecting the low separation and peak capability of HILIC-MS for a large number of QACs, in particular for hydrophobic ones.…”

“…This hydrophobicity diversity poses challenges for the LC-MS analysis. Currently, the separation of QACs of high polarity, such as choline, acetylcholine, carnitine and acetylcarnitine, are commonly conducted using hydrophilic interaction chromatography (HILIC)-MS [13,14], ion-exchange chromatography [15,16], or ionpairing reversed phase (RP) chromatography [17][18][19]. To separate carnitines of both low and high polarities, pre-column derivatization has been used to improve their separation on common RP columns [20].…”

Measuring multiple neurochemicals and related metabolites in blood and brain of the rhesus monkey by using dual microdialysis sampling and capillary hydrophilic interaction chromatography–mass spectrometry

“…The preparation of capillary columns was adopted and modified from Zhang et al [23]. To prevent the packing material from spilling out of the column a frit was formed at one end by in situ free radical polymerization modified from Viklund et al [27].…”

“…It already has proved its merits in determining peptides, proteins and small polar molecular compounds in complex matrices [21][22][23][24]. Recent reports demonstrated that HILIC-MS is well suited to determine polar neurotransmitters in EBF offering high sensitivity combined with time-saving procedures for sample preparation [23,25].…”