Determination of non-neuronal acetylcholine in human peripheral blood mononuclear cells by use of hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry.
BACKGROUND
Cholinergic components in non-neuronal tissues and cells are proposed as important in maintaining cellular proliferation and immune homeostasis. However, direct quantification of non-neuronal acetylcholine (ACh) in cells has been inefficient. Therefore, we developed a stable method for determination of intracellular ACh.


METHODS
We used ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with Waters CORTECS chromatographic columns to measure the intracellular ACh in human peripheral blood mononuclear cells (PBMCs). The presence of ACh was validated by RT-qPCR of mRNAs of cholinergic components.


RESULTS
This method successfully separated ACh from iso-ACh with highly sensitive precursor/product ion transitions and allowed a faster chromatography run time within 3.5min. The detection limit of intracellular ACh was 0.005ng/10(6) PBMCs. Intra-assay and inter-assay coefficient variations were all <7.0%. Mean recoveries in the samples were between 93.20% and 104.73%. Here, intracellular ACh was stable under multiple storage conditions, partly attributable to mutually stable relationships among cholinergic components. This method was successfully applied in a stroke study and revealed activation of the stroke-induced cholinergic anti-inflammatory pathway.


CONCLUSIONS
This method allows direct determination of intracellular ACh in various tissues. Evidences of cholinergic activity linking both the nervous system and non-neuronal system can be profiled by assessing ACh.
['Zhang|Chao|C|', 'Xia|Yiping|Y|', 'Jiang|Wei|W|', 'Wang|Chunyang|C|', 'Han|Bin|B|', 'Hao|Junwei|J|']
D000109:Acetylcholine D002853:Chromatography, Liquid D006801:Humans D057927:Hydrophobic and Hydrophilic Interactions D007249:Inflammation D007963:Leukocytes, Mononuclear D057230:Limit of Detection D016014:Linear Models D008875:Middle Aged D015203:Reproducibility of Results D053719:Tandem Mass Spectrometry
Acetylcholine Stroke Immune cells Inflammation