title: Determination of non-neuronal acetylcholine in human peripheral blood mononuclear cells by use of hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry.
abstract: 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.
authors.affiliation: Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Laboratory of Physical and Chemical Research, Tianjin Centers for Disease Control and Prevention, Tianjin, China.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Xia
authors.affiliation: Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China.
authors.forename: Chunyang
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Han
authors.affiliation: Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China. Electronic address: hjw@tijmu.edu.cn.
authors.forename: Junwei
authors.initials: J
authors.lastname: Hao
mesh_terms: D000109:Acetylcholine / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007249:Inflammation; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D057230:Limit of Detection; D016014:Linear Models; D008875:Middle Aged; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000109:Acetylcholine
keywords: Acetylcholine; Immune cells; Inflammation; Stroke