title: Surrogate based accurate quantification of endogenous acetylcholine in murine brain by hydrophilic interaction liquid chromatography-tandem mass spectrometry.
abstract: Cholinergic dysfunction is known as a hallmark feature of Alzheimer's disease (AD). Measurement of endogenous acetylcholine (ACh) in specific brain regions is important in understanding the pathology of AD and in designing and evaluating novel cholinomimetic agents for the treatment of AD. Since ACh is an endogenous neurotransmitter, there is no real blank matrix available to construct standard curves. It has been a challenging task to determine ACh in complex brain matrices. To overcome these difficulties, we employed a surrogate analyte strategy using ACh-d(4) instead of ACh to generate calibration curves and Ch-d(9) as internal standard (IS). The brain samples were deproteinized by acetonitrile with IS. Analytes and IS were separated by a HILIC column with the mobile phase composed of 20 mM ammonium formate in water-acetonitrile (30:70, v/v, adjusted to pH 3.0 with formic acid) and monitored in multiple reaction monitoring (MRM) mode using a positive electrospray source. The concentrations of endogenous ACh were calculated based on the peak area ratio of the analyte to the IS using a regression equation for the corresponding surrogate standard (ACh-d(4)). The lower limit of detection was 0.2 ng/mL and linearity was maintained over the range of 10-1000 ng/mL. Compared to other currently available methods, this approach offers improved accuracy and precision for efficient analysis of ACh. The proposed method was proved successfully by evaluating the action of typical acetylcholinesterase inhibitor huperzine A in senescence accelerated mouse prone 8 (SAMP8).
authors.affiliation: Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Peng
authors.forename: Tao
authors.initials: T
authors.lastname: Jiang
authors.forename: Zhengxing
authors.initials: Z
authors.lastname: Rong
authors.forename: Ting
authors.initials: T
authors.lastname: Liu
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.forename: Biyun
authors.initials: B
authors.lastname: Shao
authors.forename: Jian
authors.initials: J
authors.lastname: Ma
authors.forename: Lan
authors.initials: L
authors.lastname: Yang
authors.forename: Lei
authors.initials: L
authors.lastname: Kang
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Shen
authors.forename: Huafang
authors.initials: H
authors.lastname: Li
authors.forename: Hong
authors.initials: H
authors.lastname: Qi
authors.forename: Hongzhuan
authors.initials: H
authors.lastname: Chen
mesh_terms: D000109:Acetylcholine / Q000032:analysis* / Q000378:metabolism; D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D003903:Deuterium; D057927:Hydrophobic and Hydrophilic Interactions; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003903:Deuterium; D000109:Acetylcholine