Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry

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Abstract
A sensitive liquid chromatography/electrospray ionisation tandem mass spectrometric (LC/ESI‐MS/MS) method was developed for the analysis of acetylcholine and choline in microdialysis samples. A Ringer's solution that contains high (150 mM) concentrations of inorganic salts was used to extract acetylcholine and choline from a rat or mouse brain. The separation of acetylcholine, choline, an internal standard acetyl‐β‐methylcholine, endogenous compounds and inorganic cations was achieved with hydrophilic interaction chromatography using a diol column. The eluent consisted of 20 mM ammonium formate (pH 3.3) and acetonitrile (20:80) which is favourable for the ESI process. Limits of detection (signal‐to‐noise (S/N) ratio = 3) of 0.02 nM (0.2 fmol) for acetylcholine and 1 nM (10 fmol) for choline were observed using standards diluted in Ringer's solution. A good linearity was obtained from the limit of quantitation: 0.1 nM (S/N ratio = 10) to 50 nM (r = 0.999) for acetylcholine and within the concentration range of 100–3500 nM (r = 0.998) for choline. The between‐day repeatability of the method was good; RSD was 3.1% at 1 nM level of acetylcholine and 3.5% at 1000 nM level of choline. The recoveries for addition of 1 or 2.5 nM acetylcholine and 0.2 or 1 µM choline in microdialysis balancing samples were between 93 and 101% indicating that no suppressing endogenous compounds were co‐eluting with acetylcholine or choline. The developed method was applied to the analysis of microdialysis balancing samples collected from rat and mouse brains. Copyright © 2005 John Wiley & Sons, Ltd.

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