Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry.
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-beta-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 microM 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.
Uutela
Päivi
P
Faculty of Pharmacy, Division of Pharmaceutical Chemistry, P.O. Box 56, FI-00014 University of Helsinki, Finland. risto.kostiainen@helsinki.fi
Reinilä
Ruut
R
Piepponen
Petteri
P
Ketola
Raimo A
RA
Kostiainen
Risto
R
D000109:Acetylcholine / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002794:Choline / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D017022:Flow Injection Analysis / Q000379:methods*; D051379:Mice; D017551:Microdialysis / Q000379:methods*; D018377:Neurotransmitter Agents / Q000032:analysis*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D018377:Neurotransmitter Agents; D002794:Choline; D000109:Acetylcholine