Quantitative analysis of acetylcholine in rat brain microdialysates by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.
A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the quantitative analysis of acetylcholine in rat brain dialysates. The separation of acetylcholine (ACh), choline (Ch), acetyl-β-methylcholine (IS) from endogenous compounds and Ringer's salts was achieved with cation exchange chromatography. Optimization of chromatographic and mass spectrometry parameters were perfomed in order to improve sensitivity of the method. The limit of detection were 0.05 and 3.75 fmol on column with S/N ratio of 3:1 for ACh and Ch, respectively. The limit of quantitation (LOQ) for ACh and Ch measured in Ringer's solution were 0.05 nM (0.25 fmol) and 3.75 nM (18.75 fmol), respectively at S/N ratio of 10:1. Linearity of the method has been evaluated in the concentrations range between 0.05 and 5.00 nM and 3.75 and 200 nM for ACh and Ch respectively. The correlation coefficients were 0.999 and 0.995 for ACh and Ch respectively, indicating very good linearity. The LC/MS/MS method developed has been applied to evaluate the effect of oral administration of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (IDRA21), a positive modulators of AMPA receptor, on the release of ACh in the rat prefrontal cortex by microdialysis.
Carrozzo
Marina M
MM
Department of Pharmaceutical Sciencese, University of Modena and Reggio Emilia, Via Campi 183, 41125 Modena, Italy.
Cannazza
Giuseppe
G
Pinetti
Diego
D
Di Viesti
Vittoria
V
Battisti
Umberto
U
Braghiroli
Daniela
D
Parenti
Carlo
C
Baraldi
Mario
M
D000109:Acetylcholine / Q000032:analysis*; D000818:Animals; D001923:Brain Chemistry / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008297:Male; D017551:Microdialysis / Q000379:methods*; D017397:Prefrontal Cortex / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000187:drug effects; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013238:Stereotaxic Techniques; D053719:Tandem Mass Spectrometry
D007202:Indicators and Reagents; D018091:Receptors, AMPA; D000109:Acetylcholine