title: High-performance liquid chromatography/tandem mass spectrometry assay for the rapid high sensitivity measurement of basal acetylcholine from microdialysates.
abstract: A high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the analysis of acetylcholine (ACh) in brain dialysates. This separation of ACh is based on cation exchange chromatography with elution buffer consisting of a mixture of ammonium acetate, ammonium formate and acetonitrile. Using isocratic separation conditions, ACh was resolved within a minute and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for ACh was found to be 1 fmol on column with a S/N ratio of 3:1. The assay has been used routinely for the measurement of ACh in brain dialysates from awake freely moving rats. Furthermore, separation conditions were modified to allow simultaneous measurement of ACh and the acetylcholine esterase inhibitor, neostigmine.
authors.affiliation: Computational Analytical and Structural Sciences, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, CM19 5AW, Essex, UK. mark_e_hows@gsk.com
authors.forename: Mark E P
authors.initials: ME
authors.lastname: Hows
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Organ
authors.forename: Sarah
authors.initials: S
authors.lastname: Murray
authors.forename: Lee A
authors.initials: LA
authors.lastname: Dawson
authors.forename: Richard
authors.initials: R
authors.lastname: Foxton
authors.forename: Christian
authors.initials: C
authors.lastname: Heidbreder
authors.forename: Zoë A
authors.initials: ZA
authors.lastname: Hughes
authors.forename: Laurent
authors.initials: L
authors.lastname: Lacroix
authors.forename: Ajit J
authors.initials: AJ
authors.lastname: Shah
mesh_terms: D000109:Acetylcholine / Q000032:analysis*; D000779:Anesthetics, Local / Q000494:pharmacology; D000818:Animals; D001923:Brain Chemistry / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015314:Dialysis Solutions / Q000737:chemistry*; D008297:Male; D017551:Microdialysis; D009388:Neostigmine / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors
chemical_list: D000779:Anesthetics, Local; D015314:Dialysis Solutions; D009388:Neostigmine; D013779:Tetrodotoxin; D000109:Acetylcholine