Measurement of acetylcholine in rat brain microdialysates by LC-isotope dilution tandem MS

Laszlo Prokai, Petr Fryčák, Stanley M. Stevens, Vien Nguyen

Research output: Contribution to journalArticlepeer-review

23 Scopus citations


An LC-MS-MS method was developed for measuring acetylcholine (ACh) in an aqueous medium using reversed-phase ion-pair chromatography, electrospray ionization on a quadrupole ion trap instrument and a tetradeuterated analogue (ACh-1,1,2,2-d4) as an internal standard. A rapid separation was achieved on a 5-cm long octadecylsilica column (2.1 mm i.d.) by employing heptafluorobutyric acid (0.1% v/v) as an ion-pairing agent and requiring 10% v/v acetonitrile in 20 mM ammonium formate buffer under isocratic elution at 200 μl min-1 flow rate. The instrument's response was calibrated with samples containing known mole ratios of ACh and ACh-1,1,2,2-d4 in an artificial cerebrospinal fluid, which afforded the conclusion that analyte concentrations could be determined by multiplying the measured analyte to internal standard ion-current ratio with the known molar concentration of the ACh-1,1,2,2-d4 added. The rapid and simple assay was tested by measuring the basal neurotransmitter concentration in rat brain microdialysates without the use of a cholinesterase inhibitor upon sample collection.

Original languageEnglish
Pages (from-to)S101-S105
Issue numberSUPPL. 1
StatePublished - Oct 2008


  • Acetylcholine
  • Column liquid chromatography-tandem mass spectrometry
  • Electrospray ionization
  • In vivo intracranial microdialysis
  • Isotope dilution


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