A VALIDATED METHOD FOR BIOANALYSIS OF ACETYLCHOLINE FROM CEREBROSPINAL FLUIDS USING LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY

Decreased acetylcholine [ACh, (CH3)3N+CH2CH2OCOCH3] levels have been associated with several diseases of the central nervous system (CNS) such as Alzheimer's disease; therefore, it has been considered a potential biomarker. However, quantification of ACh in biological samples even in cerebrospinal fluids obtained through proximity sampling is difficult due to its often extremely low concentration owing to rapid enzyme-catalyzed hydrolysis to choline. Most limitations of previously used liquid chromatography-based (LC) assays relying on electrochemical detection have been addressed by coupling LC with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). However, numerous applications have remained challenging because of inadequate detection sensitivity achievable with most commercial instruments and columns. We improved the method using atmospheric pressure spray ionization (APSI) as an alternative to ESI and ultrahigh-performance liquid chromatography (UPLC) resulting in an about 100-fold increase in detection sensitivity over the previous LC–ESI-MS/MS assay. The presentation will feature application of the method to the sensitive determination of ACh in rodent brain microdialysates, which indicates fit for purpose to use in translational research focusing on this important neurotransmitter. The developed assay also has been rigorously validated to show that it would meet regulatory requirements for application to assess ACh as a potential clinical biomarker of CNS diseases.