Analysis of acetylcholine from extracellular fluid in brain by in vivo microdialysis and LC-ESI-MS/MS with the stable isotope-labeled internal standard

Acetylcholine (ACh) associated with Alzheimer's and Parkinson's disease is the major neurotransmitter in vertebrates. In support of clinical studies on the mechanism of the illnesses and development of medicines for these diseases, the LC-ESI-MS/MS method was developed and validated for the direct quantification of ACh in dialysate samples with acetylcholine-D9 bromide (IS) as the isotope-labeled internal standard. The analytes were separated on the Waters Hilics C18 Column (2.1 mm × 100 mm, 3 μm) on LC with mobile phase ultrapure water-200 mM ammonium formate (pH 3.04)-acetonitrile (30:5:65, vol/vol/vol) at a flow rate of 300 μL/min, and monitored with a fragment ion of m/z 87 formed from a molecular ion of m/z 146 for ACh and that of m/z 87 from m/z 155 for IS during multiple reaction monitoring (MRM) positive ion mode. The lower limit of quantitation (LLOQ) of ACh was lower than 0.1 nmol/L in dialysate samples, equivalent to 0.2 fmol injected on-column. The developed method could be utilized in the analysis of ACh in dialysate samples and these results were in good agreement with the gradient elution study.