Quantification of acetylcholine in microdialysate of subcutaneous tissue by hydrophilic interaction chromatography/tandem mass spectrometry

It has recently been suggested that acetylcholine plays an important role in the modulation of tissue inflammation. In order to further understand the newly discovered cholinergic anti-inflammatory pathway, tracking the concentration changes of acetylcholine in tissue is required. This paper describes the development of a method coupling hydrophilic interaction chromatography with electrospray ionization tandem mass spectrometry (HILIC/ESI-MS/MS) for the separation and quantification of acetylcholine in microdialysis samples of normal rats and of rats with local inflammation. The separation of acetylcholine from interferential endogenous compounds and inorganic cations was achieved with a zwitterionic stationary phase column using isocratic elution. Low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis was carried out in the positive ion mode using multiple reaction monitoring (MRM) of the following mass transitions: m/z 146 --> 87 for acetylcholine and m/z 155 --> 87 for the internal standard acetylcholine-D9. The limit of detection for acetylcholine was found to be 0.075 fmol on-column with a signal-to-noise ratio of 3:1. The lower limit of quantification was 0.25 fmol on-column. The calibration curves obtained for acetylcholine in blank microdialysates were linear in the ranges of 0.025-50 nM and 0.025-0.5 nM, with correlation coefficients equal to or greater than 0.9994 and 0.9969, respectively. The recoveries of acetylcholine for high (2 nM) and low (0.5 nM) concentrations were in the ranges of 90-96% and 95-109%, respectively. The coefficients of variation for intra-day and inter-day reproducibility were equal to or less than 7.3% and 10.4%, respectively. The method has been successfully applied in the measurement of acetylcholine in microdialysates from normal and inflamed rat tissue.