Introduction. Inflammation is characterized by complex interactions between pro- and anti- inflammatory cytokines. Recent research has probed the role of the nervous system in inflammation, part of which includes the cholinergic anti-inflammatory pathway that regulates immunologically-mediated inflammation. In this pathway, norepinephrine release from the splenic nerves binds to beta-2-adrenergic receptors on T cells, causing release of acetylcholine (ACh). ACh subsequently suppresses macrophage production and release of pro-inflammatory cytokines. In this project, we aim to develop a method to quantify the ACh release in splenocytes when challenged with different mediators in this pathway. Our method utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantification of ACh and choline (Ch) in cell culture media.

Methods. Literature review revealed that the hydrophilic interaction liquid chromatography (HILIC) is the most appropriate separation mechanism for ACh and Ch. The developed LC-MS/MS method utilizes an isocratic separation (14% 10mM ammonium formate, pH 3, and 86% acetonitrile) on an Atlantis HILIC column (2.1 x 100 mm, 3 micron). The MS operates in positive electrospray (+ESI) mode, monitoring ions specific for ACh, Ch, and their corresponding deuterium labeled internal standards. The calibration range for ACh was 0.5 - 5 micrograms/ml (3.4 - 34 mM) and 10 - 50 micrograms/ml (96 - 480 mM) for Ch. Cell culture media contained neostigmine to inhibit cholinesterase. Cell culture media samples are prepared by freeze drying, reconstituting in acetonitrile, and filtering (0.2micron). Potential loss of ACh through degradation during cell culture was evaluated by monitoring d4-labeled ACh with and without the presence of splenocytes for 4 and 24 hours.

Results. Correlation coefficients (R2) indicate linearity for ACh and Ch in acetonitrile and culture media in the aforementioned calibration range. This linearity applies to external calibration as well as calibration utilizing the deuterium labeled internal standard. Calibration curve slopes differed between samples prepared in culture media and those prepared in acetonitrile. As such, experimental samples will be matrix matched by preparing calibrants in media. During the six-min separation, ACh elutes at 3.8 min and Ch at 5.1 min. Deuterium-labeled ACh, when incubated for 4 and 24 hours, showed statistically significant loss, compared to control, of ACh after 24 hours in media and media + splenocytes. Despite this, the average loss of ACh by hydrolysis averaged less than 10%. Media and media + splenocyte samples incubated for 4 hr showed no statistically significant difference from control.

Conclusions. The developed LC-MS/MS assay for quantification of ACh and Ch in cell culture media can be applied to the investigation of the cholinergic anti-inflammatory pathway. The method provides a rapid separation of ACh and Ch, with the successful incorporation of stable-isotope labeled internal standards.