Analysis of Acetylcholine in Cerebrospinal Fluids by Liquid Chromatography–Tandem Mass Spectrometry




Nguyen, Vien
Ochoa, Samantha
Prokai-Tatrai, Katalin
Prokai, Laszlo


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Analysis of Acetylcholine in Cerebrospinal Fluids by Liquid Chromatography–Tandem Mass Spectrometry Samantha M. Brewer, Vien Nguyen, Katalin Prokai-Tatrai and Laszlo Prokai Department of Pharmacology and Neuroscience, Graduate School of Biomedical Sciences Purpose: Acetylcholine (ACh) is a neurotransmitter whose decreased levels have been associated with several diseases impacting the central nervous system. Quantification of extracellular ACh in cerebrospinal fluid (CSF) is difficult due to its low concentration (owing to its rapid hydrolysis to choline) and matric effects upon employing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantification. The purpose of this study was to develop and validate an LC-MS/MS method for the high-throughput determination of ACh in rat brain microdialysates and possibly in clinical samples in the future. Methods: All analyses were performed on a TSQ Quantum Ultra mass spectrometer connected to a Surveyor MS high-performance liquid chromatography system (Thermo Scientific, San Jose, CA). Atmospheric spray ionization was used, and analytes were detected after fragmenting their intact cations through collision-induced dissociation. Deuterium-labeled ACh (ACh-d4) was used as an internal standard for quantification. Several solvent systems were compared to identify the best conditions for the preparation of ACh and ACh-d4 stock solutions to calibrate the assay. The optimized method was used to determine the extracellular ACh levels in rat brain microdialysates using the calibration curve obtained in aCSF. Results: In order to set-up a reliable assay, the MS signals for ACh and deuterium-labeled ACh (ACh-d4) used as an internal standard had to be optimized, along with optimizing the solvent systems for the preparation of their stock solutions. The optimal solvent system afforded linear calibration and resisted signal suppression by artificial cerebrospinal fluid (aCSF). Artificial cerebrospinal fluid is used as perfusion fluid upon microdialysis as it matches the composition of actual CSF. We have established that the use of purely aqueous stock solutions of ACh and ACh-d4 afforded the best calibration in aCSF. With these measures, reliable analyses of ACh in aCSF were achieved. The obtained linear calibration could be used for quantitation of low (physiological) ACh levels in samples obtained upon in vivo intracranial microdialysis from rat cortex. Conclusions: The method presented here allows for reliable quantification of ACh in cerebrospinal fluids, and may be used to evaluate the efficiency and mode of action of potential cholinergic agents. Acknowledgment: This research has been supported in part by The Welch Foundation (endowment BK-0031). IACUC Number: 2018-0006