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Quantification of acetylcholine in cell culture systems by semi-micro high-performance liquid chromatography and
1Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1580, USA. acevedo@helix.nih.gov
Abstract:
Electrospray ionization (ESI) was used to adapt the thermospray ionization liquid chromatographic/mass spectrometric (LC/MS) method of Liberato et al. to the determination of acetylcholine and choline released from cells in culture. A clonal cell line derived from a rat pheochromocytoma (PC12) was used and cultures depolarized in the presence of cholinesterase inhibitor to maximize the recovery of acetylcholine from the culture medium and protect against its hydrolysis. Concentration of acetylcholine and choline released into the incubation medium was accomplished with Sep-Pak C16 cartridges. The preformed analyte cations were resolved by reversed-phase high-performance liquid chromatography and analyzed without derivatization by electrospray ionization mass spectrometry with the use of acetyl[2H9]choline as internal standard. LC/MS allows for the direct determination of acetylcholine and choline, which is not possible with other methods.