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Quantification of Acetylcholine in Cell Culture Systems by Semi‐micro High‐Performance Liquid Chromatography and Electrospray Ionization Mass Spectrometry
Author(s) -
Acevedo Larisa D.,
Xu Yaodong,
Zhang Xin,
Pearce Rosalyn J.,
Yergey Alfred
Publication year - 1996
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/(sici)1096-9888(199612)31:12<1399::aid-jms439>3.0.co;2-y
Subject(s) - chemistry , chromatography , thermospray , electrospray ionization , mass spectrometry , high performance liquid chromatography , electrospray , derivatization , liquid chromatography–mass spectrometry , extractive electrospray ionization , acetylcholine , analyte , tandem mass spectrometry , sample preparation in mass spectrometry , selected reaction monitoring , medicine , endocrinology
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 C 18 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[ 2 H 9 ]choline as internal standard. LC/MS allows for the direct determination of acetylcholine and choline, which is not possible with other methods.