title: LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters.
abstract: Absolute quantification of intracellular coenzyme A (CoA), coenzyme A disulfide, and short-chain acyl-coenzyme A thioesters was addressed by developing a tailored metabolite profiling method based on liquid chromatography in combination with tandem mass spectrometric detection (LC-MS/MS). A reversed phase chromatographic separation was established which is capable of separating a broad spectrum of CoA, its corresponding derivatives, and their isomers despite the fact that no ion-pairing reagent was used (which was considered as a key advantage of the method). Excellent analytical figures of merit such as high sensitivity (LODs in the nM to sub-nM range) and high repeatability (routinely 4 %; N = 15) were obtained. Method validation comprised a study on standard purity, stability, and recoveries during sample preparation. Uniformly labeled U(13)C yeast cell extracts offered ideal internal standards for validation purposes and for a quantification exercise in the rumen bacterium Megasphaera elsdenii.
authors.affiliation: Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences-BOKU, 1190, Vienna, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Neubauer
authors.forename: Dinh Binh
authors.initials: DB
authors.lastname: Chu
authors.forename: Hans
authors.initials: H
authors.lastname: Marx
authors.forename: Michael
authors.initials: M
authors.lastname: Sauer
authors.forename: Stephan
authors.initials: S
authors.lastname: Hann
authors.forename: Gunda
authors.initials: G
authors.lastname: Koellensperger
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D003065:Coenzyme A / Q000032:analysis / Q000378:metabolism*; D045851:Megasphaera / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C017145:coenzyme A disulfide; D003065:Coenzyme A