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  • LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters.

LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters.

Analytical and bioanalytical chemistry (2015-07-15)
Stefan Neubauer, Dinh Binh Chu, Hans Marx, Michael Sauer, Stephan Hann, Gunda Koellensperger
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
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Methanol, anhydrous, 99.8%
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Ammonium acetate, BioXtra, ≥98%
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Ammonium acetate solution, for molecular biology, 7.5 M
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Ammonium acetate, for molecular biology, ≥98%
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Ammonium acetate, 99.999% trace metals basis
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Methanol, NMR reference standard
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Methanol-12C, 99.95 atom % 12C
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Methanol solution, (Methanol:Dimethyl sulfoxide 1:1 (v/v))
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Acetonitrile, electronic grade, 99.999% trace metals basis
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Acetonitrile, anhydrous, 99.8%
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Malonyl coenzyme A lithium salt, ≥90% (HPLC)
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Acetoacetyl coenzyme A sodium salt hydrate, cofactor for acyl transfer