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

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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 U13C yeast cell extracts offered ideal internal standards for validation purposes and for a quantification exercise in the rumen bacterium Megasphaera elsdenii.

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Authors and Affiliations

  1. Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences-BOKU, 1190, Vienna, Austria

    Stefan Neubauer, Dinh Binh Chu & Stephan Hann

  2. Austrian Center of Industrial Biotechnology (ACIB), Muthgasse 18, 1190, Vienna, Austria

    Stefan Neubauer, Michael Sauer, Stephan Hann & Gunda Koellensperger

  3. Department of Analytical Chemistry, School of Chemical Engineering, Hanoi University of Science and Technology, No. 1 Dai Co Viet Road, Hanoi, Vietnam

    Dinh Binh Chu

  4. Department of Biotechnology, University of Natural Resources and Life Sciences-BOKU, 1190, Vienna, Austria

    Hans Marx & Michael Sauer

  5. CD Laboratory for Biotechnology of Glycerol, Muthgasse 107, 1190, Vienna, Austria

    Hans Marx & Michael Sauer

  6. Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Währinger Straße 38, 1090, Vienna, Austria

    Gunda Koellensperger

Authors
  1. Stefan Neubauer
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  2. Dinh Binh Chu
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  3. Hans Marx
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  4. Michael Sauer
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  5. Stephan Hann
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  6. Gunda Koellensperger
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Corresponding author

Correspondence to Gunda Koellensperger.

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Neubauer, S., Chu, D.B., Marx, H. et al. LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters. Anal Bioanal Chem 407, 6681–6688 (2015). https://doi.org/10.1007/s00216-015-8825-9

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  • DOI: https://doi.org/10.1007/s00216-015-8825-9

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