title: Quantitative analysis of intracellular coenzymes in Saccharomyces cerevisiae using ion pair reversed phase ultra high performance liquid chromatography tandem mass spectrometry.
abstract: A fast, sensitive and specific analytical method, based on ion pair reversed phase ultrahigh performance liquid chromatography tandem mass spectrometry, IP-RP-UHPLC-MS/MS, was developed for quantitative determination of intracellular coenzyme A (CoA), acetyl CoA, succinyl CoA, phenylacetyl CoA, flavin mononucleotide, (FMN), flavin adenine dinucleotide, (FAD), NAD, NADH, NADP, NADPH. Dibutylammonium acetate (DBAA) was used as volatile ion pair reagent in the mobile phase. Addition of DBAA to the sample solutions resulted in an enhanced sensitivity for the phosphorylated coenzymes. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP·HCl), was added to keep CoA in the reduced form. Isotope dilution mass spectrometry (IDMS) was applied for quantitative measurements for which culture derived global U-(13)C-labeled cell extract was used as internal standard. The analytical method was validated by determining the limit of detection, the limit of quantification, repeatability and intermediate precision. The method was successfully applied for quantification of coenzymes in the cell extracts of Saccharomyces cerevisiae.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands. Electronic address: r.malekiseifar@tudelft.nl.
authors.forename: Reza Maleki
authors.initials: RM
authors.lastname: Seifar
authors.forename: Cor
authors.initials: C
authors.lastname: Ras
authors.forename: Amit T
authors.initials: AT
authors.lastname: Deshmukh
authors.forename: Katelijne M
authors.initials: KM
authors.lastname: Bekers
authors.forename: Camilo A
authors.initials: CA
authors.lastname: Suarez-Mendez
authors.forename: Ana L B
authors.initials: AL
authors.lastname: da Cruz
authors.forename: Walter M
authors.initials: WM
authors.lastname: van Gulik
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Heijnen
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003067:Coenzymes / Q000032:analysis*; D055432:Metabolomics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003067:Coenzymes
keywords: Coenzymes; Isotope dilution mass spectrometry; Metabolomics; Saccharomyces cerevisiae, Ion pair LC–MS/MS; Targeted metabolite analysis