title: Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry.
abstract: A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly (13)C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology, Julianalaan 67, Delft, The Netherlands. r.malekiseifar@tudelft.nl
authors.forename: Reza M
authors.initials: RM
authors.lastname: Seifar
authors.forename: Cor
authors.initials: C
authors.lastname: Ras
authors.forename: Jan C
authors.initials: JC
authors.lastname: van Dam
authors.forename: Walter M
authors.initials: WM
authors.lastname: van Gulik
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Heijnen
authors.forename: Wouter A
authors.initials: WA
authors.lastname: van Winden
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D007554:Isotopes / Q000032:analysis*; D009711:Nucleotides / Q000032:analysis* / Q000737:chemistry; D010408:Penicillium chrysogenum / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007554:Isotopes; D009711:Nucleotides