title: Derivatization of the tricarboxylic acid cycle intermediates and analysis by online solid-phase extraction-liquid chromatography-mass spectrometry with positive-ion electrospray ionization.
abstract: The analysis of cellular metabolic processes is of fundamental biological interest. Cellular metabolites, such as the intermediates of the tricarboxylic acid (TCA) cycle, provide essential information about the metabolic state of the cell. Not only is the TCA cycle a key factor in the energy regulation within aerobic cells, it possibly also plays a role in cell signaling. This paper describes a novel derivatization strategy, using the empirically selected N-methyl-2-phenylethanamine as derivatization reagent with a carbodiimide as co-reagent, for the selective derivatization of carboxylic acids, such as the di- and tri-carboxylic acids of the TCA cycle. Optimization of the derivatization protocol is described. This procedure enables analysis of the derivatives using on-line solid-phase extraction and reversed-phase liquid chromatography in combination with sensitive positive-ion electrospray ionization mass spectrometry. The complete procedure, involving the use of core-shell silica column material, allows the rapid analysis of TCA cycle intermediates in sample matrices, here shown for pig heart tissue extracts, with a good linearity over 3-4 orders of magnitude. Detection limits range from 12 to 1000 nM, depending on the analyte.
authors.affiliation: VU University Amsterdam, Faculty of Sciences, BioMolecular Analysis Group, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: D
authors.initials: D
authors.lastname: Kloos
authors.forename: R J E
authors.initials: RJ
authors.lastname: Derks
authors.forename: M
authors.initials: M
authors.lastname: Wijtmans
authors.forename: H
authors.initials: H
authors.lastname: Lingeman
authors.forename: O A
authors.initials: OA
authors.lastname: Mayboroda
authors.forename: A M
authors.initials: AM
authors.lastname: Deelder
authors.forename: W M A
authors.initials: WM
authors.lastname: Niessen
authors.forename: M
authors.initials: M
authors.lastname: Giera
mesh_terms: D000818:Animals; D002234:Carbodiimides; D002247:Carbon Isotopes; D002264:Carboxylic Acids / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D002952:Citric Acid Cycle; D006224:Cricetinae; D007700:Kinetics; D057230:Limit of Detection; D008694:Methamphetamine / Q000031:analogs & derivatives; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013552:Swine; D053719:Tandem Mass Spectrometry; D013696:Temperature; D014020:Tissue Extracts / Q000737:chemistry
chemical_list: D002234:Carbodiimides; D002247:Carbon Isotopes; D002264:Carboxylic Acids; D014020:Tissue Extracts; C027141:N-methylphenethylamine; D008694:Methamphetamine