title: Derivatization of the tricarboxylic acid intermediates with O-benzylhydroxylamine for liquid chromatography-tandem mass spectrometry detection.
abstract: The tricarboxylic acid (TCA) cycle is an interface among glycolysis, lipid metabolism, and amino acid metabolism. Increasing interest in cancer metabolism has created a demand for rapid and sensitive methods for quantifying the TCA cycle intermediates and related organic acids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify the TCA cycle intermediates in a 96-well format after O-benzylhydroxylamine (O-BHA) derivatization under aqueous conditions. This method was validated for quantitation of all common TCA cycle intermediates with good sensitivity, including α-ketoglutarate, malate, fumarate, succinate, 2-hydroxyglutarate, citrate, oxaloacetate, pyruvate, isocitrate, and lactate using a 8-min run time in cancer cells and tissues. The method was used to detect and quantify changes in metabolite levels in cancer cells and tumor tissues treated with a pharmacological inhibitor of nicotinamide phosphoribosyl transferase (NAMPT). This method is rapid, sensitive, and reproducible, and it can be used to assess metabolic changes in cancer cells and tumor samples.
authors.affiliation: Tailored Therapeutics, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA. Electronic address: tanbo76@hotmail.com.
authors.forename: Bo
authors.initials: B
authors.lastname: Tan
authors.affiliation: Cancer Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
authors.forename: Zhaohai
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Cancer Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
authors.forename: Sucai
authors.initials: S
authors.lastname: Dong
authors.affiliation: Cancer Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
authors.forename: Genshi
authors.initials: G
authors.lastname: Zhao
authors.affiliation: Tailored Therapeutics, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
authors.forename: Ming-Shang
authors.initials: MS
authors.lastname: Kuo
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D002952:Citric Acid Cycle; D016207:Cytokines / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006898:Hydroxylamines / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D054409:Nicotinamide Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014233:Tricarboxylic Acids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016207:Cytokines; D004791:Enzyme Inhibitors; D006898:Hydroxylamines; D009363:Neoplasm Proteins; D014233:Tricarboxylic Acids; C017064:benzyloxyamine; D054409:Nicotinamide Phosphoribosyltransferase; C085455:nicotinamide phosphoribosyltransferase, human
keywords: Derivatization; LC–MS/MS; O-benzylhydroxylamine; Tricarboxylic acid cycle intermediates