Derivatization of the tricarboxylic acid intermediates with O-benzylhydroxylamine for liquid chromatography-tandem mass spectrometry detection

  • Tan B
  • Lu Z
  • Dong S
 et al. 
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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 alpha-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.

Author-supplied keywords

  • *Citric Acid Cycle
  • Cell Line, Tumor
  • Chromatography, Liquid
  • Cytokines/antagonists & inhibitors/metabolism
  • Derivatization
  • Enzyme Inhibitors/pharmacology
  • Humans
  • Hydroxylamines/*chemistry
  • Lc-ms/ms
  • Mass Spectrometry/*methods
  • Neoplasm Proteins/antagonists & inhibitors/metabol
  • Neoplasms/*metabolism/pathology
  • Nicotinamide Phosphoribosyltransferase/antagonists
  • O-benzylhydroxylamine
  • Tricarboxylic Acids/analysis/chemistry/*metabolism
  • Tricarboxylic acid cycle intermediates

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Authors

  • B Tan

  • Z Lu

  • S Dong

  • G Zhao

  • M S Kuo

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