In vitro stable isotope labeling for discovery of novel metabolites by liquid chromatography-mass spectrometry: Confirmation of γ-tocopherol metabolism in human A549 cell

Wen Chu Yang, Fred E. Regnier, Qing Jiang, Jiri Adamec

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

A general approach for discovering novel catabolic metabolites from a parent biocompound was developed and validated on the metabolism of γ-tocopherol in human A549 cell. The method is based on LC-MS analysis of in vitro stable isotope-labeled metabolites and assumes that a parent compound and its metabolites share a common functional group that can be derivatized by well-documented reagents. In this method, two equal aliquots of extracted metabolites are separately derivatized with isotope-coded (heavy) and non-isotope-coded (light) form of derivatizing reagent, mixed at 1:1 ratio and analyzed using LC-MS. The metabolites with common functional group are then easily recognized by determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to the mass difference between heavy and light form of derivatization reagent. The feasibility of this approach was demonstrated and validated by the identification of products of γ-tocopherol catabolism in human A549 cell culture media using N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NANHS) and N-methyl-d3-nicotinic acid N-hydroxysuccinimide ester (C1-d3-NANHS) derivatizing reagent. Overall four γ-tocopherol metabolites were identified including 9′-COOH, 11′-COOH, 13′-COOH and 13′-OH. In addition, the developed LC-MS method can also be used for the fast and sensitive quantitative analysis of γ-tocopherol and other forms of vitamin E related compounds.

Original languageEnglish (US)
Pages (from-to)667-675
Number of pages9
JournalJournal of Chromatography A
Volume1217
Issue number5
DOIs
StatePublished - Jan 29 2010

Keywords

  • Carboxychromanol
  • Group Specific Internal Standard Technology (GSIST)
  • LC-MS
  • Metabolite discovery
  • Stable isotope labeling
  • Tocopherol
  • Vitamin E

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

Fingerprint Dive into the research topics of 'In vitro stable isotope labeling for discovery of novel metabolites by liquid chromatography-mass spectrometry: Confirmation of γ-tocopherol metabolism in human A549 cell'. Together they form a unique fingerprint.

Cite this