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 journalArticle

31 Citations (Scopus)

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

Fingerprint

Isotope Labeling
Tocopherols
Liquid chromatography
Metabolites
Metabolism
Liquid Chromatography
Isotopes
Labeling
Mass spectrometry
Mass Spectrometry
Niacin
Esters
Light
Functional groups
Vitamin E
Culture Media
Cell Culture Techniques
Cell culture
In Vitro Techniques
A549 Cells

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

Cite this

In vitro stable isotope labeling for discovery of novel metabolites by liquid chromatography-mass spectrometry : Confirmation of γ-tocopherol metabolism in human A549 cell. / Yang, Wen Chu; Regnier, Fred E.; Jiang, Qing; Adamec, Jiri.

In: Journal of Chromatography A, Vol. 1217, No. 5, 29.01.2010, p. 667-675.

Research output: Contribution to journalArticle

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