Simultaneous analysis of retinol, all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction

Tom Teerlink, Marcel P. Copper, Ingeborg Klaassen, Boudewijn J.M. Braakhuis

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22 Citations (Scopus)

Abstract

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-transretinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.

Original languageEnglish
Pages (from-to)83-92
Number of pages10
JournalJournal of Chromatography B: Biomedical Applications
Volume694
Issue number1
DOIs
Publication statusPublished - 20 Jun 1997

Keywords

  • Retinoic acid isomers
  • Retinol
  • Vitamins

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