Gas chromatography-mass spectrometry of 4-hydroxynonenal in tissues

Frederik J.G.M. Van Kuijk, David W. Thomas, Robert J. Stephens, Edward A. Dratz

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26 Scopus citations

Abstract

In this chapter, gas chromatography–-mass spectrometry (GC-MS)-based method is discussed for the analyses of secondary lipid peroxidation products based on the formation of pentafluorobenzy (PFB) oxime derivatives from 4-hydroxyalkenals. The method improves the extraction efficiency for 4-hydroxyalkenals in biological systems by the formation of an oxime derivative, analogous to the methods used for determination of retinals. An excess of hydroxylamine or O-ethylhydroxylamine has been used to liberate vitamin A aldehydes from Schiff base binding sites. The specific procedure developed in the chapter for the 4-hydroxyalkenals is based on the use of O-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA-HCI). This reagent forms the O-pentafluorobenzyl oxime derivatives of 4-hydroxyalkenals from free aldehyde or from aldehydes bound to amino groups by Schiff base linkages. The volatility of the 4-hydroxyalkenal oxime is increased by the formation of trimethylsilyl (TMS) ethers of the hydroxyl functions. These derivatives can be detected with high sensitivity by GC-MS with negative ion chemical ionization (NICI)). This approach is inspired by the successful use of PFB ester TMS ether derivatives of hydroxy fatty acids. Isotopically labeled dideuterated analogs of 4-hydroxyalkenals are used as internal standards, which allow 4-hydroxynonenal analyses in quantitative measurements. The method is illustrated in the chapter by data derived from retinas of rats.

Original languageEnglish (US)
Pages (from-to)399-406
Number of pages8
JournalMethods in enzymology
Volume186
Issue numberC
DOIs
StatePublished - Jan 1 1990

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