Gas chromatography-mass spectrometry of 4-hydroxynonenal in tissues

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.