title: Optimized method for quantification of total F(2)-isoprostanes using gas chromatography-tandem mass spectrometry.
abstract: F2-isoprostanes are produced from the oxidative degradation of arachidonic acid and are considered the gold standard marker of lipid peroxidation in biological samples. We developed a liquid-liquid extraction method for the determination of total isoprostanes using negative chemical ionization gas chromatography-tandem mass spectrometry in plasma and tissue homogenates. Incorporating liquid-liquid extraction allows for greater sample through-put than current approaches. Here we describe the protocol and include numerous trouble-shooting suggestions. The method found healthy individuals with 150-250 pg of isoprostanes per ml of plasma and end stage kidney disease patients to have the highest measured values of up to 1100 pg/ml. This assay has an accurate working linear range of 40-1000 pg of isoprostanes (100-2500 pg/ml) and an average coefficient of variance of 7%. Tissue values for healthy mice liver were 50-70 pg/μg protein. This method provides increased ion selectivity and detection capabilities with economical sample through-put.
authors.affiliation: Exercise and Oxidative Stress Research Group, School of Human Movement Studies, University of Queensland, Brisbane, Australia; School of Medicine, University of Queensland, Brisbane, Australia.
authors.forename: David R
authors.initials: DR
authors.lastname: Briskey
authors.affiliation: Exercise and Oxidative Stress Research Group, School of Human Movement Studies, University of Queensland, Brisbane, Australia.
authors.forename: Gary R
authors.initials: GR
authors.lastname: Wilson
authors.affiliation: Exercise and Oxidative Stress Research Group, School of Human Movement Studies, University of Queensland, Brisbane, Australia.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Fassett
authors.affiliation: Exercise and Oxidative Stress Research Group, School of Human Movement Studies, University of Queensland, Brisbane, Australia. Electronic address: jcoombes@uq.edu.au.
authors.forename: Jeff S
authors.initials: JS
authors.lastname: Coombes
mesh_terms: D000328:Adult; D000818:Animals; D016022:Case-Control Studies; D028441:F2-Isoprostanes / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007676:Kidney Failure, Chronic / Q000503:physiopathology*; D015227:Lipid Peroxidation; D059625:Liquid-Liquid Extraction; D008099:Liver / Q000378:metabolism; D051379:Mice; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055815:Young Adult
chemical_list: D028441:F2-Isoprostanes
keywords: Gas chromatography; Lipid peroxidation; Liquid–liquid extraction; Oxidative stress; Tandem mass spectrometry