Optimized method for quantification of total F2-isoprostanes using gas chromatography-tandem mass spectrometry

F-2-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/mu g protein. This method provides increased ion selectivity and detection capabilities with economical sample through-put. (C) 2013 Elsevier B.V. All rights reserved.