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

• Isoprostanes were extracted from plasma and tissue using liquid–liquid extraction.
• Gas chromatography–tandem mass spectrometry utilized as the detection method.
• Healthy human samples were 150–250 pg/ml and from clinical populations >300 pg/ml.
• Results were >96% accurate with recovery >90% and a coefficient of variance of 7%.
• Extensive troubleshooting required with an unidentified interfering peak detected.

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.