Isoprostane Measurement in Plasma and Urine by Liquid Chromatography–Mass Spectrometry with One-Step Sample Preparation

Abstract

Background: Isoprostane F 2␣ (iPF 2␣ -III) concentration in plasma and urine is widely accepted as a measure of oxidative stress. Gas chromatography-mass spectrometry (GC/MS) methods for measuring iPF 2␣ -III involve several steps of sample preparation and are labor-intensive, and ELISA methods, although easier to use, are less reliable. Therefore we developed a simple and sensitive method involving 1-step sample cleanup and HPLC/MS quantification. Methods: Samples of plasma or urine were enriched with a deuterated (iPF 2␣ -III-D4) standard, treated with KOH to liberate the bound isoprostanes, then loaded onto an immunoaffinity column, and the bound isoprostane was eluted with 95% ethanol. The concentrated sample was injected onto a C-18 HPLC column, and the isoprostane was eluted with a gradient of acetonitrile in water and analyzed by electrospray negative ionization, selectively monitoring the ions 353.2 (iPF 2␣ -III) and 357.2 (iPF 2␣ -III-D4). The amount of isoprostane in the sample was calculated from the ratio of the intensities of the 2 ions. Results: The described method has a detection limit of 0.5 ng/L, with a linear dynamic range of 1-5000 ng/L. The intra-and interassay imprecisions were 4.68% and 3.88%, respectively. The values obtained correlated strongly with the GC/MS procedure (r ‫؍‬ 0.80), but the absolute values were ϳ4 -to 5-fold lower, because the present method measures specifically 1 isomer of isoprostane, whereas the GC/MS method measures 4 isomers together.

Dried blood spot (DBS) sample collection for determination of the oxidative stress biomarker 8‐epi‐PGF2α in humans using liquid chromatography/tandem mass spectrometry

“…[23] Since biomarkers and metabolites are present in low concentrations inbiological matrixes, and sample volumes are often limited, sensitive and specific techniques such as liquid chromatography/tandem mass spectrometry (LC/MS/MS) or gas chromatography/mass spectrometry (GC/MS) are the most frequently used techniques for 8-epi-PGF 2a measurements. [24] Determination of 8-epi-PGF 2a in plasma and urine has been reported in previous studies, [25][26][27] but to our knowledge there have been no other studies of 8-epi-PGF 2a in whole blood except for our recent study. [20] In this report, LC/MS/MS methods were used for 8-epi-PGF 2a detection in DBS and the stability of this marker on DBS was studied.…”

Differences in myocardial PTEN expression and Akt signalling in type 2 diabetic and nondiabetic patients undergoing coronary bypass surgery

“…In brief, 500 μl of plasma was pretreated, spiked with 15-F 2t -isoprostane-D 4 internal standard and applied to an 8-isoprostane affinity column (Cayman Chemical, Ann Arbor, MI, USA) as previously described 17 . The eluent was dried in a speedvac, and resuspended in water containing 2% acetonitrile and 0.01% NH 4 OH for subsequent LC-MS analysis.…”

EIA and GC/MS analysis of 8-isoprostane in EBC of children with problematic asthma

“…More recently, new methods for 8-iso-PGF 2a analysis, based on liquid chromatographic mass spectrometry (LC/MS), have been developed, offering the advantage of a simpler sample preparation than in GC/MS because no derivatisation of the molecule is required [30]. Despite major advances in the sensitivity of LC/MS instrumentation, one concern with these assays relates to the detection limits in biological fluids, which are often higher than those using GC/MS [30,31]. For these reasons we can infer that the GC/MS analysis can still be considered the reference analytical method.…”