Quantification of the oxidative damage biomarker 2,3-dinor-8-isoprostaglandin-F2α in human urine using liquid chromatography–tandem mass spectrometry

Abstract

F2-isoprostanes are useful biomarkers of oxidative status in humans. We developed an ultraperformance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 2,3-dinor-8-iso prostaglandin F 2α , an urinary metabolite of 8-iso-prostaglandin F 2α. Urine was purified by solid phase extraction and analyzed by UPLC-MS/MS with negative ion-electrospray ionization. The method was robust with a mean inaccuracy of 9%, interday and intraday imprecision of 7.5% or lower, and a lower limit of quantification of 0.5 μg/L, equivalent to 0.04 pmol injected onto the column. An analysis time of 6 minutes was shorter than previously published methods and amenable to large studies. Keywords Oxidative damage biomarker; UPLC-MS/MS; F2-isoprostanesVariability in the capacity of antioxidant defense systems to protect against reactive oxygen species (ROS) may contribute to individual differences in susceptibility to chronic diseases [1]. F2-isoprostanes (iPF 2α ), non-enzymatic products of arachidonic acid peroxidation, are considered sensitive and specific biomarkers of oxidative status. Among them, iPF 2α -III (8-iso-PGF 2α ) and iPF 2α -VI have been identified in human urine [2]. Increased iPF 2α -III levels have been reported in patients with cardiovascular disease [3], Alzheimer disease [4], and type 2 diabetics [5] and increased iPF 2α -VI in patients with Down syndrome [6]. Both were elevated in patients with lung disorders [7] and in heavy smokers [8;9]. 2,3-dinor-8-iso-prostaglandin F 2α (2,3-dinor-iPF 2α -III) is a metabolite of 8-iso-PGF 2α in humans with greater urinary excretion than the parent compound. It is useful as a biomarker for the overall formation of 8-iso-PGF 2α and by extension, lipid peroxidation in vivo. Traditional analytical approaches for the quantification of isoprostanes include immunological methods (ELISA and RIA) and mass Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. . The signal ratio of the primary transitions was used to quantify the analyte and the secondary transitions were used as purity checks. The peak area ratio of the primary to secondary transition was determined for each specimen and compared with a pre-determined value derived from calibration samples. This ratio remained consistent over time in calibration samples (mean ratio: 10.3±1.51; RSD =15%, n=49, over 18 weeks). Deviation from this ratio would suggest the presence of an interfering compound. SRM chromatograms corresponding to the primary and secondary transitions for 2,3-dinor-iPF 2α -III and iPF 2α -III-d 4 are shown in Fig 1A (standard mix) and 1B (contro...

The isoprostanes—25 years later

“…A multitude of papers have been published describing different analytical methods for the quantification of F 2 -IsoPs in biological fluids [34–58]. Three primary techniques are used to measure these compounds: (1) gas chromatography–mass spectrometry, (2) liquid chromatography–mass spectrometry, and (3) immunoassays (ELISAs).…”

Cross-Classification of Human Urinary Lipidome by Sex, Age, and Body Mass Index

“…Three metabolites, 9-HOTrE (hydroxyoctadecatrienoic acid), 9-KODE (oxooctadecadienoic acid,) and 9-HODE (hydroxyoctadecadienoic acid) (Fig 3a), which belong to 5-LOX pathway oxylipins [27] and putative oxidative stress markers, showed approximately 2-fold higher levels in elderly women than in elderly men. The level of 2,3-dinor-8-iso-PGF 2α , a non-enzymatic AA metabolite and an oxidative stress marker [28], was even greater in elderly women, too (Fig 3b). …”

Quantification of fatty acid oxidation products using online high-performance liquid chromatography tandem mass spectrometry

“…Additionally, one may incorporate a distinct stable isotope labeled internal standard of the parent fatty acid to monitor and control for any artificial oxidation products that form during lipid extraction[2, 8, 9, 12, 19, 23, 25, 37]. Preferably these are universally labeled 13 C unsaturated fatty acid derivatives; however, the deuterated analogs are generally less expensive and more readily available from commercial sources.…”

“…Aside from exploring the bulk oxidation, many of these oxidized lipids and their associated oxidized fatty acids have activities of significant clinical interest[3–6, 10, 14, 18, 20, 22, 26–31]. Numerous GC, LC[32], and hybrid methodologies like GC/MS[3, 5, 14, 18, 20, 22, 31] and LC/MS[2–4, 8–10, 12, 19, 23, 27, 33] exist to quantify the levels of these compounds. These markers can be used clinically to ascertain the effectiveness of antioxidant therapies; however, there are no universal guidelines regarding the scale of the analyses required for the determination of lipid oxidation (and hence fatty acid oxidation) products in a large clinical study.…”