TitleBenefits of prolonged gradient separation for high-performance liquid chromatography-tandem mass spectrometry quantitation of plasma total 15-series F-isoprostanes.
Publication TypeJournal Article
Year of Publication2006
AuthorsTaylor AW, Bruno RS, Frei B, Traber MG
JournalAnal Biochem
Volume350
Issue1
Pagination41-51
Date Published2006 Mar 01
ISSN0003-2697
KeywordsAdolescent, Adult, Arachidonic Acid, Ascorbic Acid, Chromatography, High Pressure Liquid, Dinoprost, Enzyme-Linked Immunosorbent Assay, F2-Isoprostanes, Female, Gas Chromatography-Mass Spectrometry, Humans, Mass Spectrometry, Reproducibility of Results, Tobacco Smoke Pollution
Abstract

The F(2)-isoprostanes are products of free-radical-induced oxidation of arachidonic acid (AA) that are stereoisomers of prostaglandin F(2alpha) (PGF(2alpha)). We describe a method for quantitation of several 15-series PGF isomers (15-PGFs) and AA by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). Plasma samples were subjected to alkaline hydrolysis and acidified, and total (free + esterified) 15-PGFs and AA were extracted with organic solvents. The analytes were separated by gradient reverse-phase HPLC and detected by multiple reaction monitoring on a triple-quadrupole mass spectrometer, using deuterated internal standards for quantitation. The assay had a linear range of 1-40 pg of 8-iso-PGF(2alpha) on column and can quantify as little as 40 pg/mL (0.11 nM) in plasma. Outcomes significantly correlated (p < 0.0001) with data obtained by gas chromatography-mass spectrometry GC-MS or enzyme-linked immunosorbent assay. All plasma 15-PGF isomers increased over time with in vitro cigarette smoke exposure and correlated (p < 0.0001) with each other. The same strong inter-15-PGF correlations were observed in plasma from healthy young adult subjects. The coefficients of variation of HPLC-MS-MS measurements (24-32%) were smaller than those obtained by GC-MS (53%). Thus, HPLC-MS-MS potentially offers greater precision and allows quantitation of more compounds with simpler sample preparation than existing methods. Ours is the first validated quantitative assay using HPLC-tandem MS applied to plasma total 15-PGFs.

DOI10.1016/j.ab.2005.12.003
Alternate JournalAnal. Biochem.
PubMed ID16448621
Grant ListAG05144 / AG / NIA NIH HHS / United States
AG16835 / AG / NIA NIH HHS / United States
DK59576 / DK / NIDDK NIH HHS / United States