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
Date Published2006 Mar 01
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

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.

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