Benefits of prolonged gradient separation for high-performance liquid chromatography-tandem mass spectrometry quantitation of plasma total 15-series F-isoprostanes.
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.
Taylor
Alan W
AW
Linus Pauling Institute, Oregon State University, Corvallis, 97331, USA. Alan.Taylor@oregonstate.edu
Bruno
Richard S
RS
Frei
Balz
B
Traber
Maret G
MG
D000293:Adolescent; D000328:Adult; D016718:Arachidonic Acid / Q000097:blood; D001205:Ascorbic Acid / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015237:Dinoprost / Q000031:analogs & derivatives / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D028441:F2-Isoprostanes / Q000097:blood*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D015203:Reproducibility of Results; D014028:Tobacco Smoke Pollution
D028441:F2-Isoprostanes; D014028:Tobacco Smoke Pollution; C075750:8-epi-prostaglandin F2alpha; D016718:Arachidonic Acid; D015237:Dinoprost; D001205:Ascorbic Acid