Streamlined F2-isoprostane analysis in plasma and urine with high-performance liquid chromatography and gas chromatography/mass spectroscopy

F-2-Isoprostanes in plasma and urine are generally determined by labor-intensive methods requiring sample purification by solid-phase extraction and thin-layer chromatography (TLC), A streamlined and more sensitive method for the measurement of esterified plasma F-2-isoprostanes was developed by replacing these steps with high-performance liquid chromatography (HPLC) using an amino column with a hexane/2-propanol gradient. Pentafluorobenzyl esters of F-2-isoprostanes were prepared and purified by HPLC, silylated, and then analyzed by gas chromatography (GC) with negative chemical ionization mass spectroscopy (NCI-MS). This method permits analysis with lower plasma volumes (100 mu L) and greater sensitivity (to 10 pg; allowing detection to 50 pg/mL) than provided by other methods. Urinary F-2-isoprostanes can also be efficiently quantified by this method, with 8-iso-PGF(2 alpha) being identified as a major isomer, With this procedure, esterified plasma F-2-isoprostanes were found to be 8.3 fold higher in an end-stage renal failure patient on hemodialysis and urinary 8-iso-PGF(2 alpha) was 7.1-fold higher in a cigarette smoker than respective control subjects. This method, particularly the substitution of the TLC step common to other methods with HPLC, results in a more sensitive and reproducible assay. (C) 2000 Academic Press.