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 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 2โฃ 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 2โฃ 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.